Local interleukin-12 gene transfer promotes conversion of an acute arthritis to a chronic destructive arthritis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 46, No. 5, May 2002, pp 1379–1389 DOI 10.1002/art.10233 © 2002, American College of Rheumatology Local Interleukin-12 Gene Transfer Promotes Conversion of an Acute Arthritis to a Chronic Destructive Arthritis Leo A. B. Joosten,1 Marleen Heuvelmans-Jacobs,1 Erik Lubberts,1 Fons A. J. van de Loo,1 Andrew C. Bakker,1 Monique M. A. Helsen,1 Carl D. Richards,2 and Wim B. van den Berg1 Objective. To determine whether local overexpression of interleukin-12 (IL-12), a pleiotropic cytokine that promotes the development of naive T cells into Th1 cells, could aggravate murine streptococcal cell wall (SCW)–induced arthritis, a model of acute arthritis. Methods. C57BL/6 mice were injected intraarticularly with saline or with 107 plaque-forming units of control vector (Ad5del70-3) or IL-12 vector (AdmIL12.1) into the right knee joint 1 day before intraarticular injection of 25 g of SCW fragments. The development of joint swelling, changes in chondrocyte proteoglycan (PG) synthesis, and joint destruction were examined thereafter. Results. In normal joints, high levels of IL-12 (20 ng/ml on day 1) could be detected after application of the AdmIL-12.1 vector. After 14 days, expression of IL-12 was still found locally, but IL-12 alone did not induce protracted inflammation. Local expression of IL-12, in combination with SCW, markedly aggravated SCW-induced arthritis, as determined by enhanced joint swelling and prolonged inhibition of chondrocyte PG synthesis. Histologic examination on day 21 showed a chronic inflammatory process, with persistent cartilage PG depletion, cartilage erosion, and VDIPEN neoepitope expression (indicative of metalloproteinase ac- tivation). The mixture of IL-12 with SCW fragments did not lead to a chronic destructive process in mice deficient for recombination-activating gene 2, indicating the involvement of lymphocytes. In addition, systemic flare of smoldering SCW arthritis, produced by intravenous injection of SCW fragments, was only seen in the AdmIL-12/SCW group. Conclusion. These results indicate that local overexpression of IL-12 promotes conversion of an acute arthritis to a chronic destructive immune-mediated process, which is more susceptible to flares. Originally described as cytotoxic lymphocyte maturation factor and natural killer (NK) cell stimulatory factor, interleukin-12 (IL-12) is a 70-kd heterodimeric cytokine consisting of 2 subunits, a 35-kd and a 40-kd subunit. This heterodimeric structure of IL-12 is unique among cytokines. IL-12 is produced by phagocytic cells, dendritic cells, B lymphocytes, and NK cells upon stimulation with lipopolysaccharide, bacteria, and parasites (1–4). IL-12 generates the development of naive T cells into Th1 cells and stimulates the secretion of interferon-␥ (IFN␥) by differentiated Th1 cells (5,6). Recently, it was demonstrated that the newly discovered cytokine IL-18 can act as a costimulus for IFN␥ production and NK cell cytotoxicity induced by IL-12 (7,8). However, IL-18 itself is not a strong inducer of IFN␥ in the absence of other cytokines. Through the induction of IFN␥ and tumor necrosis factor ␣ (TNF␣), IL-12 plays a crucial role in lipopolysaccharide-induced lethal shock and the generalized Shwartzman reaction (9,10). During bacterial infections, IL-12 is considered a principal protective cytokine, where it bridges innate resistance and antigen-specific immunity (11). However, IL-12 may unmask Th1-dependent autoimmune responses and may 1 Leo A. B. Joosten, PhD, Marleen Heuvelmans-Jacobs, MSc, Erik Lubberts, PhD, Fons A. J. van de Loo, PhD, Andrew C. Bakker, PhD, Monique M. A. Helsen, Wim B. van den Berg, PhD: University Medical Center Nijmegen, Nijmegen, The Netherlands; 2Carl D. Richards, PhD: McMaster University, Hamilton, Ontario, Canada. Address correspondence and reprint requests to Leo A. B. Joosten, PhD, Rheumatology Research Laboratory, University Medical Center Nijmegen, PO Box 9101, Nijmegen 6500 HB, The Netherlands. E-mail: firstname.lastname@example.org. Submitted for publication September 6, 2001; accepted in revised form December 19, 2001. 1379 1380 be a crucial intermediate in the often-suggested link between bacterial infections and the expression of autoimmune diseases (12). Rheumatoid arthritis (RA) is associated with a Th1 immune response, although typical Th1 cytokines, such as IL-2, IFN␥, and TNF␤, have been reported to be scant in synovial fluid or tissues (13). Recently, IL-12 was detected in the synovium of RA patients, and macrophage-like cells seem to be the main producers of this cytokine (14). Interestingly, exogenous IL-12 was shown to cause severe exacerbation of RA activity in an RA patient being treated for metastatic cervical cancer, indicating the disease-promoting role of IL-12 (15). This is consistent with recent data showing that IL-12 levels in both serum and synovial fluid correlated with RA disease activity (16). In murine type II collagen–induced arthritis, an autoimmune model of RA, it was clearly demonstrated that exogenous, systemically applied IL-12, either alone or in combination with IL-18, aggravated disease expression by enhancing systemic immunity against type II collagen (17–19). Both the disease-promoting and immunostimulatory roles of IL-12 in this RA model were corroborated by elegant studies in IL-12–deficient mice (20). Apart from a role in autoimmune collagen arthritis, we recently observed that arthritis induced by local injection of streptococcal cell wall (SCW) fragments (21–24) was also dependent on IL-12 (25). Blockade of endogenous IL-12 during the onset of this macrophagedriven arthritis suppressed the severity of joint inflammation, apparently by the up-regulation of IL-10. However, IL-12 production was brief, and this SCW arthritis was neither chronic nor destructive. In the present study, we investigated whether local overexpression of IL-12 could promote the conversion of an acute arthritis induced by injection of SCW fragments to a chronic joint disease. To this end, synovial lining cells were transduced with murine IL-12 just before arthritis induction. To elucidate the role of lymphocytes in the IL-12–driven conversion, mice deficient in recombination-activating gene 2 (RAG-2) were used. In addition, systemic exacerbation of the chronic arthritis was performed to confirm the hyperresponsiveness of the IL-12–transduced arthritic joint to bacterial fragments. Our findings clearly demonstrate that IL-12 can convert an acute inflammatory process into a chronic destructive process. Targeting of IL-12 may be protective against the conversion of arthritic processes to chronic destructive joint diseases as can be observed in RA in humans. JOOSTEN ET AL MATERIALS AND METHODS Animals. Male C57BL/6 mice were obtained from Charles River (Sulzfeld, Germany). Immunodeficient mice with a targeted disruption of RAG-2 (C57BL/6/Tgh [RAG-2]) were obtained from Centre National de la Recherche Scientifique CDTA (Orleans, France) (26). The mice were housed in filter-top cages, and water and food were provided ad libitum. Mice were used at 10–12 weeks of age. Materials. Bovine serum albumin was purchased from Sigma (St. Louis, MO). RPMI 1640 medium was obtained from Life Technologies (Breda, The Netherlands). IFN␥ (catalog no. CMC4034) enzyme-linked immunosorbent assay (ELISA) cytosets were obtained from BioSource (Camarillo, CA). Anti–murine IL-12 antibodies (capture 80-3891-01; detection 80-3892-01) were purchased from Genzyme (Cambridge, MA). Murine IL-12 was kindly provided by Dr. S. Wolf (Genetic Institute, Cambridge, MA). VDIPEN antibodies were kindly provided by Drs. I. I. Singer and E. K. Bayne (Merck, Rahway, NJ). SCW preparation and induction of SCW arthritis. Streptococcus pyogenes T12 organisms were cultured overnight in Todd-Hewitt broth. Cell walls were prepared as previously described (21). The supernatant resulting from centrifugation at 10,000g was used throughout the experiments. These preparations contained 11% muramic acid. Unilateral arthritis was induced by intraarticular injection of 25 g of SCW fragments (rhamnose content) in 6 l of phosphate buffered saline into the right knee joint of naive mice. Phosphate buffered saline alone was injected into the left knee joint as a control. Reactivation of SCW arthritis was performed by intravenous injection of 100 g of SCW fragments on day 28 after the initial intraarticular injection of 25 g of SCW fragments (21). Adenoviral murine IL-12 vector and intraarticular gene transfer. Recombinant adenovirus AdmIL-12.1 was constructed with insertions of the murine IL-12 p35 and p40 subunit cDNA into early region 1 (E1) and E3, respectively (27). The expression of each cDNA was driven by the human cytomegalovirus immediate early gene promoter and terminated by the polyadenylation sequence of SV40. The virus was produced by cotransfection of 293 cells with two plasmids. This double adenovirus expressed functional heterodimeric murine IL-12. Transfection with this adenoviral construct results in active production of murine IL-12, both in vitro and in vivo (18,28). As a control, we used the empty recombinant replication-defective adenovirus Ad5del70-3. Gene transfer was performed by intraarticular injection of naive mice with either 108, 107, 106, or 105 plaqueforming units (PFU) of AdmIL-12.1 or Ad5del70-3 per 6 l of saline. At different time points, patellae with adjacent tissue were dissected, and patellae washouts were used for the determination of IL-12 levels. To examine the effect of local IL-12 expression on SCW-induced arthritis, AdmIL-12.1 (107 PFU) gene transfer was performed 1 day before the injection of SCW fragments. Measurement of joint inflammation. SCW arthritis was quantified by the 99mTc-uptake method (29). This method measures by external gamma counting the accumulation of a small radioisotope at the site of inflammation due to local increased blood flow and tissue swelling. The severity of IL-12 GENE TRANSFER AND CHRONIC DESTRUCTIVE ARTHRITIS inflammation was expressed as the ratio of the 99mTc-uptake in the right (inflamed) knee joint to that in the left (control) knee joint. All values ⬎1.10 were considered to represent inflammation. Determination of chondrocyte proteoglycan (PG) synthesis. Patellae with minimal surrounding tissue were placed in RPMI 1640 medium with Glutamax, gentamicin (50 g/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 3 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 0.5 ml of LumaSolve at 65°C (Omnilabo, Breda, The Netherlands) and after the addition of 10 ml of Lipoluma (Omnilabo), the 35S-sulfate content was measured by liquid scintillation counting. Values are presented as the percentage of 35S-sulfate incorporation in the left (control) joint. Cytokine measurements. To determine the levels of IL-12 and IFN␥ in patellae washouts, patellae were isolated from inflamed knee joints as previously described (22,24,25). Patellae were cultured in RPMI 1640 medium (200 l/patella) for 1 hour at room temperature. The supernatant was then harvested and centrifuged for 5 minutes at 1,000g. Levels of IL-12 and IFN␥ were measured by ELISA. The sensitivity of the ELISA for IL-12 and IFN␥ was 20 pg/ml and 10 pg/ml, respectively. Immunohistochemical evaluation of VDIPEN expression. For immunostaining, knee joint sections were deparaffinized, rehydrated, and digested with proteinase-free chondroitinase ABC to remove the chondroitin of the PGs. Subsequently, sections were treated with 1% hydrogen peroxide, 1.5% normal goat serum, and affinity-purified rabbit anti-VDIPEN IgG (kindly provided by Drs. I. I. Singer and E. K. Bayne, Merck, Rahway, NJ). This antibody has been characterized previously (30–32). Sections were then incubated with biotinylated goat anti-rabbit IgG, and avidin– streptavidin–peroxidase (Elite kit; Vector, Burlingame, CA). Sections were counterstained with orange G (Sigma). Histologic assessment. Mice were killed by ether anesthesia, and 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 m) were stained with hematoxylin and eosin or Safranin O. Histopathologic changes were scored according to the following parameters. Cell infiltration was scored on a scale of 0–3, depending on the amount of inflammatory cells in the synovial cavity and synovial tissues. PG depletion was determined with Safranin O staining. The loss of PGs was scored on a scale of 0–3, ranging from fully stained cartilage to destained cartilage or a complete loss of articular cartilage. Cartilage destruction was scored on a scale of 0–3, ranging from no damage to fully destroyed cartilage layers. Histopathologic changes in the knee joints were scored in the region of the patella/femur on 5 semiserial sections of the joint, spaced 70 m apart. Scoring was performed on decoded slides by 2 observers (LABJ and MMAH), as described elsewhere (17,31,32). Statistical analysis. Differences between experimental groups were tested using the Mann-Whitney U test, unless stated otherwise. 1381 RESULTS Local IL-12 overexpression in knee joints of naive mice. To examine IL-12 protein overexpression in the joint, naive mice were injected intraarticularly with 108 PFU of AdmIL-12.1 adenovirus. The highest IL-12 protein expression was found on day 1 (up to 20 ng/ml). Considerable amounts of IL-12 could be detected in synovial washouts up to day 14 after AdmIL-12.1 gene transfer (Figure 1A). To determine whether overexpression of IL-12 in naive joints leads to inflammation or inhibition of chondrocyte function, a dose-range experiment was performed with 108, 107, 106, or 105 PFU of AdmIL-12.1 virus particles. On day 2, we measured local levels of IL-12, the degree of joint inflammation, and levels of chondrocyte PG synthesis. As shown in Figure 1B, there was a clear dose-range effect of the virus concentration on the amount of IL-12 produced. IL-12 produced by the 108 PFU dose induced a mild joint inflammation on day 2, but histologic examination on day 7 showed no persistent inflammation (data not shown). Analysis of chondrocyte function on day 2 revealed that injection of 108 PFU induced a 30% inhibition of chondrocyte PG synthesis. Chondrocyte PG synthesis was completely restored on days 4 and 7 after injection of the AdmIL12.1 virus. The 107 PFU dose of AdmIL-12.1 resulted in significant local levels of IL-12 without any signs of inflammation or inhibition of chondrocyte synthetic function. This dose was used for IL-12 gene transfer in combination with SCW arthritis. Aggravation of SCW-induced arthritis by IL-12 gene transfer. To investigate the disease-promoting effect of local IL-12 expression, we injected 107 PFU of AdmIL-12.1 adenovirus intraarticularly 1 day before induction of SCW arthritis in the same joint. As shown in Figure 2A, there was markedly enhanced joint swelling during the first 2 weeks in the AdmIL-12.1–injected group compared with the group injected with Ad5del70-3 or with saline. Significantly increased joint swelling was still noted on day 21. Intraarticular injection of 107 PFU of Ad5del70-3 control virus did not significantly influence joint swelling. Inhibition of chondrocyte PG synthesis is one of the deleterious effects of joint inflammation on cartilage. Severe inhibition of matrix synthesis can be seen in the first stages of SCW-induced arthritis (Figure 2B). At later time points (days 7 and 14), an overshoot phenomenon can be observed. Injection of Ad5del70-3 control adenoviral vector before arthritis induction showed re- 1382 JOOSTEN ET AL IFN␥ to get insight into the local production of these cytokines after induction of arthritis. At several time points, synovial tissue washouts were analyzed for these Figure 1. Intraarticular transfer of murine interleukin-12 (IL-12) gene. A, To determine the duration of IL-12 overexpression, patellae with surrounding tissue were isolated at different time points after intraarticular gene application with 108 plaque-forming units (PFU) of AdmIL-12.1 adenoviral vector. Patellae were cultured for 1 hour in RPMI 1640 medium, and IL-12 levels were determined by enzyme-linked immunosorbent assay. Values are the mean ⫾ SD of 6 patellae washouts. B, Levels of IL-12 overexpression after injection of different doses of AdmIL-12.1 adenoviral vector. On day 2 after gene transfer, patellae were isolated, washouts were prepared, and IL-12 levels were measured as above. Values are the mean and SD of at least 5 mice per group. sults similar to those with saline injection. In contrast, long-lasting inhibition of chondrocyte PG synthesis was seen in patellar cartilage after AdmIL-12.1 injection. Severe disturbance of chondrocyte function was noted up to day 21. We also determined protein levels of IL-12 and Figure 2. Deterioration of streptococcal cell wall (SCW)–induced arthritis by interleukin-12 (IL-12) gene transfer. A, Effect of IL-12 overexpression on joint swelling. On day ⫺1, saline or 107 plaqueforming units of either Ad5del70-3 or AdmIL-12.1 was injected into the right knee joint of naive mice. One day later, arthritis was induced in the same joint by intraarticular injection of 25 g of SCW fragments. Joint swelling was determined at different time points after induction of arthritis. Values are the mean and SD of at least 6 mice per group. B, Prolonged inhibition of patellar cartilage chondrocyte proteoglycan synthesis. At different time points, patellae were isolated from the inflamed (right) and control (left) knee joints and chondrocyte synthetic function was determined by 35S-sulfate incorporation. Values are the mean and SD percentage of proteoglycan (PG) synthesis in the inflamed joint compared with the control joint in at least 6 mice per group. The experiment was repeated twice, with similar results. ⴱ ⫽ P ⬍ 0.05 versus Ad5del70-3, by Mann-Whitney U test. IL-12 GENE TRANSFER AND CHRONIC DESTRUCTIVE ARTHRITIS Table 1. 1383 IL-12 and IFN␥ levels in transduced knee joints with SCW arthritis* Day 2 Saline Ad5del70-3 AdmIL-12.1 Day 7 Day 14 IL-12 IFN␥ IL-12 IFN␥ IL-12 IFN␥ 120 ⫾ 20 140 ⫾ 30 580 ⫾ 40† 40 ⫾ 20 80 ⫾ 20 290 ⫾ 80† ⬍20 ⬍20 439 ⫾ 70† ⬍10 ⬍10 170 ⫾ 40† ⬍20 ⬍20 410 ⫾ 170† ⬍10 ⬍10 70 ⫾ 10† * Local levels of interleukin-12 (IL-12) and interferon-␥ (IFN␥) were determined in patellae washouts at several time points after induction of arthritis. One day before the injection of 25 g of streptococcal cell wall (SCW) fragments, knee joints were transduced with 107 plaque-forming units of Ad5del70-3 or AdmIL-12.1. IL-12 and IFN␥ levels were measured by enzyme-linked immunosorbent assay (sensitivity 20 pg/ml and 10 pg/ml, respectively). Values are the mean ⫾ SD pg/ml in 6 patellae washout samples per time point. † P ⬍ 0.01 versus Ad5del70-3, by Mann-Whitney U test. Th1-directing cytokines. As shown in Table 1, during the early stage (day 2), both IL-12 and IFN␥ could be detected in synovial washouts of mice injected with saline, Ad5del70-3, and AdmIL-12.1. However, in the AdmIL-12.1 group, markedly higher levels of IL-12 and IFN␥ were found at this time point. At later stages, only AdmIL-12.1–injected animals expressed detectable levels of IL-12 and IFN␥. The results indicate that local overexpression of IL-12 generates prolonged production of proinflammatory cytokines. Induction of cartilage damage by local overexpression of IL-12. Histologic analysis of whole knee joint sections revealed that IL-12 gene transfer leads to a chronic destructive SCW arthritis. As shown in Table 2, Table 2. the number of inflammatory cells in the synovial tissue was significantly enhanced compared with control virus Ad5del70-3 or with saline on days 7 and 21. Detailed analysis of cellular influx revealed that predominately lymphocytes were present in the synovial tissues of the IL-12-transduced joints (Figure 3). In addition, loss of matrix PG due to the inflammatory process was strongly deteriorated after IL-12 overexpression. Furthermore, cartilage destruction, determined as chondrocyte death and cartilage erosions, was increased in the AdmIL-12.1–injected knee joints, especially on day 21 (Table 2). Figure 4 illustrates the increased number of dead chondrocytes in the cartilage due to overexpression of IL-12. Joint changes on day 21 of SCW arthritis after IL-12 gene transfer* Day 7 Saline Ad5del70-3 AdmIL-12.1 Day 21 Saline Ad5del70-3 AdmIL-12.1 Infiltration of cells Chondrocyte death Proteoglycan depletion Cartilage erosion 0.8 ⫾ 0.3 1.1 ⫾ 0.3 2.4 ⫾ 0.4† 0.0 ⫾ 0.0 0.0 ⫾ 0.0 0.2 ⫾ 0.3 1.0 ⫾ 0.6 1.6 ⫾ 0.5 2.8 ⫾ 0.4† 0.0 ⫾ 0.0 0.0 ⫾ 0.0 0.0 ⫾ 0.0 0.1 ⫾ 0.1 0.0 ⫾ 0.2 0.9 ⫾ 0.3† 0.0 ⫾ 0.0 0.1 ⫾ 0.1 1.2 ⫾ 0.4† 0.0 ⫾ 0.0 0.3 ⫾ 0.4 1.5 ⫾ 0.6† 0.0 ⫾ 0.0 0.0 ⫾ 0.0 0.4 ⫾ 0.1† * Histopathologic features of arthritic knee joints after gene transfer with Ad5del70-3 or AdmIL-12.1 virus. On day ⫺1, saline or 107 plaque-forming units of the adenovirus was injected intraarticularly into the right knee joint. One day later, 25 g of streptococcal cell wall (SCW) fragments was injected into the same knee joint. On days 7 and 21, knee joints were obtained and processed for histologic analysis. Histopathologic features were scored on a scale of 0–3, as indicated in Materials and Methods. Values are the mean ⫾ SD of at least 6 knee joints per group. IL-12 ⫽ interleukin-12. † P ⬍ 0.01 versus Ad5del70-3, by Mann-Whitney U test. 1384 JOOSTEN ET AL demonstrate the destructive character of the chronic inflammation caused by the IL-12 overexpression, sections were stained for this neoepitope. VDIPEN was highly expressed throughout the patellar cartilage layer in the AdmIL-12.1–treated group (Figure 5C). In contrast, no VDIPEN expression was noted in the Figure 3. Histopathologic features of streptococcal cell wall (SCW)– induced arthritis in combination with interleukin-12 (IL-12) gene transfer. Mouse knee joints were injected with A, saline, B, Ad5del70-3 (107 plaque-forming units [PFU]), or C, AdmIL-12.1 (107 PFU) and SCW fragments and were harvested and examined on day 21 after arthritis induction. Note the chronic inflammation and the cellular infiltrate in the AdmIL-12.1–injected group. (Hematoxylin and eosin stained; original magnification ⫻ 400.) VDIPEN neoepitope is a marker of metalloproteinase-mediated cleavage of aggrecan, the major PG of articular cartilage. Previous studies have revealed that the VDIPEN neoepitope was more abundant at sites and stages of advanced damage (31,32). To further Figure 4. Chondrocyte damage after IL-12 gene transfer. Mouse knee joints were injected with A, saline, B, Ad5del70-3, or C, AdmIL-12.1 as in Figure 3. Note the severe chondrocyte death in the cartilage layers of the patella and femur in the AdmIL-12.1–injected group. (Hematoxylin and eosin stained; original magnification ⫻ 100.) See Figure 3 for definitions. IL-12 GENE TRANSFER AND CHRONIC DESTRUCTIVE ARTHRITIS 1385 Ad5del70-3–injected (Figure 5B) or the saline-injected (Figure 5A) groups. Lymphocyte dependence of the IL-12 gene transfer–induced conversion to chronic destructive arthritis. To investigate whether a local immune response is involved in the process of switching from an acute SCW arthritis to a chronic destructive arthritis, we performed experiments in RAG-2–deficient mice. These mice do not establish immune responses to antigens because their T cells and B cells are not functional (26). Figure 6. Role of functional lymphocytes in the conversion of an acute arthritis to a chronic streptococcal cell wall (SCW)–induced arthritis. Wild-type (C57BL/6) and recombination-activating gene 2 (RAG-2)–deficient mice were injected intraarticularly injected 107 plaque-forming units of AdmIL-12.1 virus. One day later, 25 g of SCW fragments was injected in the same joint. Joint swelling was determined on days 3, 14, and 21. Values are the mean and SD of at least 7 mice per group. ⴱ ⫽ P ⬍ 0.05 versus wild-type mice, by Mann-Whitney U test. Figure 5. Metalloproteinase activity as visualized by VDIPEN staining. Shown are immunostained paraffin sections of mouse knee joints injected with A, saline, B, Ad5del70-3, and C, AdmIL-12.1 (see Figure 3 for details). VDIPEN neoepitope expression was seen only in cartilage layers of the AdmIL-12.1–injected group. (Original magnification ⫻ 200.) The results (Figure 6) demonstrated that IL-12 overexpression leads to a local immune-mediated arthritic process. Overexpression of IL-12 in C57BL/6 mice induced a chronic inflammatory joint disease after injection of SCW fragments, whereas RAG-2–deficient mice did not develop chronic arthritis. The initial arthritis was equivalent in the two mouse strains, as determined on day 3. The results of histologic assessments corroborated the finding that RAG-2–deficient mice did not generate a chronic destructive arthritis (Table 3). Systemic reactivation of chronic SCW arthritis. In line with the above findings, we investigated whether the local chronic inflammatory process induced by IL-12 gene transfer could be experiencing a flare. Earlier studies have shown that chronically inflamed joints bearing a specific T cell infiltrate can be reactivated with small amounts of homologous antigen. To this end, mice were challenged intravenously with 100 g of SCW fragments on day 28. SCW arthritis in mice of the saline-injected and the Ad5del70-3–injected groups did not show a significant flare (Figure 7). In contrast, intravenous administration of SCW fragments reactivated inflammation in the AdmIL-12.1–injected group (Figure 7). These data indicate that local overexpression of 1386 JOOSTEN ET AL Table 3. Joint changes in C57BL/6 and RAG-2⫺/⫺ mice after IL-12 gene transfer* C57BL/6 mice RAG-2⫺/⫺ mice Infiltration of cells Chondrocyte death Proteoglycan depletion Cartilage erosion 1.0 ⫾ 0.3 0.2 ⫾ 0.1† 1.1 ⫾ 0.5 0.1 ⫾ 0.2† 1.4 ⫾ 0.4 0.2 ⫾ 0.3† 0.5 ⫾ 0.2 0.0 ⫾ 0.0† * Histopathologic changes in C57BL/6 mice and recombination-activating gene 2 (RAG-2)– deficient mice after gene transfer with AdmIL-12.1 virus in combination with streptococcal cell wall (SCW) fragments. On day ⫺1, 107 plaque-forming units of the adenovirus was injected intraarticularly into the right knee joint. One day later, 25 g of SCW fragments was injected into the same knee joint. On day 21, knee joints were obtained and processed for histologic analysis. Histopathologic features were scored on a scale of 0–3, as indicated in Materials and Methods. Values are the mean ⫾ SD of at least 6 knee joints per group. IL-12 ⫽ interleukin-12. † P ⬍ 0.01 versus C57BL/6 mice, by Mann-Whitney U test. IL-12 leads to a chronic T cell–dependent hyperresponsive state. Histologic sections obtained before and after reactivation revealed increased numbers of inflammatory cells, such as polymorphonuclear granulocytes and monocytes, in the synovial cavity and tissue of the AdmIL-12.1–injected group that had been rechallenged with SCW fragments (Figure 8). Figure 7. Systemic reactivation of SCW-induced arthritis after IL-12 gene transfer. Mice were injected with saline, Ad5del70-3 (107 PFU), or AdmIL-12.1 (107 PFU) 1 day before the induction of SCW arthritis. On day 28, joint swelling was examined by a 99mTc-uptake method. An arthritis flare was then induced by intravenous (i.v.) injection of 100 g of SCW fragments, and 24 hours later (day 29), joint swelling in the inflamed (right) and control (left) knee joints was measured. Values are the mean and SD of at least 6 mice per group. ⴱ ⫽ P ⬍ 0.05 versus the Ad5del70-3 group, by Mann-Whitney U test. The experiment was repeated, and similar results were obtained. See Figure 3 for other definitions. DISCUSSION In the present study, we demonstrated that local overexpression of IL-12 changed an acute joint inflammation induced by bacterial fragments to a chronic destructive arthritic process. This conversion was shown to be mediated by lymphocytes, since RAG-2–deficient mice did not develop a chronic stage of arthritis after IL-12 gene transfer. RA is a disease that is characterized by Th1associated pathologic changes. IL-12 is expressed in the synovial tissue of RA patients and may be involved in the IFN␥-dominant cytokine production by infiltrating T cells in joints with chronic arthritis. As previously demonstrated, the major producers of IL-12 in human RA synovium are mainly CD68-positive macrophages (18,33). Several cell types, including macrophages and NK cells, produce IL-12 after in vitro triggering with a range of stimuli (1–3). Bacteria or fragments of bacteria are the most potent inducers of IL-12 production by monocyte/ macrophages. Interestingly, gram-positive bacteria are potent inducers of IL-12 production, whereas gram-negative bacteria are inducers of IL-10 (34). IL-12 alone, as well as in synergy with IL-18, stimulates IFN␥ production. It has been demonstrated that bacteria, in combination with IL-12 or IFN␥, induce a strong elevation of TNF␣ and nitric oxide production by macrophages (35). Recently, we demonstrated that fragments of gram-positive streptococci generated enhanced levels of TNF␣, IL-1, IL-12, and IFN␥ when injected intraarticularly into the knee joints of naive mice (25). Apart from bacterial cell wall fragments, intraarticular application of CpG motifs of bacterial DNA into the mouse knee joint can induce arthritis. Analysis of cytokine messenger RNA (mRNA) expression revealed IL-12 GENE TRANSFER AND CHRONIC DESTRUCTIVE ARTHRITIS 1387 Figure 8. Influx of inflammatory cells after reactivation of chronic arthritis induced by interleukin-12 (IL-12) gene transfer. A, Ad5del70-3–injected joint on day 28. B, Ad5del70-3–injected joint on day 29 (1 day after reactivation of arthritis). C, AdmIL-12.1–injected joint on day 28. D, AdmIL-12.1–injected joint on day 29 (1 day after reactivation of arthritis) (see Figure 7 for details). Note the influx of predominantly polymorphonuclear cells in the synovial tissue and joint cavity in the AdmIL-12.1–injected knee after rechallenge (day 29). (Hematoxylin and eosin stained; original magnification ⫻ 100.) that IL-12 was up-regulated for several days after injection of CpG oligonucleotide (36). Furthermore, murine synovial explants cultured for 24 hours in vitro with CpG oligonucleotides showed an up-regulation of mRNA levels for IL-12, IL-15, and IL-18 (Joosten LAB, et al: unpublished observations). These data indicate that bacterial cell wall or DNA fragments can induce the production of Th1-directing cytokines in synovial tissues. As previously shown, IL-12 is involved in SCWinduced arthritis. Blockade of endogenous IL-12 by use of anti–murine IL-12 antibodies suppressed joint swelling in SCW-induced arthritis. In addition, reduced levels of IFN␥ and IL-1 were found in washouts of synovial tissue explants (25). IL-1 is a pleiotropic cytokine that exerts a direct cartilage-destructive property as well as an immune-stimulatory activity. It has been shown that IL-1␤ is a potent costimulus of IFN␥ production by a subset of NK cells following infectious insult (37). The important role of IL-1 in the development of chronic arthritis was nicely demonstrated in IL-1␣/␤ deficient mice (23). It is unlikely that IL-12 plays a direct role in the production of IL-1, since IL-12 administration to mice did not result in elevated serum levels of IL-1␣ or IL-1␤ (38). Gene transfer by adenoviral vectors has been shown to be an effective way to generate overexpression of certain cytokines or cytokine inhibitors (14,39). Synovial cells can be targeted by adenoviral constructs to achieve high levels of cytokines, such as IL-4, IL-10, or IL-17 (40,41). In the present study, we showed that high levels of IL-12 were produced up to day 14 after local application of 1 ⫻ 107 PFU of AdmIL-12.1. Interestingly, apart from IL-12, enhanced levels of IFN␥ were found in transduced knee joints (Table 1). IL-12 is thought to play a pivotal role in inducing Th1 cell–mediated organ-specific autoimmune diseases, 1388 as described previously (42). The role of IL-12 in the induction of chronic Th1-mediated diseases, such as experimental colitis and arthritis, is well known. IL-12 exposure or the use of IL-12–deficient mice has revealed that IL-12 is a crucial immune-stimulatory cytokine for the development of experimentally induced arthritis (13,15,16). This is the first study showing that local overexpression of IL-12 in combination with an acute inflammation generates a chronic immune-mediated destructive process. It has recently been shown that bacteria or fragments of bacteria activate nuclear factor B directly and, via induction of cytokines, activate signal transducer and activator of transcription 1 (STAT-1) and STAT-3 (43) in macrophages. This is consistent with our previous findings that SCW fragments generate the production of cytokines, such as TNF␣, IL-1, IL-12, IL-18, and IFN␥, when injected directly into the mouse knee joint (25,44). To corroborate the finding that local lymphocytes were involved, we performed experiments in immunodeficient mice with a targeted disruption in the recombination-activation gene 2, which is expressed only in lymphocytes (26). These mice lack functional T and B cells, as shown in several models of experimental autoimmune diseases (45,46). We found that local overexpression of IL-12 did not convert acute SCW-induced arthritis to a chronic arthritis in the RAG-2–deficient mice, indicating that T and B cells are involved in the generation of the chronic destructive arthritis (Figure 6). Involvement of a local IL-12–induced hyperreactive immune response was further investigated by reactivation of the arthritic process by intravenous challenge with SCW fragments (Figures 7 and 8). We demonstrated previously that this latter reactivation was dependent on lymphocytes, since antilymphocyte treatment abrogated the exacerbation of arthritis (21). Taken together, these findings demonstrate that IL-12 can convert modest joint inflammation to a T cell–mediated destructive joint pathology. Whether IL-12 contributes to the development of the chronic destructive process in human RA is still not known, since anti–IL-12 treatment is not performed in the clinic. However, it is known that several bacterial infections correlate with arthritis. Apart from septic arthritis, arthritis occurs in patients with Lyme disease and infections of the throat and gastrointestinal tract. IL-12 levels are elevated in patients with infections, and it might be that IL-12 is the important cytokine that generates a local autoimmune disease. It has been postulated that a possible cause of joint destruction in septic arthritis is the presence of CpG motifs in the joint JOOSTEN ET AL (47). CpG motifs can induce local production of IL-12, and the acute bacterial-induced inflammation could be transformed to a destructive arthritis. Furthermore, CpG motifs present in Chlamydia can act as an adjuvant to trigger immune responses to self antigens, causing heart diseases (48). Enhanced local levels of IL-12, due to the presence of bacterial fragments or CpG motifs, may be the driving force responsible for the development of an autoimmune response to antigens, such as PGs, type II collagen, or human cartilage gp-39. IL-12 may be a novel target in human RA, since IL-12 may promote the spreading of autoimmune processes to other joints. However, IL-12 inhibition should be limited to the joint to obtain an efficient innate immune response. REFERENCES 1. Stern AS, Podlaski FJ, Hulmes JD, Pan EY, Quinn PM, Wolitzky AG, et al. Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells. Proc Natl Acad Sci U S A 1990;87:6808–12. 2. Kobayashi M, Fitz L, Ryan M, Hewick RM, Clark SC, Chan SH, et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 1989;170:827–45. 3. D’Andrea A, Rengaraju M, Valiante NM, Chehimi J, Kubin M, Aste M, et al. Production of natural killer cell stimulatory factor (interleukin-12) by peripheral blood mononuclear cells. J Exp Med 1992;176:1387–98. 4. Gazzinelli RT, Hieny S, Wynn TA, Wolf SF, Sher A. IL-12 is required for the T-lymphocyte-independent induction of interferon-␥ by an intracellular parasite and induces resistance in T-celldeficient hosts. Proc Natl Acad Sci U S A 1993;90:6115–9. 5. Seder RA, Gazzinelli RT, Sher A, Paul WE. Interleukin-12 acts directly on CD4⫹ T-cells to enhance priming for interferon-␥ production and diminished interleukin-4 inhibition of such priming. Proc Natl Acad Sci U S A 1993;90:10188–92. 6. Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996;383:787–93. 7. Okamura H, Tsutsui H, Kashiwamura SI, Yoshimoto T, Nakanishi K. Interleukin-18: a novel cytokine that augments both innate and acquired immunity. Adv Immunol 1998;70:281–312. 8. Munder M, Mallo M, Eichmann K, Modolell M. Murine macrophages secrete interferon ␥ upon combined stimulation with interleukin (IL)-12 and IL-18: a novel pathway of autocrine macrophage activation. J Exp Med 1998;187:2103–8. 9. Wysocka M, Kubin M, Vieira LQ, Ozmen L, Garotta G, Scott P, et al. Interleukin-12 is required for interferon-␥ production and lethality in lipopolysaccharide-induced shock in mice. Eur J Immunol 1995;25:672–6. 10. Ozmen L, Pericin M, Hakimi J, Chizzonite RA, Wysocka M, Trinchieri G, et al. Interleukin-12, interferon-␥ and tumor necrosis factor ␣ are the key cytokines of the generalized Shwartzman reaction. J Exp Med 1994;180:907–15. 11. Trinchieri G. IL-12: a proinflammatory cytokine with immunoregulatory functions that bridges innate resistance and antigen specific adaptive immunity. Annu Rev Immunol 1995;13:251–76. 12. Segal BM, Shevach EM. IL-12 unmasks autoimmune disease in resistant mice. J Exp Med 1996;184:771–5. IL-12 GENE TRANSFER AND CHRONIC DESTRUCTIVE ARTHRITIS 13. Chen E, Keystone EC, Fish EN. Restricted cytokine expression in rheumatoid arthritis. Arthritis Rheum 1993;36:901–10. 14. Morita Y, Yamamura M, Nishida K, Harada S, Okamoto H, Inoue M, et al. Expression of interleukin-12 in synovial tissue from patients with rheumatoid arthritis. Arthritis Rheum 1998;41: 306–14. 15. Peeva E, Fishman AD, Goddard G, Wadler S, Barland P. Rheumatoid arthritis exacerbation caused by exogenous interleukin-12. Arthritis Rheum 2000;43:461–3. 16. Kim WU, Min SY, Cho ML, Youn J, Min JK, Lee SH, et al. The role of IL-12 in inflammatory activity of patients with rheumatoid arthritis (RA). Clin Exp Immunol 2000;119:175–81. 17. Joosten LAB, Lubberts E, Helsen MMA, van den Berg WB. Dual role of interleukin-12 in early and late stages of murine collagen type II arthritis. J Immunol 1997;159:4094–102. 18. Parks E, Strieter R, Lukacs NW, Gauldie J, Hitt M, Graham FL, et al. Transient gene transfer of IL-12 regulates chemokine expression and disease severity in experimental arthritis. J Immunol 1998:160:4615–9. 19. Leung BP, McInnes IB, Esfandiari E, Wei X, Liew FY. Combined effects of IL-12 and IL-18 on the induction of collagen-induced arthritis. J Immunol 2000;164:6495–502. 20. McIntyre KW, Schuster DJ, Gillooly KM, Warrier RR, Connaughton SE, Hall LB, et al. Reduced incidence and severity of collagen-induced arthritis in IL-12-deficient mice. Eur J Immunol 1996;26:2933–8. 21. Van den Broek MF, van den Berg WB, van de Putte LBA, Severijnen AJ. Streptococcal cell wall induced arthritis and flare-up reactions in mice induced by homologous and heterologous cell walls. Am J Pathol 1988;133:139–49. 22. Kuiper S, Joosten LAB, Bendele AM, Edwards CK III, Arntz OJ, Helsen MMA, et al. Different roles for tumour necrosis factor ␣ and interleukin-1 in murine streptococcal cell wall arthritis. Cytokine 1998;10:690–702. 23. Van den Berg WB, Joosten LAB, Kollias G, van de Loo FAJ. Role of tumour necrosis factor ␣ in experimental arthritis: separate activity of interleukin-1␤ in chronicity and cartilage destruction. Ann Rheum Dis 1999;58 Suppl 1:I40–8. 24. Lubberts E, Joosten LAB, Helsen MMA, van den Berg WB. Regulatory role of interleukin 10 in joint inflammation and cartilage destruction in murine streptococcal cell wall (SCW) arthritis: more therapeutic benefit with IL-4/IL-10 combination therapy than with IL-10 treatment alone. Cytokine 1998;10:361–9. 25. Joosten LAB, Helsen MMA, van den Berg WB. Blockade of endogenous interleukin-12 results in suppression of murine streptococcal cell wall arthritis by enhancement of interleukin-10 and interleukin-1Ra. Ann Rheum Dis 2000;59:196–205. 26. Shinkai Y, Rathbun G, Lam K-P, Oltz EM, Stewart V, Mendelsohn M, et al. RAG-2–deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 1992;68: 855–67. 27. Bramson J, Hitt M, Gallichan WS, Rosenthal KL, Gauldie J, Graham FL. Construction of a double recombinant adenovirus vector expressing a heterodimeric cytokine: in vitro and in vivo production of biologically active interleukin-12. Hum Gene Ther 1996;7:333–42. 28. Chen Y, Song K, Eck SL, Chen Y. An intra-Peyer’s patch gene transfer model for studying mucosal tolerance: distinct roles of B7 and IL-12 in mucosal T cell tolerance. J Immunol 2000;165:3124–53. 29. Kruijsen MWM, van den Berg WB, van den Putte LBA, van den Broek WJM. Detection of and quantification of experimental joint inflammation in mice by measurements of 99mTc-pertechnetate uptake. Agents Actions 1981;11:640–2. 30. Singer II, Kawka DW, Bayne EK, Donatelli SA, Weidner JR, Williams HR, et al. VDIPEN, a metalloproteinase-generated neoepitope, is induced and immunolocalized in articular cartilage during inflammatory arthritis. J Clin Invest 1995;95:2178–84. 1389 31. Van Meurs JBJ, van Lent PLEM, Singer II, Bayne EK, van de Loo FAJ, van den Berg WB. Interleukin-1 receptor antagonist prevents expression of the metalloproteinase-generated neoepitope VDIPEN in antigen-induced arthritis. Arthritis Rheum 1998;41:647–56. 32. Joosten LAB, Helsen MMA, Saxne T, van de Loo FAJ, Heinegård D, van den Berg WB. IL-1␤ blockade prevents cartilage and bone destruction in murine type II collagen-induced arthritis, whereas TNF␣ blockade only ameliorates joint inflammation. J Immunol 1999;163:5049–55. 33. Sakkas LI, Johanson NA, Scanzello CR, Platsoucas DC. Interleukin-12 is expressed by infiltrating macrophages and synovial lining cells in rheumatoid arthritis and osteoarthritis. Cell Immunol 1998;188:105–10. 34. Hessle C, Andersson B, Wold AE. Gram-positive bacteria are potent inducers of monocytic interleukin-12 (IL-12) while gramnegative bacteria preferentially stimulate IL-10 production. Infect Immun 2000;68:3581–6. 35. Xing Z, Zganiacz A, Santosuosso M. Role of IL-12 in macrophage activation during intracellular infection: IL-12 and mycobacteria synergistically release TNF␣ and nitric oxide from macrophages via IFN-␥ induction. J Leukoc Biol 2000;68:897–902. 36. Deng GM, Nilson IM, Verdrengh M, Collins LV, Tarkowski A. Intra-articular localized bacterial DNA containing CpG motifs induces arthritis. Nat Med 1999;5:702–5. 37. Cooper MA, Fehniger TA, Ponnappan A, Mehta V, Wewers MD, Caligiuri MA. Interleukin-1␤ costimulates interferon-␥ production by human natural killer cells. Eur J Immunol 2001;31:792–801. 38. Nakamura S, Otani T, Ijiri Y, Motoda R, Kurimoto M, Orita K. IFN-␥-dependent and -independent mechanisms in adverse effects caused by concomitant administration of IL-18 and IL-12. J Immunol 2000;164:3330–6. 39. Bakker AC, van de Loo FAJ, Joosten LA, Varley AW, Munford RS, van den Berg WB. Disease inducible IL-1Ra overexpression results in a more efficient inhibition of the collagen induced arthritis model than constitutive overexpression of IL-1Ra [abstract]. Arthritis Rheum 2000;43 Suppl 9:S169. 40. Lubberts E, Joosten LAB, Chabaud M, van den Bersselaar LAM, Coenen-de Roo CCJ, Richards CD, et al. IL-4 gene therapy for collagen arthritis suppresses synovial IL-17 and osteoprotegerin ligand and prevents bone erosion. J Clin Invest 2000;105:697–710. 41. Lubberts E, Joosten LAB, van den Bersselaar LAM, Helsen MMA, Bakker AC, van Meurs JBL, et al. Adenoviral vectormediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction. J Immunol 1999;163:4546–56. 42. Trembleau S, Germann T, Gately MK, Adorini L. The role of IL-12 in the induction of organ-specific autoimmune diseases. Immunol Today 1995;68:333–6. 43. Miettinen M, Lehtonen A, Julkunen I, Matikainen S. Lactobacilli and streptococci activate NFB and STAT signaling pathways in human macrophages. J Immunol 2000;164:3733–40. 44. Joosten LAB, van de Loo FAJ, Lubberts E, Helsen MMA, Netea MG, van der Meer JWM, et al. An IFN-␥ independent proinflammatory role of IL-18 in murine streptococcal cell wall arthritis. J Immunol 2000;165:6553–8. 45. McKisic MD, Redmond WL, Barthold S. T cell-mediated pathology in murine Lyme borreliosis. J Immunol 2000;164:6096–9. 46. Simpson SJ, Shag S, Comiskey M, de Jong YP, Wang B, Mizoguchi E, et al. T cell-mediated pathology in two models of experimental colitis depends predominantly on the interleukin-12/signal transducer and activator of transcription (Stat)-4 pathway, but is not conditional on interferon ␥ expression by T cells. J Exp Med 1998;187:1225–34. 47. Krieg AM. A possible cause of joint destruction in septic arthritis. Arthritis Res 1999;1:3–4. 48. Bachmaier K, Neu N, de la Maza LM, Pal S, Hessel A, Penninger JM. Chlamydia infections and heart disease linked through antigenic mimicry. Science 1999;283:1335–9.