Interleukin-18 Promotes Joint Inflammation and Induces Interleukin-1-Driven Cartilage Destruction
2004; Elsevier BV; Volume: 165; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63357-3
ISSN1525-2191
AutoresLeo A. B. Joosten, Ruben L. Smeets, Marije I. Koenders, L. A. M. Van Den Bersselaar, Monique M. Helsen, Birgitte Oppers‐Walgreen, Erik Lubberts, Yoichiro Iwakura, Fons A. J. van de Loo, Wim B. van den Berg,
Tópico(s)Autoimmune and Inflammatory Disorders Research
ResumoInterleukin (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-1β 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 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-1β 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. 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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.18Kalina U Kauschat D Koyama N Nuernberger H Ballas K Koschmieder S Bug G Hofmann WK Hoelzer D Ottmann OG IL-18 activates STAT3 in the natural killer cell line 92, augments cytotoxic activity, and mediates IFN-gamma production by the stress kinase p38 and by the extracellular regulated kinases p44erk-1 and p42erk-21.J Immunol. 2000; 165: 1307-1313Crossref PubMed Scopus (110) Google Scholar, 19Morel JC Park CC Kumar P Koch AE Interleukin-18 induces rheumatoid arthritis synovial fibroblast CXC chemokine production through NFkappaB activation.Lab Invest. 2001; 81: 1371-1383Crossref PubMed Scopus (79) Google Scholar, 20Morel JC Park CC Zhu K Kumar P Ruth JH Koch AE Signal transduction pathways involved in rheumatoid arthritis synovial fibroblast interleukin-18-induced vascular cell adhesion molecule-1 expression.J Biol Chem. 2002; 277: 34679-34691Crossref PubMed Scopus (103) Google Scholar IL-18 signaling is regulated in vivo by naturally occurring IL-18 binding protein (IL-18BP) that binds and neutralizes IL-18.21Novick D Kim SH Fantuzzi G Reznikov LL Dinarello CA Rubinstein M Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response.Immunity. 1999; 10: 127-136Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar The fact that there are several splice variants of IL-18BP may indicate a complex extracellular regulation of IL-18 signaling.22Kim SH Eisenstein M Reznikov L Fantuzzi G Novick D Rubinstein M Dinarello CA Structural requirements of six naturally occurring isoforms of the IL-18 binding protein to inhibit IL-18.Proc Natl Acad Sci USA. 2000; 97: 1190-1195Crossref PubMed Scopus (265) Google Scholar 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.23Gracie JA Forsey RJ Chan WL Gilmour A Leung BP Greer MR Kennedy K Carter R Wei XQ Xu D Field M Foulis A Liew FY McInnes IB A proinflammatory role for IL-18 in rheumatoid arthritis.J Clin Invest. 1999; 104: 1393-1401Crossref PubMed Scopus (565) Google Scholar, 24Pizarro TT Michie MH Bentz M Woraratanadharm J Smith Jr, MF Foley E Moskaluk CA Bickston SJ Cominelli F IL-18, a novel immunoregulatory cytokine, is up-regulated in Crohn's disease: expression and localization in intestinal mucosal cells.J Immunol. 1999; 162: 6829-6835PubMed Google Scholar IL-18 induces tumor necrosis factor (TNF)-α, GM-CSF, interferon-γ, and nitric oxide (NO) production by synovial cells isolated from patients with rheumatoid arthritis through a direct, interferon-γ-independent pathway, via constitutive IL-18Rα expression.23Gracie JA Forsey RJ Chan WL Gilmour A Leung BP Greer MR Kennedy K Carter R Wei XQ Xu D Field M Foulis A Liew FY McInnes IB A proinflammatory role for IL-18 in rheumatoid arthritis.J Clin Invest. 1999; 104: 1393-1401Crossref PubMed Scopus (565) Google Scholar 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-β. Furthermore, IL-18 expression in synovial tissue biopsies from rheumatoid arthritis patients with clinically active disease is associated with enhanced IL-1β and TNF-α levels.25Joosten LA Radstake TR Lubberts E van den Bersselaar LA van Riel PL vanLent PL Barrera P van den Berg WB Association of interleukin-18 expression with enhanced levels of both interleukin-1beta and tumor necrosis factor alpha in knee synovial tissue of patients with rheumatoid arthritis.Arthritis Rheum. 2003; 48: 339-347Crossref PubMed Scopus (106) Google Scholar 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-κB-dependent mechanisms.19Morel JC Park CC Kumar P Koch AE Interleukin-18 induces rheumatoid arthritis synovial fibroblast CXC chemokine production through NFkappaB activation.Lab Invest. 2001; 81: 1371-1383Crossref PubMed Scopus (79) Google Scholar, 26Leung BP Culshaw S Gracie JA Hunter D Canetti CA Campbell C Cunha F Liew FY McInnes IB A role for IL-18 in neutrophil activation.J Immunol. 2001; 167: 2879-2886PubMed Google Scholar, 27Park CC Morel JC Amin MA Connors MA Harlow LA Koch AE Evidence of IL-18 as a novel angiogenic mediator.J Immunol. 2001; 167: 1644-1653PubMed Google Scholar, 28Komai-Koma M Gracie JA Wei XQ Xu D Thomson N McInnes IB Liew FY Chemoattraction of human T cells by IL-18.J Immunol. 2003; 170: 1084-1090PubMed Google Scholar Preclinical studies have shown that IL-18 is a primary cytokine that promotes both systemic and local cytokine production.29Joosten LA van De Loo FA Lubberts E Helsen MM Netea MG van Der Meer JW Dinarello CA van Den Berg WB An IFN-gamma-independent proinflammatory role of IL-18 in murine streptococcal cell wall arthritis.J Immunol. 2000; 165: 6553-6558PubMed Google Scholar, 30Takeda K Tsutsui H Yoshimoto T Adachi O Yoshida N Kishimoto T Okamura H Nakanishi K Akira S Defective NK cell activity and Th1 response in IL-18-deficient mice.Immunity. 1998; 8: 383-390Abstract Full Text Full Text PDF PubMed Scopus (770) Google Scholar Administration of IL-18 alone or in combination with IL-12 increased the severity of murine type II collagen (CIA) arthritis.31Wei XQ Leung BP Arthur HM McInnes IB Liew FY Reduced incidence and severity of collagen-induced arthritis in mice lacking IL-18.J Immunol. 2001; 166: 517-521PubMed Google Scholar Blockade of endogenous IL-18 during the onset of disease in an acute model of joint inflammation, significantly reduced local TNF-α and IL-1β levels.30Takeda K Tsutsui H Yoshimoto T Adachi O Yoshida N Kishimoto T Okamura H Nakanishi K Akira S Defective NK cell activity and Th1 response in IL-18-deficient mice.Immunity. 1998; 8: 383-390Abstract Full Text Full Text PDF PubMed Scopus (770) Google Scholar 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.32Plater-Zyberk C Joosten LA Helsen MM Sattonnet-Roche P Siegfried C Alouani S van De Loo FA Graber P Aloni S Cirillo R Lubberts E Dinarello CA van Den Berg WB Chvatchko Y Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis.J Clin Invest. 2001; 108: 1825-1832Crossref PubMed Scopus (204) Google Scholar 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-α and IL-6 levels in the joint.33Smeets RL van de Loo FA Arntz OJ Bennink MB Joosten LA van den Berg WB Adenoviral delivery of IL-18 binding protein C ameliorates collagen-induced arthritis in mice.Gene Ther. 2003; 10: 1004-1011Crossref PubMed Scopus (80) Google Scholar 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.15Olee T Hashimoto S Quach J Lotz M IL-18 is produced by articular chondrocytes and induces proinflammatory and catabolic responses.J Immunol. 1999; 162: 1096-1100PubMed Google Scholar 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 PGE2.34Reznikov LL Kim SH Westcott JY Frishman J Fantuzzi G Novick D Rubinstein M Dinarello CA IL-18 binding protein increases spontaneous and IL-1-induced prostaglandin production via inhibition of IFN-gamma.Proc Natl Acad Sci USA. 2000; 97: 2174-2179Crossref PubMed Scopus (70) Google Scholar 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. Male C57/BL6 mice were obtained from Charles River, Sulzfeld, Germany. Breeder pairs of TNF-α-deficient mice were kindly provided by Prof. Dr. G. Kollias, Athens, Greece.35Pasparakis M Alexopoulou L Episkopou V Kollias G Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response.J Exp Med. 1996; 184: 1397-1411Crossref PubMed Scopus (1003) Google Scholar IL-1β gene-deficient mice36Zheng H Fletcher D Kozak W Jiang M Hofmann KJ Conn CA Soszynski D Grabiec C Trumbauer ME Shaw A Resistance to fever induction and impaired acute-phase response in interleukin-1 beta-deficient mice.Immunity. 1995; 3: 9-19Abstract Full Text PDF PubMed Scopus (339) Google Scholar were a kind gift from Merck, Rahway, NJ. Breeder pairs of IL-1α,β-deficient mice were provided by Prof. Dr. Y. Iwakura, Tokyo, Japan.37Horai R Asano M Sudo K Kanuka H Suzuki M Nishihara M Takahashi M Iwakura Y Production of mice deficient in genes for interleukin (IL)-1alpha, IL-1beta, IL-1alpha/beta, and IL-1 receptor antagonist shows that IL-1beta is crucial in turpentine-induced fever development and glucocorticoid secretion.J Exp Med. 1998; 187: 1463-1475Crossref PubMed Scopus (512) Google Scholar 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. 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-1β, IL-1Ra, IL-18, and TNF-α 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. 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.38He TC Zhou S da Costa LT Yu J Kinzler KW Vogelstein B A simplified system for generating recombinant adenoviruses.Proc Natl Acad Sci USA. 1998; 95: 2509-2514Crossref PubMed Scopus (3221) Google Scholar 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 107Schonbeck U Mach F Libby P Generation of biologically active IL-1 beta by matrix metalloproteinases: a novel caspase-1-independent pathway of IL-1 beta processing.J Immunol. 1998; 161: 3340-3346PubMed Google Scholar 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-1β, or TNF-α levels. In addition, we examined joint swelling (days 2, 4, and 7) and histopathology (days 7 and 14) after mIL-18 gene transfer. Joint inflammation was quantified by 99mTc-uptake method.39Lens JW van den Berg WB van de Putte LB Quantitation of arthritis by 99mTc-uptake measurements in the mouse knee-joint: correlation with histological joint inflammation scores.Agents Actions. 1984; 14: 723-728Crossref PubMed Scopus (35) Google Scholar 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. To determine levels of several cytokines, including IL-1β, IL-18, and TNF-α in patellae washouts, patellae were isolated from inflamed knee joints as previously described.40Joosten LA Koenders MI Smeets RL Heuvelmans-Jacobs M Helsen MM Takeda K Akira S Lubberts E van de Loo FA van den Berg WB Toll-like receptor 2 pathway drives streptococcal cell wall-induced joint inflammation: critical role of myeloid differentiation factor 88.J Immunol. 2003; 171: 6145-6153PubMed Google Scholar 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 × g. Cytokine levels were determined using the Luminex multianalyte technology.41De Jager W Te Velthuis H Prakken BJ Kuis W Rijkers GT Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells.Clin Diagn Lab Immunol. 2003; 10: 133-139Crossref PubMed Scopus (497) Google Scholar 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-1β, IL-18, or TNF-α 5, 20, and 5 pg/ml, respectively. 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.42Joosten LA Heuvelmans-Jacobs M Lubberts E van de Loo FA Bakker AC Helsen MM Richards CD van den Berg WB Local interleukin-12 gene transfer promotes conversion of an acute arthritis to a chronic destructive arthritis.Arthritis Rheum. 2002; 46: 1379-1389Crossref PubMed Scopus (15) Google Scholar, 43Joosten LA Helsen MM Saxne T van De Loo FA Heinegard D van Den Berg WB IL-1 alpha beta blockade prevents cartilage and bone destruction in murine type II collagen-induced arthritis, whereas TNF-alpha blockade only ameliorates joint inflammation.J Immunol. 1999; 163: 5049-5055PubMed Google Scholar Patellae with minimal surrounding tissue were isolated from knee joints of naive C57/BL6 mice.29Joosten LA van De Loo FA Lubberts E Helsen MM Netea MG van Der Meer JW Dinarello CA van Den Berg WB An IFN-gamma-independent proinflammatory role of IL-18 in murine streptococcal cell wall arthritis.J Immunol. 2000; 165: 6553-6558PubMed Google Scholar, 44De Vries BJ van den Berg WB van de Putte LB Salicylate-induced depletion of endogenous inorganic sulfate. Potential role in the suppression of sulfated glycosaminoglycan synthesis in murine articular cartilage.Arthritis Rheum. 1985; 8: 922-929Crossref Scopus (38) Google Scholar 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. 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-1β 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. Differences between experimental groups were tested using the Mann-Whitney U-test unless stated otherwise. 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-1β and TNF-α 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-1β, IL-18, or TNF-α, 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-1β and TNF-α levels found in patellar washouts after injection of admIL-18 virus.Table 1Local Cytokine Production after IL-18 Gene TransferIL-18IL-1βTNF-αAdmIL-18Ad5Del70-3AdmIL-18Ad5Del70-3AdmIL-18Ad5Del70-3Day 1820 ± 12249 ± 2645 ± 1521 ± 14100 ± 3439 ± 24Day 2512 ± 78N.D.76 ± 34N.D.74 ± 23N.D.Day 4325 ± 56N.D.178 ± 65N.D.56 ± 41N.D.Day 7200 ± 49N.D.137 ± 41N.D.34 ± 29N.D.Day 14125 ± 23N.D.32 ± 28N.D.21 ± 18N.D.Male C57BL/6 mice were intra-articularly injected at day 0 with 1.107 pfu of either AdmIL-18 or Ad5Del70-3. At several time points, patellae with adjacent synovial tissue were isolated and cultured for 1 hour at room temperature in RPMI 1640 medium (completed with 1% bovine serum albumin). Thereafter the levels IL-18, IL-1β, or TNF were determined by using the Luminex bead array system. The sensitivity of the kits was <10 pg/ml for each cytokine. The data represents the mean ± SD of six patellae washout per time point. ND, not detectable. Open table in a new tab
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