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(American Journal of Pathology. 2004;165:959-967.)
© 2004 American Society for Investigative Pathology

Interleukin-18 Promotes Joint Inflammation and Induces Interleukin-1-Driven Cartilage Destruction

Leo A.B. Joosten*, Ruben L. Smeets*, Marije I. Koenders*, Liduine A.M. van den Bersselaar*, Monique M.A. Helsen*, Birgitte Oppers-Walgreen*, Erik Lubberts*, Yoichiro Iwakura{dagger}, Fons A.J. van de Loo* and Wim B. van den Berg*

From the Rheumatology Research Laboratory and Advanced Therapeutics,* University Medical Center Nijmegen, Nijmegen, The Netherlands; and the Center for Experimental Medicine,{dagger} Institute of Medical Science, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Interleukin (IL)-18 is a member of the IL-1 family of proteins that exerts proinflammatory effects and is a pivotal cytokine for the development of Th1 responses. The goal of the present study was to investigate whether IL-18 induces joint inflammation and joint destruction directly or via induction of other cytokines such as IL-1 and tumor necrosis factor (TNF). To this end we performed both in vitro and in vivo kinetic studies. For in vivo IL-18 exposure studies C57BL/6, TNF-deficient, and IL-1-deficient mice were injected intra-articularly with 1.107 pfu mIL-18 adenovirus followed by histopathological examination. Local overexpression of IL-18 resulted in pronounced joint inflammation and cartilage proteoglycan loss in control mice. Of high interest, IL-18 gene transfer in IL-1-deficient mice did not show cartilage damage, although joint inflammation was similar to that in wild-type animals. Overexpression of IL-18 in TNF-deficient mice showed that TNF was partly involved in IL-18-induced joint swelling and influx of inflammatory cells, but cartilage proteoglycan loss occurred independent of TNF. In vitro cartilage degradation by IL-18 was found after a 72-hour culture period. Blocking of IL-1 with IL-1Ra or an ICE-inhibitor resulted in complete protection against IL-18-mediated cartilage degradation. The present study demonstrated that IL-18 induces joint inflammation independently of IL-1. In addition, we showed that IL-1ß generation, because of IL-18 exposure, was essential for marked cartilage degradation both in vitro and in vivo. These findings implicate that IL-18, in contrast to TNF, contributes through separate pathways to joint inflammation and cartilage destruction.

Interleukin (IL)-18 is a recently discovered cytokine and is a member of the IL-1 cytokine family, originally identified as interferon-{gamma}-inducing factor.1-3 The biologically inactive precursor of IL-18 is intracellularly cleaved by IL-1ß-converting enzyme (ICE or caspase-1) to yield an active 18-kd glycoprotein.4-6 Pro-IL-18 can also be activated by ICE-independent mechanisms, such as enzymatic cleavage by gelatinase-B (MMP-9) and neutrophil proteinase-3.7-9 In addition, FasL-stimulated macrophages produce active IL-18 in the absence of ICE or caspase-1.10 IL-18 expression has been detected in several cell types including activated macrophages, Kupffer cells, dendritic cells, Langerhans cells, as well as osteoblasts and chondrocytes.11-15 The receptor for IL-18, IL-18R, is comprised of unique {alpha} (IL-1rp) and nonbinding ß (AcPL) signaling chains. This receptor is expressed on many cell types, involved in both innate and adaptive immune responses, and signals through a pathway that involves MyD88 (myeloid differentiation 88), IRAK-1/4 (IL-1 receptor-associated kinases), TRAF6 (tumor necrosis factor receptor-associated factor 6), and nuclear factor-{kappa}ß.16,17 Recently, it was demonstrated that IL-18 could activate gene transcription independently from the MyD88/IRAK pathway. PI3K/Akt (phosphatidylinositol 3 kinase) as well as mitogen-activated protein kinases (MAPK) Erk-1 and Erk-2 signaling cascades seem to be involved in IL-18 signaling.18-20 IL-18 signaling is regulated in vivo by naturally occurring IL-18 binding protein (IL-18BP) that binds and neutralizes IL-18.21 The fact that there are several splice variants of IL-18BP may indicate a complex extracellular regulation of IL-18 signaling.22

Several studies have elucidated a broad spectrum of effector functions beyond lymphocyte activation that implicate IL-18 as an important regulator of chronic inflammation in human autoimmune diseases.23,24 IL-18 induces tumor necrosis factor (TNF)-{alpha}, GM-CSF, interferon-{gamma}, and nitric oxide (NO) production by synovial cells isolated from patients with rheumatoid arthritis through a direct, interferon-{gamma}-independent pathway, via constitutive IL-18R{alpha} expression.23 The IL-18-induced cytokine production by synovial macrophages was potentiated by IL-12 and/or IL-15, and suppressed by IL-10 and transforming growth factor-ß. Furthermore, IL-18 expression in synovial tissue biopsies from rheumatoid arthritis patients with clinically active disease is associated with enhanced IL-1ß and TNF-{alpha} levels.25 In addition, IL-18 induces the expression of CXC chemokines by synovial fibroblasts, stimulates angiogenesis, and is involved in leukocyte recruitment by up-regulation of vascular adhesion molecule-1 through nuclear factor-{kappa}B-dependent mechanisms.19,26-28

Preclinical studies have shown that IL-18 is a primary cytokine that promotes both systemic and local cytokine production.29,30 Administration of IL-18 alone or in combination with IL-12 increased the severity of murine type II collagen (CIA) arthritis.31 Blockade of endogenous IL-18 during the onset of disease in an acute model of joint inflammation, significantly reduced local TNF-{alpha} and IL-1ß levels.30 In line with these findings, blockade of IL-18 with antibodies or with the endogenous inhibitor IL-18BP suppressed the disease activity in murine CIA.32 Interestingly, intra-articular overexpression of IL-18BP using an adenoviral vector for murine IL-18BPc ameliorated disease activity and suppressed joint destruction in CIA. Neutralization of local IL-18 activity was accompanied by reduction of TNF-{alpha} and IL-6 levels in the joint.33

It was demonstrated previously that IL-18 induces chondrocyte proliferation; up-regulates mRNA expression of inducible nitric oxide synthetase, stromelysin (MMP-3), and cyclooxygenase 2 (COX2) in cultured chondrocytes; and increases cartilage glycosaminoglycan release in vitro.15 In contrast to these observations, several investigations have shown that IL-18 exposure of different cell types, such as peripheral blood mononuclear cells and macrophages, did not lead to the production of NO, COX-2, or PGE2.34 In the present study we examined whether IL-18 induces inhibition of chondrocyte proteoglycan synthesis and cartilage proteoglycan depletion directly or via induction of other mediators, such as IL-1 and TNF. Local gene transfer technology was used to explore the direct proinflammatory role of IL-18 in naïve murine knee joints. In vitro studies with cartilage explants were performed to get more insight in IL-18-driven cartilage destruction.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male C57/BL6 mice were obtained from Charles River, Sulzfeld, Germany. Breeder pairs of TNF-{alpha}-deficient mice were kindly provided by Prof. Dr. G. Kollias, Athens, Greece.35 IL-1ß gene-deficient mice36 were a kind gift from Merck, Rahway, NJ. Breeder pairs of IL-1{alpha},ß-deficient mice were provided by Prof. Dr. Y. Iwakura, Tokyo, Japan.37 The breeder pairs were controlled for cytokine deficiency by genotyping, according standard protocols. The mice were housed in filter top cages, and water and food were provided ad libitum. The mice were used at the age of 10 to 12 weeks. Care was taken to house all of the deficient and control littermate mice under identical conditions. All animal experiments conducted in this study were cared for in accordance with the institutional ethics committee.

Materials

Bovine serum albumin was purchased from Sigma Chemical Co., St. Louis, MO. RPMI 1640 medium was obtained from Life Technologies, Breda, The Netherlands. Recombinant murine IL-1ß, IL-1Ra, IL-18, and TNF-{alpha} were purchased from R&D Systems, Abingdon, UK. The ICE inhibitor Boc-Asp(Obz)-CMK (N-1430) was obtained from Bachem AG, Bubendorf, Switzerland. Recombinant human IGF-1 was purchased from Preprotech, Rocky Hill, NJ. Radioactive 35S-sulfate was purchased from NEN Life Sciences Products, Boston, MSA. Bioplex kits for multicytokine determination were purchased from Bio-Rad, Hercules, CA.

Adenoviral mIL-18 Vector and Intra-Articular Gene Transfer

Recombinant adenovirus AdmIL-18 was constructed with insertion of the murine pro-IL-18 cDNA in the early regions 1 (E1) and 3 (E3), respectively.38 Expression of cDNA was driven by the human cytomegalovirus immediate early gene promoter and terminated by the polyadenylation sequence of SV40. The virus was produced by co-transfection of 293 cells with the plasmid. Large scale production was performed in conjunction with Prof. Dr. J. Kolls from the Department of Medicine, Louisiana State University, New Orleans, LA. Transfection with this adenoviral construct results in active mIL-18 production, both in vitro and in vivo. As a control we used the empty recombinant replication-defective adenovirus Ad5del70-3.Gene transfer was performed by intra-articular injection of naive mice with 107 pfu/6 µl of AdmIL-18 or Ad5del70-3. At different time points, patellae with adjacent tissue were dissected and patellae washouts were used for the determination of IL-18, IL-1ß, or TNF-{alpha} levels. In addition, we examined joint swelling (days 2, 4, and 7) and histopathology (days 7 and 14) after mIL-18 gene transfer.

Measurement of Joint Inflammation

Joint inflammation was quantified by 99mTc-uptake method.39 This method measures by external gamma counting the accumulation of a small radioisotope at the site of inflammation because of local increased blood flow and tissue swelling. The severity of inflammation is expressed as the ratio of the 99mTc-uptake in the right (inflamed) over the left (control) knee joint. All values exceeding 1.10 were assigned as inflammation.

Cytokine Measurements

To determine levels of several cytokines, including IL-1ß, IL-18, and TNF-{alpha} in patellae washouts, patellae were isolated from inflamed knee joints as previously described.40 Patellae were cultured in RPMI 1640 medium containing 0.1% bovine serum albumin (200 µl/patella) for 1 hour at room temperature. Thereafter supernatant was harvested and centrifuged for 5 minutes at 1000 x g. Cytokine levels were determined using the Luminex multianalyte technology.41 The BioPlex system in combination with multiplex cytokine kits was used. Cytokines were measured in 50 µl of patellae washout medium. The sensitivity of the multiplex kit was for IL-1ß, IL-18, or TNF-{alpha} 5, 20, and 5 pg/ml, respectively.

Histology

Mice were sacrificed by ether anesthesia. Thereafter, whole knee joints were removed and fixed for 4 days in 4% formaldehyde. After decalcification in 5% formic acid the specimens were processed for paraffin embedding. Tissue sections (7 µm) were stained with hematoxylin and eosin (cell influx) or Safranin O (cartilage proteoglycan depletion). Histopathological changes were scored using the following parameters. Infiltration of cells was scored on a scale of 0 to 3, depending on the amount of inflammatory cells in the synovial cavity and synovial tissues. The loss of proteoglycans was scored on a scale of 0 to 3, ranging from full stained cartilage to destained cartilage or complete loss of articular cartilage. Histopathological changes in the knee joints were scored in the patella/femur region on five semiserial sections of the joint, spaced 70 µm apart. Scoring was performed on decoded slides by two observers, as described previously.42,43

Chondrocyte Proteoglycan Synthesis

Patellae with minimal surrounding tissue were isolated from knee joints of naive C57/BL6 mice.29,44 Thereafter, patellae were cultured in RPMI 1640 medium, glutamax, and gentamycin (50 µg/ml) supplemented with rhIGF-1 (250 ng/ml) with or without IL-1 (10 ng/ml) or IL-18 (10 to 100 ng/ml) for either 24, 48, or 72 hours. Thereafter the patellae were placed in RPMI 1640 medium with glutamax, gentamycin (50 µg/ml), and 35S-sulfate (0.74 MBq/ml). After 3 hours of incubation at 37°C in a CO2 incubator, patellae were washed in saline three times, fixed in 4% formaldehyde, and subsequently decalcified in 5% formic acid for 4 hours. Patellae were punched out of the adjacent tissue, dissolved in 0.25 ml of LumaSolve at 65°C (Ominlabo, Breda, The Netherlands), and after addition of 1 ml of Lipoluma (Omnilabo) the 35S content was measured by liquid scintillation counting (Trilux 1450 microbeta; EG&G Wallac, Turku, Finland). Values are presented as percentage of 35S incorporation of the left control joint.

In Vitro Proteoglycan Degradation Assay

Patellae with minimal surrounding tissue were placed in RPMI 1640 medium with glutamax, gentamycin (50 µg/ml), and 35S-sulfate (0.74 MBq/ml). After 3 hours of incubation at 37°C in a CO2 incubator, patellae were extensively washed in sterile saline three times and were cultured for 24, 48, or 72 hours in either RPMI 1640 medium or 0.1% bovine serum albumin and 250 ng/ml of recombinant human IGF-1. Patellae were exposed to IL-1ß or IL-18 with or without inhibitors/antagonists. Thereafter, patellae were fixed in 4% formaldehyde and subsequently decalcified in 5% formic acid for 4 hours. Patellae were punched out of the adjacent tissue, dissolved in 0.25 ml LumaSolve at 65°C (Ominlabo), and after addition of 1 ml of Lipoluma (Omnilabo) the 35S content was measured by liquid scintillation counting. Values are presented as percentage of 35S incorporation of the left control joint.

Statistical Analysis

Differences between experimental groups were tested using the Mann-Whitney U-test unless stated otherwise.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Intra-Articular Overexpression of IL-18 Results in Delayed Joint Swelling and Suppression of Chondrocyte Metabolic Function

To investigate the effect of prolonged IL-18 exposure in vivo, an adenovirus coding for murine IL-18 was injected in the right knee joint of naive C57/BL6 mice. Table 1 shows that IL-18 was highly expressed in a mouse knee joint after adenoviral gene transfer, using 1.107 pfu of AdmIL-18. Enhanced levels of IL-18 could be found up to day 14 after injection of the adenovirus coding for IL-18. Although not vehemently, levels of both IL-1ß and TNF-{alpha} were locally elevated after injection of AdmIL-18 (Table 1) . In contrast, injection of control adenovirus (Ad5del70-3) did lead to slightly enhanced levels of IL-1ß, IL-18, or TNF-{alpha}, only detectable at day 1 after virus injection (Table 1) . Local overexpression of IL-18 in wild-type mice resulted in protracted joint inflammation, as determined by joint swelling assessment (Figure 1, A and B) . A gradually increased joint swelling was observed after local injection of AdmIL-18. At day 2 we noted a right/left ratio of 1.2 ± 0.05 that increased to 1.4 ± 0.08 at day 14. In addition, we analyzed the suppressive effect of high IL-18 levels on chondrocyte proteoglycan metabolism. Despite high IL-18 levels at days 1 and 2, no inhibition of chondrocyte proteoglycan synthesis was noted (Figure 1A) . Significant suppression of chondrocyte proteoglycan synthesis was found after day 4. This was in line with the increasing IL-1ß and TNF-{alpha} levels found in patellar washouts after injection of admIL-18 virus.


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  		Table 1. Local Cytokine Production after IL-18 Gene Transfer

 


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  		Figure 1. IL-18-driven joint inflammation in wild-type, IL-1–/–, and TNF–/– mice. IL-18 was overexpressed in the right knee of C57/BL6 mice by intra-articular injection of 1.107 pfu of AdmIL-18. A: At several time points both joint swelling (99mTc-uptake) and chondrocyte proteoglycan synthesis (35S-sulfate incorporation) were assessed. B: To examine the role of either IL-1 or TNF, IL-18 was overexpressed in IL-1- or TNF-deficient mice. Data expressed the mean ± SD of at least eight mice per group. The experiment was repeated once with approximately the same outcome. *, P < 0.01, Mann-Whitney U-test compared to C57/BL6 wild-type mice control group.

 
To examine the role of either IL-1 or TNF in the IL-18-driven joint swelling we injected 1.107 pfu of admIL-18 in IL-1- or TNF-{alpha}-deficient mice. Lack of TNF-{alpha} resulted in suppressed joint swelling after local injection of the IL-18 adenoviral vector, as visualized in Figure 1B . In contrast to TNF-deficient mice, mice lacking both forms of IL-1 did not differ from the wild-type animals at all examined days after application of the IL-18 vector, indicating no role of IL-1{alpha} or IL-1ß in the IL-18-driven joint swelling (Figure 1B) .

IL-18 Drives Influx of Inflammatory Cells and Cartilage Degradation by Separate Pathways

Because extended IL-18 exposure in vivo caused enhanced joint swelling we examined whether IL-18 overexpression initiated influx of inflammatory cells and cartilage degradation. Figure 2B shows clearly that IL-18 generated an influx of cells in the joint cavity of wild-type C57/BL6 mice. Both at days 7 and 14 enhanced cell influx was noted, although the number of cells was less pronounced at day 14 (data not shown). Remarkably lower numbers of inflammatory cells were found in the joint cavity of TNF-deficient mice compared to wild-type animals (Figure 2D) . In contrast to TNF gene knockout mice, overexpression of IL-18 in IL-1{alpha},ß-deficient mice resulted in a similar influx of inflammatory cells in synovial tissue as in wild control mice (Figure 2C) . These data indicate that influx of inflammatory cells, induced by local IL-18 application, is driven mainly by TNF-{alpha}.



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  		Figure 2. Joint inflammation induced by prolonged IL-18 overexpression. At day 0 1.107 pfu of control virus (Ad5del70-3) or 1.107 pfu of AdmIL-18 was intra-articularly injected in wild-type, IL-1-deficient, or TNF-deficient mice. At day 7 knee joints were removed and processed for histological analysis. A: Inflammation found after injection of Ad5del70-3 control virus. B: IL-18-induced joint inflammation in a wild-type mouse. C: IL-18-induced joint inflammation in IL-1 gene-deficient mouse. D: IL-18-induced joint inflammation in TNF knockout mouse. P, patella; F, femur; JC, joint cavity; and C, cartilage. H&E staining. Original magnifications, x200.

 
In addition, we analyzed the catabolic effect of extended IL-18 exposure in vivo on cartilage. Figure 3B demonstrates nicely that IL-18 gene transfer resulted in loss of matrix proteoglycans from the cartilage of a wild-type mouse. Both at days 7 and 14 enhanced cartilage proteoglycan degradation was seen, although the strongest proteoglycan depletion was found at 14 days after intra-articular injection of adenoviral vector coding for IL-18. In contrast to the role of TNF-{alpha} in the attraction of proinflammatory cells, provoked by IL-18 overexpression, we noted no differences in cartilage proteoglycan depletion between wild-type and TNF-{alpha}-deficient mice (Figure 3D) . Of high interest, IL-1{alpha},ß gene-deficient mice were almost completely protected against cartilage proteoglycan loss, induced by extended IL-18 exposure in vivo (Figure 3C) .



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  		Figure 3. Cartilage degradation after IL-18 gene transfer in wild-type, IL-1-deficient, or TNF-deficient mice. IL-18-driven cartilage proteoglycan loss was analyzed on day 14 after intra-articular injection of 1.107 pfu of Ad5del70-3 or AdmIL-18. A: Wild-type mouse injected with control virus. B: AdmIL-18 injection in a wild-type mouse. C: IL-18 overexpression in a IL-1{alpha},ß-deficient mouse. D: IL-18 overexpression in a TNF-deficient mouse. For details see Figure 2 . Note the enhanced cartilage proteoglycan loss in wild-type and TNF-deficient mice (arrows). Safranin O staining. Original magnifications, x400.

 
IL-18 Induces Delayed Cartilage Degradation in Vitro

To obtain more insight in the mechanism of IL-18-mediated cartilage damage we investigated the effect of IL-18 on both chondrocyte proteoglycan synthesis and cartilage matrix degradation. Therefore, we exposed patellar cartilage explants with minimal adjacent synovial tissue to IL-18 in an in vitro culture system. Figure 4A shows that IL-18 did not induce significant inhibition of chondrocyte proteoglycan synthesis in vitro after a 72-hour culture period, even at a concentration of 100 ng/ml of IL-18. In contrast to IL-18, IL-1ß strongly inhibits chondrocyte proteoglycan synthesis (50% inhibition compared to IGF-1 control) already after 24 hours of culture. Previous reports indicated that IL-18 induces catabolic responses in chondrocytes,15 but we found no suppressive effect on chondrocyte metabolic function, determined as chondrocyte proteoglycan synthesis. In additional studies, we examined the catabolic effect of IL-18 on in vitro cartilage degradation. 35Sulfate-prelabeled patellar cartilage explants were cultured up to 72 hours with either IL-18 or IL-1ß. Figure 4B shows late cartilage degradation after exposure to both 10 and 100 ng/ml of IL-18. We found that nearly 40% of the prelabeled cartilage was released after a 72-hour culture period. Interestingly, no cartilage proteoglycan-loss was observed after 24 or 48 hours of exposure with IL-18. In contrast to IL-18, IL-1ß already induced cartilage degradation after 24 hours of stimulation (Figure 4B) . Interestingly, the degree of cartilage proteoglycan release was comparable at 72 hours between IL-18 (100 ng/ml) and IL-1ß (10 ng/ml).



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  		Figure 4. Prolonged exposure to IL-18 induced cartilage degradation in vitro. A: Patellar cartilage explants were isolated from naïve knee joints of C57/BL6 mice and cultured for 24 to 72 hours in RPMI 1640 medium supplemented with recombinant hIGF-1 (250 ng/ml) with or without mIL-18. Thereafter chondrocyte proteoglycan synthesis was determined by 35S-sulfate incorporation. For details see Materials and Methods. B: For cartilage degradation studies cartilage explants were prelabeled with 35S-sulfate. Data expressed the mean ± SD of six patellae per group. The experiments were repeated twice with similar outcome. *, P < 0.01, Mann-Whitney U-test compared to IGF-1 control group.

 
IL-18-Driven Cartilage Degradation in Vitro is IL-1ß-Dependent

To investigate whether IL-1{alpha} or IL-1ß was involved in the IL-18-driven cartilage degradation, we blocked IL-1 receptor signaling and de novo production of IL-1ß. To this end we added either IL-1Ra or an IL-1-converting enzyme (ICE) inhibitor to the in vitro cultures. Figure 5A shows that addition of 1 µg/ml of human IL-1Ra completely blocked the IL-18-driven cartilage proteoglycan loss as found after 72 hours of culture. Inhibition of IL-1ß-induced cartilage proteoglycan degradation in vitro by IL-1Ra confirmed the efficacy of used concentration of IL-1Ra. Using the ICE inhibitor we demonstrated that newly processed IL-1ß was responsible for the observed IL-18-driven cartilage degradation in vitro (Figure 5B) .



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  		Figure 5. In vitro cartilage degradation by IL-18 is mediated by IL-1. Patellar cartilage explants were prelabeled as indicated in Materials and Methods. Thereafter, cartilage explants were cultured for 72 hours with IGF-1-containing medium with either IL-18 (10 or 100 ng/ml) or IL-1ß (10 ng/ml). To block IL-1 we added either recombinant mIL-1Ra (A, 10 µg/ml) or ICE-inhibitor (B, 2.5 µmol/L) to the culture medium. C: To confirm the ICE data and examine the role of TNF in IL-18-driven proteoglycan loss we use patellar explants from both IL-1ß- and TNF-{alpha}-deficient mice. For details see Figure 4 . *, P < 0.01, Mann-Whitney U-test compared to C57/BL6 x 129Sv wild-type mice control group.

 
To exclude that TNF-{alpha}, induced by IL-18 exposure, was involved in the observed proteoglycan degradation we used cartilage explants from TNF-{alpha}-deficient mice. Figure 5C indicates that TNF-{alpha} was not a crucial cytokine in the IL-18-induced cartilage proteoglycan loss. We found no difference in IL-18-mediated cartilage degradation between the wild-type and TNF-{alpha}-deficient mice after 72 hours of IL-18 exposure. Using patellae from IL-1ß knockout mice confirmed the findings with the ICE inhibitor, that IL-18-induced cartilage degradation is mediated by IL-1ß generation (Figure 5C) .


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The present study was performed to investigate the direct role of IL-18 in joint inflammation and/or cartilage damage. To this end we overexpressed murine IL-18 locally and analyzed joint inflammation and cartilage damage. The role of either IL-1 or TNF in IL-18-driven joint inflammation was studied by using mice deficient for either IL-1 or TNF. The in vivo observations were confirmed by in vitro cartilage degradation studies. Therefore, we exposed murine cartilage explants to IL-18 and analyzed the catabolic effect of IL-18. Using IL-1 inhibitors and/or cartilage explants from IL-1ß or TNF-deficient mice we clearly demonstrated that IL-1ß was the pivotal second mediator in IL-18-driven cartilage destruction.

Prolonged overexpression of IL-18 in naïve murine knee joint, using an adenovirus coding for murine IL-18, resulted in mild joint inflammation and cartilage proteoglycan loss (Figures 1, 2, and 3) . Recent reports indicated a role of IL-18 in the pathogenesis of several human inflammatory diseases. Elevated IL-18 levels can be found in psoriasis, inflammatory bowel disease, and sacroidoses.45 Several preclinical studies have shown that co-administration of IL-18 enhanced the inflammatory response to antigens such as collagen type II.26,46 This is the first study that showed that IL-18 overexpression causes joint inflammation that accumulates in time (Figures 1A and 2) . In line with previous findings, IL-18 attracts predominantly neutrophils into the joint tissues, although at later stages (day 14) for the most part monocytes/macrophages were seen in the synovial lining (data not shown). By using TNF-deficient mice we found that IL-18-driven joint inflammation was partly TNF-dependent. Joint swelling was completely absent and the influx of inflammatory cells was reduced in TNF gene-deficient mice. We have shown previously that TNF is the pivotal cytokine that drives swelling in acute SCW-induced joint inflammation.40,42,47 IL-18 itself can induce chemokines and chemoattractant factors (eg, IL-8 and LTB4) that may explain the partly TNF-independent cell influx.19,28,48 Interestingly, overexpression of IL-18 in a naive knee joint leads to cartilage damage, determined as loss of matrix proteoglycans. The loss of matrix proteoglycans was IL-1-dependent because IL-18 overexpression in IL-1{alpha},ß-deficient mice did not result in cartilage damage. It is well known that IL-1 is the crucial cytokine that promotes cartilage destruction via induction of several catabolic mediators in synovial lining cells as well as in chondrocytes. IL-1 was originally identified as catabolin because it could induce cartilage destruction.49,50 Blocking studies with antibodies or IL-1 receptor antagonist (IL-1Ra) in models of arthritis clearly showed that IL-1 drives cartilage and bone destruction.43,51 Recently, it was demonstrated that intra-articular injection of IL-1Ra in patients with painful knee osteoarthritis had a dramatic therapeutic response, indicating once more the pivotal role of IL-1 in cartilage catabolism.52

Here we showed that prolonged exposure to IL-18 in vitro, up to 72 hours, did not result in inhibition of chondrocyte proteoglycan synthesis. Even at high concentrations (100 ng/ml) no suppressive effects of IL-18 were noted (Figure 2B) . In contrast, IL-1ß induced substantial inhibition of chondrocyte metabolic function already at 24 hours and at low concentrations (1 ng/ml). Because it is known that NO is the causative agent for suppression of chondrocyte proteoglycan synthesis51 we analyzed NO production of cartilage explants after IL-18 exposure. In contrast to IL-1, we found low levels of NO in supernatants after 48 hours or 72 hours of culture with 10 ng/ml or 100 ng/ml IL-18 (data not shown). This is in line with a previous study indicating that IL-18 could produce NO in chondrocytes,15 although the levels of NO were higher in cultured chondrocytes then in cartilage explants. Despite the enhanced NO levels in IL-18-exposed cartilage explants, no inhibition of chondrocyte proteoglycan synthesis was found. This indicates that although the signaling pathway of IL-18 and IL-1 are similar, IL-18 seems to generate a protective mechanism against NO-mediated suppression of chondrocyte metabolic function. It is demonstrated that heme oxygenase-1 (HO-1) plays a crucial role in NO production, because this enzyme catalyzes heme protein, which is a co-factor for the NOS-2 enzyme. More recently, it was shown that IL-1 and IL-17 down-regulate HO-1 activity, which may explain the increased NO production by chondrocytes and the induction of inhibition of chondrocyte proteoglycan synthesis.53 IL-18 regulates induction of HO-1 expression via both Erk/MAPK and PI3K/Akt pathways that can be activated by IL-18 receptor signaling.54,55 At the moment, investigations are performed to unravel the IL-18-induced protection on chondrocyte anabolic metabolism.

IL-18 induces degradation of cartilage matrix molecules after extended exposure (Figure 4B) . Olee and colleagues15 showed that IL-18 could stimulate release of proteoglycans from human articular cartilage. Whether this was because of increased degradation or enhanced synthesis of proteoglycans was not investigated. Although IL-18 exposure of cartilage resulted in rapidly increased mRNA levels of several catabolic mediators, such as MMP-3 and MMP-9, no degradation of proteoglycans was noted up to 48 hours of culture. Here we report that production of a second messenger molecule was responsible for the delayed cartilage destruction by IL-18. Blockade of IL-1ß or the activation of pro-IL-1ß was sufficient to protect the cartilage explants from proteoglycan release (Figure 5, A and B) . It is known that IL-18 initiates production of several cytokines, including IL-1 and TNF, in monocytes and neutrophils.26,56 In addition, very recently it was shown that IL-18 stimulates monocyte IL-1 and TNF production, induced by contact to activated T cells isolated from rheumatoid arthritis synovium.57 Although IL-18 can induce TNF mRNA production in chondrocytes (data not shown), TNF is not involved in IL-18-mediated cartilage proteoglycan loss in vitro (Figure 5C) .

In conclusion, this study indicates that IL-18 promotes cartilage proteoglycan loss, both in vitro and in vivo, mediated by IL-1 generation. Although IL-18 can induce both IL-1 and NO production in chondrocytes it is not competent to generate inhibition of chondrocyte proteoglycan synthesis. Local overexpression of IL-18 resulted in a delayed influx of inflammatory cells that is partly TNF-dependent. These data implicate that IL-18 can contribute to joint inflammation and cartilage destruction by separate pathways. Targeting of IL-18 during inflammatory joint diseases, such as rheumatoid arthritis, may provide a novel therapy because IL-18 promotes development of immunity and contributes to cartilage destruction via IL-1 production.


   Acknowledgements
 
We thank Prof. Dr. Jay Kolls (Department of Medicine, Louisiana State University, New Orleans, LA) for helping us with the large scale production of AdmIL-18.


   Footnotes
 
Address reprint requests to Leo A.B. Joosten, Ph.D., Rheumatology Research Laboratory and Advanced Therapeutics, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: l.joosten{at}reuma.umcn.nl

Supported by the Dutch Arthritis Association (grant NR 99-2-403).

Accepted for publication June 1, 2004.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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