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Originally published online as doi:10.1189/jlb.0503230 on August 11, 2003

Published online before print August 11, 2003
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(Journal of Leukocyte Biology. 2003;74:889-896.)
© 2003 by Society for Leukocyte Biology

Generation and characterization of mice transgenic for human IL-18-binding protein isoform a

Giamila Fantuzzi*,1, Nirmal K. Banda{dagger}, Carla Guthridge{dagger}, Andrea Vondracek{dagger}, Soo-Hyun Kim*, Britta Siegmund*,2, Tania Azam*, Joseph A. Sennello*, Charles A. Dinarello* and William P. Arend{dagger}

Divisions of
* Infectious Diseases and
{dagger} Rheumatology, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO

1Correspondence: University of Colorado Health Sciences Center, 4200 East Ninth Avenue B168, Denver, CO 80262. E-mail: Giamila.Fantuzzi{at}UCHSC.edu


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ABSTRACT
 
Interleukin (IL)-18 binding protein (IL-18BP) is a natural inhibitor of the pleiotropic cytokine IL-18. To study the role of IL-18BP in modulating inflammatory responses in vivo, mice transgenic for human IL-18BP isoform a (IL-18BP-Tg) were generated. The transgene was expressed at high levels in each organ examined. High levels of bioactive human IL-18BPa were detectable in the circulation of IL-18BP-Tg mice, which were viable, fertile, and had no tissue or organ abnormality. The high levels of IL-18BP in the transgenic mice were able to completely neutralize the interferon-{gamma} (IFN-{gamma})-inducing activity of exogenously administered IL-18. Following administration of endotoxin, with or without prior sensitization with heat-inactivated Propionibacterium acnes, IL-18BP-Tg mice produced significantly lower serum levels of IFN-{gamma} and macrophage-inflammatory protein-2 compared with nontransgenic littermates. Significantly reduced production of IFN-{gamma} in response to endotoxin was also observed in cultures of IL-18BP-Tg splenocytes. Finally, IL-18BP-Tg mice were completely protected in a model of hepatotoxicity induced by administration of concanavalin A. These results indicate that high endogenous levels of IL-18BP in trangenic mice effectively neutralize IL-18 and are protective in response to different inflammatory stimuli.

Key Words: cytokines • interferon • endotoxin • hepatitis


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INTRODUCTION
 
Interleukin (IL)-18 is a pleiotropic cytokine produced by a variety of cell types, including monocytes, macrophages, dendritic cells, and epithelial cells [1 ]. In addition to acting as a potent costimulus for production of interferon-{gamma} (IFN-{gamma}) by natural killer (NK) and T cells, IL-18 induces production of several proinflammatory cytokines and chemokines [2 3 4 ]. Other activities of IL-18 include the potentiation of T and NK cell cytotoxicity, mediated by Fas and/or perforin [5 6 7 ]. IL-18 has been implicated in various inflammatory and autoimmune conditions. Thus, neutralization experiments have demonstrated a critical role for IL-18 in lipopolysaccharide (LPS)-induced liver injury in mice primed with heat-inactivated Propionibacterium acnes [2 ]. Furthermore, neutralization of IL-18 or blocking of IL-18 processing by caspase-1 confers resistance to LPS-induced lethality in mice [8 ]. In this model, protection is associated with reduced production of IFN-{gamma} and the chemokine macrophage inflammatory protein-2 (MIP-2). Among other diseases, IL-18 blockade is protective in models of immune-mediated liver injury and colitis [9 10 11 12 13 ].

The extracellular domain of most cytokine receptors is cleaved from the cell surface by proteases and is found in the circulation and in the urine [14 ]. In the case of the IL-1 family of receptors, soluble IL-1 type I and type II receptors are each found in the circulation of healthy humans, and recombinant forms can be used to neutralize IL-1 activity. Even the extracellular domain of the IL-1 orphan receptor T1/ST2 is shed [15 ], although its ligand has not been identified. It is also likely that the extracellular domains of the IL-18 receptor (IL-18R){alpha} and IL-18Rß chains are present in the serum of healthy subjects, although there are presently no published studies indicating their levels. Recombinant forms of the IL-18R{alpha} and IL-18Rß have been studied for their ability to neutralize IL-18 [16 ] but require relatively high concentrations compared with neutralization by the naturally occurring IL-18 binding protein (IL-18BP), which is a unique molecule in that its affinity for IL-18 is greater than the binding of IL-18 to cell-bound IL-18Rs [17 , 18 ]. IL-18BP is constitutively secreted [14 , 19 ] and neutralizes IL-18 activity at equimolar ratios [17 ], and its amino acid sequence is only distantly related to the ligand-binding chain IL-18R{alpha} [20 ]. It appears that IL-18BP may have evolved as a member of the IL-1 receptor family but lost its transmembrane domain and retained only a single immunoglobulin (Ig)-like domain, whereas the other members of the IL-1R family contain three Ig-like domains [21 ]. There is another member of the IL-1R family with a single Ig-like domain [22 ].

Four isoforms of IL-18BP have been described in humans and two in mice; these isoforms derive from alternative splicing of a single gene in each species [23 ]. In humans, only isoforms a and b, which contain an intact Ig domain, display IL-18-inhibiting activity [23 ]. Isoform a has the highest affinity for IL-18 [17 ] and recombinant IL-18BPa has therefore been used to neutralize IL-18 in various models of disease [9 , 24 ]. IL-18BPa is also a potential pharmacological therapy for inhibiting IL-18 in humans.

Although administration of exogenous IL-18BP to reduce the biological effects of endogenous IL-18 in short-term models of disease is a valid strategy, this approach has limitations in mouse models of chronic disease, such as collagen-induced arthritis [25 ], nonobese diabetic mice [26 ], atherosclerosis in apolipoprotein-1-deficient mice [27 ], or graft versus host disease [28 ]. These and other chronic disease models are easier to study using mice with high levels of endogenous IL-18BP, which do not require repeated injections of recombinant mouse IL-18BP. Furthermore, as administration of IL-18BP is a therapeutic strategy in treating human disease, the effect of chronic reduction in endogenous IL-18 in host resistance to infection can be best studied in mice overexpressing IL-18BP rather than in mice deficient in IL-18 [29 ]. To evaluate whether high levels of endogenous IL-18BP could block IL-18 activity and therefore display anti-inflammatory activities, we generated transgenic mice overexpressing human IL-BP isoform a (IL-18BP-Tg mice). In the present report, the generation of these mice is described together with their characterization in terms of transgene expression and ability to neutralize IL-18-mediated effects.


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MATERIALS AND METHODS
 
Generation of IL-18BP-Tg mice
The Animal Care and Use Committee of the University of Colorado Health Sciences Center (Denver) approved all studies. To generate IL-18BP-Tg mice, a 592-base pair (bp) fragment (bases 51–643) of the human IL-18BP a gene was cloned into the EcoRI sites of the pCAGGs expression vector. The sequence of the IL-18BPa insert was confirmed by automated sequencing. The pCAGGs/IL-18BPa vector was maxiprepped, isolated by CsCl gradients, and transiently transfected into RAW264.7 cells to confirm expression. The IL-18BPa genomic fragment was released from the vector backbone using SalI/HindIII digestion and then separated from the pCAGGs vector using low melt-point agar and transverse alternating field-gel electrophoresis. The band of interest was visualized and removed under long-range UV light. The fragment was purified by liquefying the agarose at 70°C, adding an equal volume of phenol, and centrifuging for 10 min at maximum speed. The purified 2878-bp fragment was then injected into oocytes from hepatitis B virus mice using standard protocols.

Genetic screening
To screen for potential IL-18BP-Tg founders in the population of putative transgenic pups, DNA was extracted from tail samples by phenol/chloroform extraction. The DNA was purified and then analyzed by Southern blot after digestion with 5 U EcoRI overnight at 37°C. Using Ready-to-Go DNA labeling beads (Amersham Pharmacia Biotech, Piscataway, NJ), a probe was prepared by adding 45 µl heat-denatured human IL-18BPa DNA to a microcentrifuge tube containing a reaction mix bead, 5 µl [32P]dCTP (3000 ci/mmol) and 5 µl dH2O. Confirmed founders were then back-crossed into DBA/1J mice. As detailed below, pups were subsequently tested by polymerase chain reaction (PCR), Western blot, enzyme-linked immunosorbent assay (ELISA), and bioassay to confirm transmission of the transgene.

PCR genotyping
For PCR analysis, tail DNA was obtained using the Quantum Prep AquaPure genomic DNA kit (Biorad, Hercules, CA) according to the manufacturer’s instructions. RNA was isolated from different tissues using TRIzol reagent (Life Technologies, Gaithersburg, MD). Control RNA from four non-Tg littermates was processed in an identical manner. cDNA was generated from 5 µg total RNA by reverse transcriptase (RT)-PCR using oligo-dt primers. PCR was performed with 5 µl generated cDNA using 1 µl dNTPs (10 mM), 1 µl 3'-primer, 1 µl 5'-primer, 1 µl 50 mM MgCl2, 5 µl PCR buffer, 0.5 µl Taq polymerase, and 35 µl H2O. The PCR conditions included 30 cycles of 1.5 min denaturation at 94°C, 2 min annealing at 62°C, and 2.5 min extension at 72°C. The following primers were used: 5' primer, 5'-ACA CCT GTC TCG CAG ACC AC-3', and 3' primer, 5'-TCA GCT GCT CCA GCA CCA A-3'.

Western blot
The human IL-18BP amino acid sequence 143-STNFSCVLVDPEQVVQRHVVL-163 was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Immunizing rabbits with the KLH-conjugated synthetic peptide (Research Genetics, Huntsville, AL) generated a polyclonal anti-IL-18BP antibody. This antibody was affinity-purified and used as the primary antibody in Western blot analyses at a 1:500 dilution overnight. After washing, membranes were then incubated with a donkey anti-rabbit horseradish peroxidase (HRP)-conjugated antibody, and the reaction was visualized using a Super Signal enhanced chemiluminescence kit (Pierce, Rockford, IL).

ELISA for IL-18BPa
An ELISA was developed to quantify levels of human IL-18BPa in the serum of Tg mice. Plates were coated overnight with an affinity-purified goat anti-human IL-18BPa IgG preparation (400 ng/ml, R&D Systems, Minneapolis, MN). After washing, plates were incubated for 1 h with samples and standards. Plates were then washed and incubated for 1 h with 100 ng/ml biotinylated affinity-purified goat anti-human IL-18BPa IgG (R&D Systems). After washing, HRP-conjugated streptavidin was added. The reaction was stopped, and the plates were read at 450 nm. The ELISA did not cross-react with any of the murine IL-18BP isoforms.

IL-18BP bioassay
To evaluate IL-18BP inhibitory activity, a bioassay in which IL-18 induces production of IFN-{gamma} was used [23 ]. NK cells (a subclone of the human NK92 line, kindly provided by Tomoaki Hoshino [30 ]) were maintained in supplemented RPMI-1640 medium containing 10% fetal bovine serum, 50 pg/ml IL-2, and 200 pg/ml IL-15. The bioassay was performed in 96-well microtiter plates at a volume of 200 µl. Briefly, IL-18 (20 ng/ml) was preincubated with 5 µl IL-18BP-Tg and non-Tg mouse serum in 0.1 ml RPMI. NK cells (1x106/ml) were added in 0.1 ml RPMI with 0.5 ng/ml IL-12 and 10 ng/ml IL-18 (both from Peprotech, Inc., Rocky Hill, NJ). After 16–20 h incubation at 37°C in humidified air with 5% CO2, the culture supernatant was collected for IFN-{gamma} measurement.

Histologic analysis
Mice were anesthetized with isofluorane, and blood samples were collected for determination of complete blood counts and clinical chemistry. Eleven organs were removed and fixed in formalin for histological analysis. The liver and spleen of each animal were weighed, and organ to body-weight ratios were calculated. An individual blinded to the genetic status of the animals performed all evaluations.

In vivo studies
Responses to the combination of IL-12 and IL-18 were studied in mice receiving four daily intraperitoneal injections of murine recombinant IL-12 (200 ng/mouse/day, Peprotech) plus murine recombinant IL-18 (1000 ng/mouse day, Peprotech). Control mice received saline. Mice were weighed daily. Two hours after the fourth injection, mice were bled, and sera were prepared for evaluation of IFN-{gamma} levels by ELISA (BD PharMingen, San Diego, CA).

For evaluation of responses to P. acnes and LPS, mice received an intravenous (i.v.) injection of 1 mg/mouse of heat-killed P. acnes in the tail vein (Ribi Immuno-Chem Research, Hamilton, MT). One week later, the mice received an i.v. injection of 2 µg/mouse Escherichia coli LPS O55:B5 (Sigma Chemical Co., St. Louis, MO). Six hours later, mice were bled from the retro-orbital plexus under isofluorane anesthesia, and serum was prepared for determination of IFN-{gamma} and MIP-2 levels.

To investigate the response to LPS alone, mice received an i.v. injection of 600 µg/mouse E. coli LPS O55:B5. Control mice received an i.v. injection of saline. Mice were bled and killed 6 h after injection for measurement of serum IFN-{gamma} and MIP-2 levels.

The effect of high levels of IL-18BPa on the response to concanavalin A (Con A) was studied in mice treated with an i.v. injection of 200 µg/mouse Con A (Sigma Chemical Co.). Control mice received saline. Mice were bled 24 h later for evaluation of serum alanine aminotransferase (ALT) levels.

In vitro studies
Age-matched IL-18BP-Tg and non-Tg mice were killed, and spleens were removed. Single-cell suspensions were prepared in RPMI supplemented with 10% fetal calf serum. Cells were washed, resuspended at 2 x 106/ml, and then incubated in triplicate for 24 or 48 h with increasing concentrations of LPS. IFN-{gamma} levels were measured in the culture supernatants.


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RESULTS
 
Generation of IL-18BP-Tg mice
Following injection of the human IL-18BPa DNA fragment into oocytes, 144 transgenic embryos were created and transplanted into pseudo-pregnant FVB/N mothers. Of the 20 resultant pups, three mice were positive by Southern blot analysis. It was also confirmed that these mice contained circulating, bioactive IL-18BP. These three founding mice were each back-crossed into the DBA/1J background for six generations. The transgene was successfully transmitted to ~50% of the pups from each litter, as evaluated by Southern blot analysis, PCR analysis, ELISA, and bioassay. The male/female ratio was ~50% in IL-18BP-Tg and non-Tg littermates.

Expression of human IL-18BPa in Tg mice
RT-PCR analysis, performed in mice from the F3 and F6 generations, demonstrated that the transgene was expressed in the IL-18BP-Tg mice in every organ evaluated (liver, brain, small intestine, large intestine, lungs, skin, spleen, heart, kidney, stomach, muscle; Fig. 1A ). Serum obtained from IL-18BP-Tg mice was positive for IL-18BPa, as evaluated by Western blot analysis (Fig. 1B) . Quantification of human IL-18BPa levels in the sera of Tg mice using an ELISA indicated the presence of varying levels. The range of human IL-18BPa serum levels was predominantly between 500 and 2500 ng/ml, and several animals reached an excess of 5000 ng/ml. As expected, human IL-18BPa was below detection limits in sera obtained from non-Tg littermates. Evaluation of IL-18BP bioactivity revealed the presence of a strong IFN-{gamma}-inhibitory activity in the serum of IL-18BP-Tg mice but not in non-Tg littermates (Fig. 2 ).



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  		Figure 1. Expression of human IL-18BP in Tg mice. (A) Total RNA extracted from the organs of IL-18BP-Tg mice was analyzed by RT-PCR for expression of the human IL-18BPa transgene. Expression was positive in each organ evaluated. -ve control, Empty vector; +ve control, vector containing the IL-18BP transgene; THP-1 cells, RNA extracted from the human myelomonocytic cell line THP-1 (negative control). (B) Western blot analysis of serum obtained from IL-18BP-Tg and non-Tg mice. The numbers correspond to eartag numbers of individual mice. The symbols + or - indicate the genotype as evaluated by PCR (+, IL-18BP-Tg; -, non-Tg).



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  		Figure 2. Neutralization of IL-18 by IL-18BP-Tg serum. The IFN-{gamma}-inhibitory activity of sera obtained from five IL-18BP-Tg (+) and five non-Tg (-) mice was analyzed in a bioassay as described in Materials and Methods. The third column represents the IFN-{gamma}-inducing ability of a combination of IL-12 + IL-18 without any mouse serum added. Sera obtained from IL-18BP-Tg mice indicated decreased IFN-{gamma} production in response to a combination of IL-12 and IL-18 (fourth column). In contrast, sera obtained from non-Tg mice indicated no significant change in IFN-{gamma} production (fifth column). Data are expressed as the mean ± SEM. ***, P < 0.001, by ANOVA.

Histologic analysis
Organs obtained from three IL-18BP-Tg and three non-Tg littermates were histologically evaluated and found to be clinically normal. Spleen and liver weight relative to body weight did not differ between Tg and non-Tg mice. Clinical chemistry values and hematology findings did not differ between Tg and non-Tg mice.

Neutralization of exogenously administered IL-18 in vivo in IL-18BP-Tg mice
To determine whether the high levels of IL-18BPa present in Tg mice were able to neutralize the bioactivity of exogenously administered IL-18 in vivo, mice were injected with a combination of a low dose of murine recombinant IL-12 and murine recombinant IL-18, as described previously and with minor modifications [31 , 32 ]. Coadministration of a low dose of IL-12 is necessary, as administration of IL-18 alone fails to induce IFN-{gamma} [33 ]. During the course of the 4-day treatment with IL-12 and IL-18, non-Tg mice had a 7% body-weight loss, whereas no reduction in body weight was observed in IL-18BP-Tg mice (Fig. 3A ). Accordingly, at the end of the treatment period, high serum IFN-{gamma} levels were present in non-Tg mice, whereas IFN-{gamma} remained below detection limits in the sera of IL-18BP-Tg mice (Fig. 3B) . These data indicate that the high levels of human IL-18BPa present in Tg mice were able to effectively neutralize exogenously administered IL-18.



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  		Figure 3. Response of IL-18BP-Tg mice to IL-12 and IL-18 administration. IL–18BP-Tg (open symbols) and non-Tg (filled symbols) mice received daily injections of IL-12 and IL-18 as described in Materials and Methods. (A) Body weight. (B) Two hours after the last injection, mice were bled, and serum IFN-{gamma} levels were measured. Data are expressed as the mean ± SEM of five mice per group. *, P < 0.05, and ***, P < 0.001, by ANOVA.

Reduced in vivo IFN-{gamma} and MIP-2 production in IL-18BP-Tg mice
Induction of IFN-{gamma} and of the chemokine MIP-2 in response to LPS is dependent on endogenous production of IL-18 [8 ]. To determine whether the high levels of IL-18BPa present in Tg mice were able to neutralize the bioactivity of endogenously produced IL-18 in vivo, two separate experimental models were used. In the first set of experiments, mice were injected with P. acnes and subsequently with LPS, as described in Materials and Methods. As shown in Figure 4A and 4B , this treatment induced very high serum levels of IFN-{gamma} and MIP-2 in non-Tg mice. Levels of these two cytokines were markedly and significantly reduced in the sera of IL-18BP-Tg mice. Accordingly, a more severe liver damage was observed in non-Tg compared with IL-18BP-Tg mice, as evaluated by serum ALT levels (257±123 vs. 89±14 in non-Tg vs. IL-18BP-Tg mice, respectively; P<0.05 by Student’s t-test).



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  		Figure 4. Serum levels of IFN-{gamma} and MIP-2 in IL-18BP-Tg mice receiving P. acnes and LPS. IL–18BP-Tg (open bars) and non-Tg (solid bars) mice received an i.v. injection of P. acnes. One week later, mice were injected with LPS, and sera were obtained 6 h later. (A) Serum levels of IFN-{gamma}; (B) serum levels of MIP-2. Data are expressed as the mean ± SEM of five mice per group. *, P < 0.05, and **, P < 0.01, by ANOVA.

The effect of IL-18BPa overexpression on cytokine levels induced by administration of LPS alone was also evaluated. Figure 5A and 5B , shows that induction of IFN-{gamma} and MIP-2 by LPS was significantly reduced in IL-18BP-Tg mice compared with non-Tg mice receiving LPS.



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  		Figure 5. Serum levels of IFN-{gamma} and MIP-2 in IL-18BP-Tg mice in response to LPS. IL-18BP-Tg (open bars) and non-Tg (solid bars) mice received an i.v. injection of LPS. Sera were obtained 6 h later. (A) Serum levels of IFN-{gamma}; (B) serum levels of MIP-2. Data are expressed as the mean ± SEM of five mice per group. *, P < 0.05, by ANOVA.

Reduced in vitro IFN-{gamma} production in splenocytes from IL-18BP-Tg mice
Production of IFN-{gamma} by spleen cells stimulated with LPS is dependent on endogenously produced IL-18 [34 , 35 ]. Splenocytes from IL-18BP-Tg and non-Tg mice were stimulated in vitro with LPS, and production of IFN-{gamma} was evaluated. As shown in Figure 6 , when stimulated with LPS for 24 h, spleen cells from IL-18BP-Tg mice produced significantly lower amounts of IFN-{gamma} compared with cells obtained from non-Tg mice. Comparable results were obtained with cells stimulated for 48 h (data not shown).



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  		Figure 6. LPS-induced IFN-{gamma} production from IL-18BP-Tg splenocytes in vitro. Splenocytes were obtained from IL-18BP-Tg (open symbols) and non-Tg (filled symbols) mice. Cells were cultured for 24 h in the presence of increasing concentrations of LPS. IFN-{gamma} levels were measured in the culture supernatants. Data are expressed as the mean ± SEM of three mice per group. *, P < 0.05, by ANOVA.

Protection from Con A-induced liver necrosis in IL-18BP-Tg mice
In vivo administration of Con A induces hepatotoxicity [36 ]. In this model, liver damage is dependent on IL-18 but not on IFN-{gamma} [10 ]. To determine whether the high levels of IL-18BPa present in Tg mice could neutralize IFN-{gamma}-independent effects of IL-18, IL-18BP-Tg and non-Tg mice were injected with Con A, and their serum ALT levels were measured 24 h later. As indicated in Figure 7 , the Con A-induced increase in serum levels of ALT was completely suppressed in IL-18BP-Tg mice.



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  		Figure 7. IL-18BP-Tg mice are resistant to Con A-induced hepatotoxicity. IL-18BP-Tg (open bar) and non-Tg (solid bar) mice received an i.v. injection of Con A. Twenty-four hours later, mice were bled, and serum ALT levels were measured. Data are expressed as the mean ± SEM of five mice per group. ***, P < 0.001, by ANOVA.


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DISCUSSION
 
In the present article, we report the generation and characterization of transgenic mice overexpressing human IL-18BP isoform a. The transgene was expressed at high levels in each organ analyzed, and high levels of bioactive IL-18BP were detectable in the circulation. Yet, IL-18BP-Tg mice were viable and did not show any tissue or organ abnormality. This observation indicates that high levels of bioactive IL-18BP do not influence fertility, embryonic development, or metabolic and tissue homeostasis, thus implying that IL-18 activity is not necessary for mouse development, as previously shown in IL-18 knockout (KO) mice [37 ]. Similar to IL-18 KO mice, IL-18BP-Tg mice also did not develop infections or spontaneous pathological changes, indicating that IL-18 activity is dispensable for immune homeostasis under unchallenged conditions [37 ]. It was only upon challenge with inflammatory stimuli that differences between IL-18BP-Tg and non-Tg mice became evident. However, we have only analyzed IL-18BP-Tg mice on a DBA/1J background. Strain-related differences in development of spontaneous pathology have been described for IL-1 receptor antagonist-deficient mice [38 , 39 ]. Therefore, the possibility remains that a phenotype might develop when IL-18BP-Tg mice are back-crossed into a different strain. Studies are currently in progess to investigate this issue.

We initially determined that the IL-18BP produced by the transgene was bioactive. The ability of IL-18BP-Tg serum to neutralize IL-18 in the NK cell bioassay indicated in vitro bioactivity. In addition, the lack of IFN-{gamma} production and body-weight loss in IL-18BP-Tg mice injected with IL-12 and IL-18 was clear evidence that circulating, endogenously produced transgenic human IL-18BP was able to completely neutralize exogenously administered IL-18 in vivo. Hence, these results clearly indicate that the high levels of human IL-18BP present in Tg mice maintain full bioactivity.

Once the bioactivity of transgenic IL-18BP was verified, we examined whether the presence of high levels of this endogenous inhibitor could protect mice from IL-18-mediated inflammatory pathologies. To this aim, we chose three acute models in which a critical role for IL-18 has been previously demonstrated using anti-IL-18 antibodies, administration of exogenous recombinant IL-18BP, and/or IL-18 KO mice.

IL-18 was originally isolated from the livers of mice pretreated with P. acnes and subsequently challenged with LPS [40 ]. Under these conditions, liver Kupffer cells produce massive amounts of IL-18, which then synergize with IL-12 in stimulating high levels of IFN-{gamma} and other proinflammatory cytokines, with eventual induction of liver necrosis [6 , 7 ]. When challenged with P. acnes/LPS, IL-18 KO mice do not develop hepatotoxicity and exhibit significantly reduced serum levels of IFN-{gamma} [41 ]. Furthermore, neutralization of IL-18 with exogenous IL-18BP provides protection against P. acnes/LPS-induced hepatotoxicity and reduces induction of IFN-{gamma} [9 ]. In agreement with these results, in the present report, we show that high levels of endogenous human IL-18BPa are able to significantly protect mice from P. acnes/LPS-induced production of IFN-{gamma} and MIP-2.

The second model evaluated consisted of a single injection of LPS without prior sensitization. In mice, high doses of LPS induce lethality through induction of tumor necrosis factor {alpha} and IFN-{gamma}. Neutralization of either of these cytokines or their gene deletion confers significant protection [42 , 43 ]. Previous studies indicated that IL-18 is also a critical player in the inflammatory response to LPS. In fact, IL-18 blockade using neutralizing antibodies or the use of caspase-1-deficient mice, in which production of bioactive IL-18 does not take place, demonstrated that IL-18 is necessary for LPS to fully exert its toxicity [8 ]. The likely mechanism by which IL-18 neutralization protects from LPS-induced lethality involves an absence of synergism with IL-12, leading to a reduction in IFN-{gamma} production, although other mechanisms might also be operative [3 ]. Accordingly, our present data indicate that serum levels of IFN-{gamma} and the chemokine MIP-2 are significantly lower in IL-18BP-Tg versus non-Tg mice. However, it is important to note that in agreement with previous reports, IL-18 seems to play a more important role in the P. acnes/LPS model compared with the single LPS injection model. This difference is likely attributable to the marked activation of Kupffer cells, which are major producers of IL-18, by the P. acnes sensitization step.

When isolated splenocytes were stimulated in vitro with LPS, cultures obtained from IL-18BP-Tg mice produced significantly lower levels of IFN-{gamma} compared with non-Tg spleen cell cultures. Splenocyte cultures contain a mixture of immune cell types. LPS initially stimulates monocyte/macrophages to produce IL-12 and IL-18, which then act on T lymphocytes and NK cells to induce synthesis of IFN-{gamma}. We had previously demonstrated that in this system, neutralization of endogenous IL-18 strongly inhibits IFN-{gamma} production [34 ]. Initial evaluation of Tg mice has shown that the transgene is expressed in every organ analyzed. Our present data thus indicate that when placed in culture for up to 48 h, IL-18BP-Tg spleen cells continue to produce high levels of bioactive IL-18BP.

We also evaluated the effects of IL-18BP-Tg in the model of Con A-induced hepatotoxicity, in which direct T cell activation by Con A is followed by production of IL-18 by Kupffer cells [44 ]. This is a well-established model of fulminant hepatitis. Although the pathogenic role of IFN-{gamma} in Con A-induced liver damage is controversial [10 , 45 , 46 ], the role of IL-18 has been widely demonstrated using a multplicity of approaches. Hence, blockade of IL-18 using administration of neutralizing antibodies, exogenous IL-18BP, or inhibitors of caspase-1 protects mice from liver necrosis [9 , 10 , 47 ]. When IL-18BP-Tg mice were injected with Con A, they were completely protected from hepatotoxicity, whereas the non-Tg mice were highly susceptible. These results confirm the critical role played by IL-18 in acute immune-mediated liver necrosis.

In conclusion, the present report indicates that transgenic expression of human IL-18BP in mice leads to production of a highly bioactive molecule, which is able to protect mice in the setting of IL-18-mediated inflammatory models. In particular, high levels of endogenous IL-18BP are effective in reducing inflammatory mediators in models involving Kupffer cell-derived IL-18, further stressing the important role of the IL-18/IL-18BP system in hepatic inflammation. The availability of IL-18BP-Tg mice will be a critical tool in the evaluation of the role of this cytokine inhibitor in experimental animal models of acute and chronic inflammatory conditions.


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ACKNOWLEDGEMENTS
 
A grant from the Cystic Fibrosis Foundation (to G. F.), National Institutes of Health Grants AI46374 (to W. P. A.) and AI15614 and HL68743 (to C. A. D.), and the Deutsche Forschungsgemeeinschaft Grants DFG SI 749/2-1 and 749/3-1 (to B. S.) supported this work.


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FOOTNOTES
 
2 Current address: Universitätsklinikum Benjamin Franklin, Medizinische Klinik I, Hindenburgdamm 30, 12200 Berlin, Germany. Back

Received May 19, 2003; revised July 14, 2003; accepted July 18, 2003.


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