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Amgen, Inc., Thousand Oaks, CA 91320
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production
by KG1 cells (EC50 = 0.3 µg/ml). In mice challenged
with an LD90 of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg)
administered 10 min before LPS blocks IFN- production and protects
against lethality. IL-18BP-Fc administered 10 min before LPS blocks
IFN- production induced by LPS (5 mg/kg) with ED50 of
0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5
mg/kg)-induced IFN- production even when administered 6 days before
LPS but shows no effect when administered 9 or 12 days before LPS.
Given 10 min before LPS challenge to mice primed 12 days in advance
with heat-killed Propionibacterium acnes, IL-18BP-Fc
prevents LPS-induced liver damage and IFN- and Fas ligand
expression. Given at the moment of priming with P.
acnes, IL-18BP-Fc decreases P. acnes-induced
granuloma formation, macrophage-inflammatory protein-1
and
macrophage-inflammatory protein-2 production and prevents sensitization
to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-
and Fas ligand expression as well as liver damage induced by
Pseudomonas aeruginosa exotoxin A or by anti-Fas
agonistic Ab. In conclusion, IL-18BP can be engineered and produced in
recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed
with remarkable in vitro and in vivo properties of binding and
neutralizing IL-18. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by NK and T cells (2, 3), especially in synergy
with IL-12 (4), which has been shown to up-regulate
IL-18R (5). IL-18 enhances Fas ligand
(FasL)2 expression and
cytotoxicity of NK and T cells, which mediate IL-18 antitumoral
activity (6). In addition to directing the Th1 response,
IL-18 is a macrophage activator, triggering the production of various
proinflammatory cytokines, including TNF, IL-1, and the chemokines IL-8
and macrophage-inflammatory protein (MIP)-1 (7, 8, 9). In
vivo, IL-18 has been shown to be involved in both physiological and
pathological processes, playing important roles in host defense and
inflammation. Experiments with anti-IL-18 Abs have demonstrated
that IL-18 is critical for LPS-induced liver injury in primed mice with
heat-killed Propionibacterium acnes (2).
Furthermore, IL-18 is a pivotal mediator of endotoxic shock, because
IL-18 blockade has been shown to confer resistance to LPS-induced
lethality in mice (10). Recently, IL-18 has also been
shown to act as a primary proinflammatory cytokine during the onset
of streptococcal cell wall-induced arthritis (11). In this
condition, IL-18 blockade ameliorates joint pathology by reducing TNF
and IL-1 levels (11). IL-18-binding protein (IL-18BP) is a naturally occurring protein that binds and neutralizes IL-18 (12). IL-18BP has a single Ig domain and has limited homology to the members of the IL-1 receptor family. However, IL-18BP is not a receptor molecule and does not have a transmembrane domain. IL-18BP is a constitutively secreted protein, and it is not cleaved from the cell surface. IL-18BP binds IL-18 with specificity and high affinity and blocks its biological activities (12). Four isoforms resulting from mRNA splicing have been described for human IL-18BP (13). The binding site resides in the Ig domain. As a consequence, only two isoforms, a and b, which possess the complete Ig domains, neutralize IL-18 with high affinity. The two other isoforms, c and d, are unable to neutralize IL-18 (13). Isoform a, the one with highest affinity for IL-18, represents an ideal candidate for blocking IL-18 to treat human disease (14).
We have prepared a construct in which a sequence encoding isoform a of human IL-18BP is attached to a sequence encoding human IgG1 Fc. This construct was expressed in mammalian cells to produce IL-18BP-Fc. In this study, we have investigated the ability of IL-18BP-Fc to bind and neutralize IL-18 in vitro and in vivo to establish whether this construct has the potential to be used in patients to treat IL-18-mediated disorders. We have particularly focused on the ability of IL-18BP-Fc to treat Fas/FasL-mediated models of liver disease in mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A DNA sequence encoding the entire molecule (aa 1–192) of human
IL-18BP isoform a (12) was designed,
synthesized by in vitro amplification of a template of DNA
oligonucleotides, and fused in frame to either a sequence encoding the
C-terminal 235 aa of human IgG1 Fc for IL-18BP-Fc or a sequence
encoding a six-histidine tag for IL-18BP-His. The resulting constructs
were ligated within the pDSR2–1 expression vector containing the
dehydrofolate reductase (DHFR) gene (Amgen, Thousand Oaks, CA) and
transfected into DHFR-deficient Chinese hamster ovary cells (Amgen)
using the Invitrogen calcium phosphate transfection kit (Invitrogen
Life Technologies, Carlsbad, CA). Transfected cells were selected for
DHFR expression and subcloned. The resulting clones were screened for
IL-18BP-Fc or IL-18BP-His secretion by Western blot of the culture
medium with a polyclonal anti-IL-18BP Ab (Amgen). Clones showing
high levels of secretion were expanded into roller bottles with the
production of conditioned medium containing IL-18BP-Fc or IL-18BP-His.
IL-18BP-Fc was purified by chromatography on protein G-Sepharose
(Amersham Pharmacia Biotech, Piscataway, NJ) and found to be a 150-kDa
homodimer >95% pure by SDS-PAGE and endotoxin free by the
Limulus amebocyte lysate test. IL-18BP-His was purified by
successive chromatographies on HiTrap Chelating Sepharose and HiTrap
Phenyl Sepharose HP (both from Amersham Pharmacia Biotech) and found to
be a 40-kDa molecule also >95% pure and endotoxin free. The sequence
encoding the C-terminal 235 aa of human IgG1 Fc was also expressed
alone to generate Fc for use as a control.
BIAcore affinity determination
The binding affinities of IL-18BP-Fc for human, mouse, and rat IL-18 were determined using a BIAcore 2000 apparatus (BIAcore, Uppsala, Sweden). Human, mouse, and rat IL-18 were obtained as Escherichia coli-expressed recombinant proteins from R&D Systems (Minneapolis, MN). Determinations were conducted at 25°C using PBS containing 0.005% Surfactant P20 as running buffer. Protein G was immobilized onto a CM5 chip following manufacturer’s instructions to capture IL-18BP-Fc. IL-18 was serially diluted from 0.3 µM to 0.14 nM in sample buffer (PBS with 0.005% Surfactant P20 and 100 µg/ml BSA) before injection over the surface with the captured IL-18BP-Fc at 50 µl/min for 3 min. IL-18 samples were also injected over a blank protein G surface to subtract any nonspecific binding background. The protein G surfaces were regenerated with sequential injection of 100 µl ImmunoPure IgG elution buffer (Pierce, Rockford, IL) and 100 µl 1 M NaCl containing 8 mM glycine, pH 1.5, at 50 µl/min between cycles. Binding affinities (KD) of IL-18BP-Fc for IL-18 were determined by nonlinear regression analysis using BIAevaluation 3.1 software (BIAcore).
In vitro IL-18 bioassay
KG1 human myeloid leukemia cells (American Type Culture
Collection, Manassas, VA) were cultured at the concentration of 3
x 106/ml in complete RPMI 1640 (Life
Technologies, Gaithersburg, MD) with 10% FBS (Life Technologies) and
25 x 10-3 M HEPES for 24 h at 37°C
and stimulated in triplicates to produce IFN- with E.
coli-expressed recombinant IL-18 (100 ng/ml; Amgen) and TNF (20
ng/ml; Amgen) in the presence or absence of different concentrations of
IL-18BP-Fc. After culture, cells were frozen and thawed twice, and
supernatant was collected and tested for IFN- by ELISA.
Animals
Female BALB/c and C57BL/6 mice, weighing 
20 g, were obtained
from Charles River Laboratories (Wilmington, MA) and The Jackson
Laboratory (Bar Harbor, ME), respectively. Male Lewis rats, weighing
350 g, were obtained from Charles River Laboratories. Animals were
housed in rooms maintained at constant temperature and humidity under
12 h light/dark cycle and fed with normal rodent chow and water
ad libitum.
LPS-induced IFN- production and lethality
For lethality studies, BALB/c mice were injected i.p. with 15
mg/kg LPS (E. coli 0111:B4; List Biological Laboratories,
Campbell, CA) and monitored for survival. Ten minutes before LPS, mice
received 5 mg/kg IL-18BP-Fc i.p. or Fc as a control. For IFN-
production studies, BALB/c mice were injected i.p. with LPS, and blood
was collected 6 h after LPS. In the dose-response experiment,
doses of IL-18BP-Fc or IL-18BP-His between 0 and 5 mg/kg were injected
i.p. 10 min before LPS (5 mg/kg). In the time course experiment, mice
were given 5 mg/kg IL-18BP-Fc or IL-18BP-His i.p. on day 12, 9, 6, 3,
or 1 before LPS (5 mg/kg). Control mice received no IL-18BP-Fc or
IL-18BP-His before LPS. Rats were injected i.v. with 1 mg/kg LPS and
bled 6 h later; 2 mg/kg IL-18BP-Fc or Fc were administered i.p. 10
min before LPS.
P. acnes/LPS-, Con A-, Pseudomonas aeruginosa exotoxin A (PEA)-, and anti-Fas agonistic Ab-induced liver damage
To induce liver damage with LPS after priming with P.
acnes, BALB/c mice were given 500 µg/mouse i.v. of heat-killed
P. acnes (Ribi Immuno-Chem Research, Hamilton, MT), and
12 days later they were challenged with 50 µg/kg LPS i.v. IL-18BP-Fc
(5 mg/kg) was administered i.p. either at the time of P.
acnes administration or 10 min before LPS challenge. To induce
liver damage with Con A or PEA, C57BL/6 mice received i.v. either 7.5
mg/kg Con A or 300 µg/kg PEA. To induce liver damage with
anti-Fas agonistic Ab, BALB/c mice were injected i.v. with the
hamster anti-mouse Fas mAb Jo2 (BD PharMingen, San Diego, Ca) at
doses between 1 and 10 µg/mouse. IL-18BP-Fc (5 mg/kg) was
administered i.p. 10 min before Con A or PEA or anti-Fas agonistic
Ab. Control mice received Fc in all experiments. Mice were monitored
for survival or sacrificed to collect livers for histological
examination, mRNA and chemokine measurements, and blood for serum
IFN- and transaminase measurements at various times, as indicated,
after the administration of P. acnes, LPS, Con A, PEA, or
anti-Fas agonistic Ab.
Histological examination
Blocks of liver tissue were fixed in zinc formalin, embedded in paraffin, and sectioned. P. acnes-induced granulomas were counted by light microscopy. Tissue sections were stained for the F4/80 Ag by indirect immunoperoxidase technique using a rat mAb (Serotec, Raleigh, NC) and diaminobenzidine as the chromogen, and counterstained with hematoxylin. For each mouse, one section was used to prepare six adjacent images using a microscope with a 10x objective interfaced to a digital camera (Spot Camera; Diagnostic Instruments, Sterling Heights, MI). For each image, granulomas (F4/80-positive) and parenchyma were traced, and the number of granulomas per square millimeter of parenchyma area was determined by image analysis using Metamorph software (version 4.5r4; Universal Imaging, West Chester, PA). Con A-induced liver damage was evaluated by light microscopy. Tissue sections were stained with H&E. One section per animal was subjectively scored for hepatocellular necrosis using the following designations: 0 for normal appearance; 1 for minimal; 2 for mild; 3 for moderate; and 4 for marked necrosis.
Measurement of IFN- and FasL mRNA
IFN- and FasL mRNA were measured by RNase protection assay in
livers collected 6 h after the administration of LPS or Con A.
After homogenization of the livers, total RNA was extracted using the
RNA Stat-60 solution (Tel-Test, Friendswood, TX). Extracted RNA was
quantitated by spectrophotometry. Antisense riboprobes were prepared by
in vitro transcription of cloned DNA templates with SP6 (for IFN-
and FasL mRNA) or T3 (for cyclophilin mRNA) RNA polymerases (Ambion,
Austin, TX), labeled with [32P]UTP, and
purified by PAGE and elution in ammonium acetate buffer containing EDTA
and SDS. Organ-extracted RNA (5 µg) was hybridized with
105 cpm of each labeled riboprobe. Unhybridized
RNA was digested with RNases A and T1 (Ambion). Hybridized and
RNase-protected RNA was precipitated, washed, and electrophoresed on
polyacrylamide gel. Hybrids containing cytokine mRNA were
electrophoresed with hybrids containing cyclophilin mRNA as
housekeeping RNA. The radioactivity of the riboprobes in the hybrids
was quantitated by a PhosphorImager (Molecular Dynamics, Sunnyvale,
CA), and cytokine-housekeeping riboprobe radioactivity ratios were
calculated.
Cytokine and transaminase measurement
IFN- was measured in serum and in culture supernatants by
ELISA using commercially available kits and following manufacturers’
instructions (R&D Systems for human and mouse IFN- and Biosource,
Camarillo, CA, for rat IFN-). MIP-1 and MIP-2 were measured in
liver homogenates by ELISA using commercially available kits as above
(R&D Systems). Livers were homogenized in 4 volumes PBS containing
0.2% Tween 20 and centrifuged for 5 min at 13,000 rpm. Supernatants
were collected for chemokine and total protein measurements. Total
protein content was determined by the Bradford assay (Bio-Rad
Laboratories, Hercules, CA). Aspartate transaminase (AST) and alanine
transaminase (ALT) were measured in serum by enzymatic assays using
commercially available kits and following manufacturer’s instruction
(Hoffman-LaRoche, Basel, Switzerland).
Statistical analysis
Results are expressed as mean ± SE. Parameters were analyzed with the Student t test, but survival was analyzed with the Fisher exact test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The binding affinity of IL-18BP-Fc for IL-18 was determined with a
BIAcore-based method and using sensor chips with immobilized protein G
to capture IL-18BP-Fc. IL-18BP-Fc binds human, mouse, and rat IL-18
with high affinity, as indicated by the rapid association rate, the
slow dissociation rate, and the low dissociation constant (Table I
).
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The ability of IL-18BP-Fc to neutralize human IL-18 was assessed
using a bioassay based on the production of IFN- by KG1 cells in
response to the combination of IL-18 and TNF. As shown in Fig. 1, IL-18BP-Fc inhibits the production of
IFN- in a dose-dependent manner. The dose-response curve is sigmoid.
Doses of IL-18BP-Fc in the range 1–10 µg/ml inhibit IFN-
production by 80%. The EC50 is 0.3
µg/ml. Doses of IL-18BP-Fc in the range 0.01–0.1 µg/ml inhibit
IFN- production by 10%. Doses lower that 0.01 µg/ml are
ineffective.
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production and lethality in
vivo
To test whether IL-18BP-Fc neutralizes IL-18 in vivo, we first
studied the effects of IL-18BP-Fc on LPS-induced IFN- production and
lethality in mice. Administration of IL-18BP-Fc (5 mg/kg) inhibits by
81% the production of IFN- (Fig. 2A) and reduces to 20% the
lethality (Fig. 2B) induced by a dose of LPS (15 mg/kg)
equivalent to LD90. The inhibitory effect of
IL-18BP-Fc on LPS-induced IFN- production is dose dependent and
still evident at low doses. As shown in Fig. 2C, at doses of
0.5 and 5 mg/kg, IL-18BP-Fc inhibits the production of IFN- induced
by 5 mg/kg LPS by 90%. At 0.05 mg/kg, IL-18BP-Fc inhibits by 75%;
and at 0.005 mg/kg it inhibits by 50%. The inhibitory effect of
IL-18BP-Fc on LPS-induced IFN- production is also long lasting.
IL-18BP-Fc (5 mg/kg) inhibits IFN- production by >90% when
administered up to 6 days before LPS challenge but shows no effect when
administered 9 or 12 days before LPS (Fig. 2D). The
inhibitory effect of IL-18BP-Fc is similar to that of IL-18BP-His with
respect to dose efficacy but not duration. IL-18BP-Fc and IL-18BP-His
show similar dose dependency profiles and ED50
(Fig. 2C), but IL-18BP-His inhibits LPS-induced IFN-
production by >90% only when administered 1 day before LPS challenge
(Fig. 2D). When administered 2 days before LPS, IL-18BP-His
inhibits only by 54% and shows no effect when given 3 days or more
before LPS (Fig. 2D).
IL-18BP-Fc (2 mg/kg) also abrogates the production of IFN- induced
in rats by 1 mg/kg LPS (serum IFN- levels were 3.9 ± 1.9 pg/ml
in IL-18BP-Fc-treated rats compared with 333.6 ± 55.4 pg/ml in
Fc-treated controls, n = 5, p <
0.05).
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We then studied the effects of IL-18BP-Fc on LPS challenge after
P. acnes-priming. Mice were primed with P. acnes
(500 µg/mouse i.v.) and 12 days later challenged with LPS (50 µg/kg
i.v.). IL-18BP-Fc or Fc were administered 10 min before LPS challenge.
After LPS treatment, livers were collected for mRNA measurements, and
blood was collected for IFN- and transaminase measurements.
IL-18BP-Fc reduces liver damage induced by LPS in P.
acnes-primed mice. As shown in Fig. 3A, IL-18BP-Fc-treated mice
have serum AST and ALT levels 69 and 63% lower, respectively,
than Fc-treated controls. IL-18BP-Fc also inhibits the production of
IFN- triggered by LPS challenge by 77% and prevents the increase in
IFN- and FasL mRNA levels in the liver (Fig. 3, B–D).
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We also studied the effects of IL-18BP-Fc on P. acnes
priming and therefore injected IL-18BP-Fc at the time of P.
acnes administration. Livers were collected every 3 days until day
12 for histological examination and chemokine measurements. Twelve days
after priming, mice were challenged with LPS and monitored for survival
or bled for IFN- measurement. We first assessed the effects of
IL-18BP-Fc on granuloma formation in the liver after administration of
P. acnes. IL-18BP-Fc reduces the number of granulomas/liver
area compared with control Fc by 35% (Fig. 4A). To investigate whether
chemokines are involved in granuloma formation, we measured the levels
of MIP-1 and MIP-2 in the liver after the administration of P.
acnes. As shown in Fig. 4, B and C, MIP-1
and MIP-2 levels are increased by injection of P. acnes with
peak levels on day 9. IL-18BP-Fc reduces MIP-1 and MIP-2 levels on
day 9 compared with Fc by 65 and 80%, respectively. This reduction is
no longer detectable, however, when MIP-1 and MIP-2 levels are
measured 12 days after P. acnes administration (Fig. 4, B and C). We next investigated the effect of
IL-18BP-Fc administration at the time of P. acnes priming on
sensitization to LPS. As shown in Fig. 4D, the levels of
IFN- induced by LPS challenge are reduced in the IL-18BP-Fc-treated
mice compared with controls by 42% at the 6th h and by 68% at 10th h
after LPS. Also, the lethality caused by LPS challenge is decreased in
the mice treated with IL-18BP-Fc at the time of priming with P.
acnes compared with controls treated with Fc. Although all the
IL-18BP-Fc-treated mice survived after LPS challenge, only 10% of the
Fc-treated controls did (Fig. 4E).
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We next investigated whether IL-18BP-Fc protects against Con
A-induced hepatotoxicity, which is dependent on T cells. IL-18BP-Fc
protects against Con A-induced liver damage decreasing the serum levels
of AST and ALT by 91 and 93%, respectively, in comparison with control
Fc (Fig. 5A). IL-18BP-Fc does
not change significantly (p = 0.069) the serum
levels of IFN- (Fig. 5B) but reduces the liver levels of
IFN- and FasL mRNA (Fig. 5, C and D).
IL-18BP-Fc reduces the severity of hepatocellular apoptosis as assessed
by histological examination. Foci of apoptosis characterized by the
presence of hepatocytes with hypereosinophilic cytoplasm and pyknotic
nuclei and typically located between the centrolobular vein and the
portal area are hallmarks of Con A-induced liver damage (Fig. 5, E and F). IL-18BP-Fc treatment reduces the size
and the number of these foci compared with control treatment (Fig. 5, F and G). Liver scores were 0.75 ± 0.31 in
IL-18BP-Fc-treated mice compared with 3.12 ± 0.29 in Fc-treated
controls (n = 8, p < 0.01).
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). This reduction is by 51 and 54% at
the 8th h and by 44 and 48% at the 16th h for AST and ALT,
respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production and that prevents liver damage in a series
of models of Fas/FasL-mediated liver disease. Thus, IL-18BP-Fc is a
potential therapeutic for treatment of IL-18-mediated disease in the
clinics. Binding proteins, including soluble receptors, are important regulators of cytokine activities and thereby of the immune response (15). Several viruses produce molecules homologous to binding proteins and use them to escape host immunity (16). Poxviruses in particular have been found to encode proteins homologous to IL-18BP, underscoring its significance as an immunoregulator (17, 18). Binding proteins may either agonize or antagonize the activity of the cytokines they bind. For example, soluble IL-6R binds IL-6 to form a complex that confers responsiveness to IL-6 onto a variety of cells, such as endothelial cells, that do not express transmembrane IL-6R but do express gp130 (19). In contrast, soluble TNFRs act as antagonists of TNF activity (20, 21), and recombinant human TNFRII-Fc (etanercept) has been developed successfully for the therapeutic neutralization of TNF in patients with rheumatoid arthritis (22, 23). To develop a molecule capable of therapeutic neutralization of IL-18, we have engineered and produced in recombinant form a fusion protein comprising the isoform of human IL-18BP endowed with the highest binding affinity for IL-18, namely isoform a, and human IgG1 Fc.
IL-18BP-Fc binds IL-18 with high affinity in human, mouse, and rat
species and inhibits human IL-18 in vitro with low
EC50. IL-18BP-Fc is also very active in vivo.
IL-18BP-Fc protects against LPS-induced lethality, confirming the
essential role of IL-18 in endotoxemia (10, 24, 25, 26). This
protection is associated with strong inhibition of the production of
IFN-, underscoring the importance of IL-18 as an IFN--inducing
factor (2). IL-18BP-Fc displays remarkable ability in
blocking LPS-induced IFN- production, being effective at low doses
and for long periods of time, a consequence of the high binding
affinity and probable long half-life in the circulation, two properties
that the two components of the IL-18BP-Fc molecule would respectively
contribute. Relevant to this regard are the results of the
dose-response and time course experiments in which the inhibitory
effect of IL-18BP-Fc on LPS-induced IFN- production was compared
with that of IL-18BP-His. When administered at varying doses 10 min
before LPS challenge, IL-18BP-Fc and IL-18-His show a similar
dose-dependent effect. However, when administered at a fixed dose at
different times before LPS, IL-18BP-Fc shows a much more prolonged
effect than IL-18BP-His. IL-18BP-Fc virtually abrogates IFN-
production when given up to 6 days before LPS, whereas IL-18BP-His does
this only when given 1 day before LPS. These results indicate that the
addition of an IgG Fc fragment succeeds in modifying IL-18BP
pharmacodynamic properties and in conferring onto it important duration
of action. Only specific pharmacokinetic studies will, however,
establish whether this modification effectively makes IL-18BP more
advantageous in view of a possible use as a therapeutic agent.
IL-18BP-Fc also prevents the development of other models in which IL-18 has been show to play a crucial role, i.e., liver disease models induced by P. acnes/LPS, Con A, PEA, and anti-Fas agonistic Ab (2, 27, 28, 29). Liver damage in these models is Fas mediated and triggered by the induction of FasL expression on local NK and T cells (27, 28, 29, 30, 31, 32). We chose to study these models because the Fas/FasL interaction is thought to be central to the pathogenesis of liver damage that occurs in many varieties of hepatitis in the clinics (33, 34, 35).
IL-18 was originally identified as IFN inducing factor in the serum
and liver of mice treated with LPS after P. acnes priming
(2). This model of liver damage develops in two phases:
the priming phase, induced by P. acnes administration; and
the eliciting phase, which follows LPS challenge (36).
During the priming phase, P. acnes injection induces the
formation of intrahepatic granulomas, mainly composed by activated
lymphocytes and macrophages. Sensitization to LPS is entailed by this
process. During the eliciting phase, LPS-induced liver damage develops.
LPS initially triggers the production of IL-12 and IL-18 by
macrophages. IL-12 and IL-18 stimulate in turn NK and T cells to
produce IFN-. Then IFN-, in combination with IL-18, stimulates
these cytotoxic cells to express FasL, which finally triggers
hepatocyte apoptosis (27, 37). A pivotal role for IL-18 in
the eliciting phase of the P. acnes/LPS-induced liver injury
has previously been demonstrated using either neutralizing
anti-IL-18 Ab or IL-18-deficient mice (2, 26, 27).
Consistently, IL-18BP-Fc is found in this study to inhibit liver
toxicity triggered by LPS after P. acnes priming, as
indicated by the transaminase measurements. At the same time, the
induction of the circulating levels of IFN- is decreased as well as
the expression of IFN- and FasL in the liver, confirming that the
development of hepatic injury in this model depends on IL-18 for the
induction of both critical effectors IFN- and FasL (27, 37). We also show in this study that IL-18BP-Fc beneficially
affects the priming phase of the model. This is an original
demonstration that reveals the role of IL-18 in the processes of
granuloma formation and sensitization to LPS. IL-18BP-Fc decreases the
number of granulomas in the liver induced by P. acnes
administration. This effect may be due, at least in part, to the
inhibition by IL-18BP-Fc of the peak production in the liver of the
chemokines MIP-1 and MIP-2. Chemokines indeed mediate the
recruitment of dendritic cells into liver granulomas after injection of
P. acnes and are able to regulate Kupffer cell mobility
(38). This finding is in agreement with the observation
that IL-18 plays a role in the recruitment of neutrophils in liver and
lungs (10). The inhibitory effect of IL-18BP-Fc on
MIP-1 and MIP-2 production is detectable on day 9 after P.
acnes administration, when the levels of these chemokines reach a
peak, but not on days 3 and 6, when levels are modest. As indicated by
the results of the time course experiment involving LPS-induced IFN-
production, IL-18BP-Fc is still active on day 6 but not day 9 of
administration. These observations suggest that IL-18 is not
responsible for the early production of MIP-1 and MIP-2 but
presumably for other early events following P. acnes
injection that permit MIP-1 and MIP-2 peak production on day 9.
IL-18BP-Fc also decreases sensitization to LPS, as indicated by the
reduced production of IFN- and blocked lethality after LPS challenge
in P. acnes-primed mice treated with IL-18BP-Fc at priming
time. The results from the time course experiment rule out the
possibility that IL-18BP-Fc given at priming time is effective at the
time of LPS challenge, i.e., 12 days after priming, making it therefore
legitimate to draw the conclusion that IL-18BP-Fc inhibits the process
of sensitization of LPS along with that of granuloma formation.
The beneficial effect observed in this study of IL-18BP-Fc against the
liver damage induced by two T cell activators, Con A and PEA, is
consistent with data showing that an anti-IL-18 Ab protects against
Con A- and PEA-induced liver injury (28). Administration
of Con A and PEA induces a cytokine cascade similar to the one
triggered by LPS challenge in P. acnes-primed mice
(28, 31, 39). However, Con A and PEA directly stimulate T
cells to produce IFN- and TNF, which in turn stimulate the
production of IL-18 by macrophages. Then IL-18, in combination with
IFN-, induces FasL expression on cytotoxic cells and causes
hepatotoxicity (27, 37). As observed in P.
acnes-primed mice challenged with LPS, IL-18BP-Fc also reduces the
serum levels of IFN- and the levels of IFN- and FasL mRNA in the
liver after Con A administration. These results indicate that the liver
damage triggered by Con A and PEA is mediated by IL-18 via a mechanism
involving both IFN- production and FasL expression. In contrast,
IL-18BP-Fc only marginally reduces IFN- production after Con A,
whereas completely inhibits LPS-induced IFN- levels. This is likely
to be a consequence of the fact that Con A directly stimulates T cells
to produce IFN-, with no requirement for IL-18 mediation
(40).
We also tested IL-18BP-Fc in a model of liver disease induced by an
anti-Fas agonistic Ab (Jo2). In vivo administration of this Ab in
mice has been shown to trigger severe liver damage leading to fulminant
hepatitis and death through direct Fas ligation on hepatocytes
(41). In contrast, Fas engagement can also stimulate
macrophages to produce IL-18 and cause nonlethal liver injury that both
IL-18 and IFN- mediate with downstream involvement of FasL
(29). We show that IL-18BP-Fc inhibits the liver damage
induced by a low dose of anti-Fas agonistic Ab, demonstrating the
role of IL-18 also in this type of liver injury. These data indicate
the potential therapeutic value of IL-18BP-Fc in the treatment of liver
diseases where organ damage is Fas/FasL-mediated. IL-18BP-Fc fails to
show beneficial effects when the Ab is used at high lethal dose,
probably because in this case the direct apoptotic action of the Ab is
overwhelming and masks the one that proceeds from macrophage activation
via IL-18 and then IFN- and FasL.
Serum levels of IL-18 are elevated in several pathological conditions. IL-18 levels are elevated in the serum of septic patients and of patients with liver diseases such as hepatitis C virus hepatitis, autoimmune hepatitis, and primary biliary cirrhosis, where they have been found to correlate with disease severity (42, 43, 44, 45). IL-18 levels are high in the intestinal mucosa from patients with Crohn’s disease and also in the synovium of patients with rheumatoid arthritis (46, 47, 48). These findings indicate that IL-18 may be a mediator of inflammatory pathology in humans (49, 50), an inhibitor of which would be a desirable therapeutic (14). Natural cytokine inhibitors, such as soluble TNFRs and IL-1R antagonist, are in clinical use in the context of an anti-cytokine strategy (23, 51). Our data indicate that IL-18BP-Fc may represent a useful therapeutic to apply to a variety of inflammatory diseases and prompt its evaluation in patients with hepatitis.
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2 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; IL-18BP, IL-18-binding protein; FasL, Fas ligand; DHFR, dehydrofolate reductase; PEA, Pseudomonas aeruginosa exotoxin A; AST, aspartate transaminase; ALT, alanine transaminase.
Received for publication April 17, 2001. Accepted for publication September 7, 2001.
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