Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation cartilage destruction and bone erosion.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 50, No. 2, February 2004, pp 650–659 DOI 10.1002/art.20001 © 2004, American College of Rheumatology Treatment With a Neutralizing Anti-Murine Interleukin-17 Antibody After the Onset of Collagen-Induced Arthritis Reduces Joint Inflammation, Cartilage Destruction, and Bone Erosion Erik Lubberts,1 Marije I. Koenders,1 Birgitte Oppers-Walgreen,1 Liduine van den Bersselaar,1 Christina J. J. Coenen-de Roo,2 Leo A. B. Joosten,1 and Wim B. van den Berg1 Objective. Interleukin-17 (IL-17) is a proinflammatory cytokine that is expressed in the synovium of rheumatoid arthritis (RA) patients. This T cell cytokine is implicated in the initiation phase of arthritis. However, the role of IL-17 during the effector phase of arthritis has still not been identified; this was the objective of the present study. Methods. Mice with collagen-induced arthritis (CIA) were treated with polyclonal rabbit anti-murine IL-17 (anti–IL-17) antibody–positive serum or normal rabbit serum after the first signs of arthritis. In addition, during a later stage of CIA mice were selected and treated with anti–IL-17 antibody or control serum. Arthritis was monitored visually, and joint pathology was examined radiologically and histologically. Systemic IL-6 levels were measured by enzyme-linked immunosorbent assay, and local synovial IL-1 and receptor activator of NF-B ligand (RANKL) expression was analyzed using specific immunohistochemistry. Results. Treatment with a neutralizing anti–IL-17 antibody after the onset of CIA significantly reduced the severity of CIA. Radiographic analysis revealed marked suppression of joint damage in the knee and ankle joints. Histologic analysis confirmed the suppression of joint inflammation and showed prevention of cartilage and bone destruction after anti–IL-17 antibody therapy. Systemic IL-6 levels were significantly reduced after anti–IL-17 antibody treatment. Moreover, fewer IL-1␤– positive and RANKL-positive cells were detected in the synovium after treatment with neutralizing IL-17. Interestingly, initiation of anti–IL-17 antibody therapy during a later stage of CIA, using mice with higher clinical arthritis scores, still significantly slowed the progression of the disease. Conclusion. IL-17 plays a role in early stages of arthritis, but also later during disease progression. Systemic IL-6 was reduced and fewer synovial IL-1– positive and RANKL-positive cells were detected after neutralizing endogenous IL-17 treatment, suggesting both IL-1–dependent and IL-1–independent mechanisms of action. Our data strongly indicate that IL-17 neutralization could provide an additional therapeutic strategy for RA, particularly in situations in which elevated IL-17 may attenuate the response to anti–tumor necrosis factor/anti–IL-1 therapy. Supported by a grant from the Dutch Arthritis Association (NR-00-1-302). Dr. Lubberts’ work was supported by a Veni Fellowship of the Netherlands Organization for Scientific Research (NWO grant 906-02-038). 1 Erik Lubberts, PhD, Marije I. Koenders, MSc, Birgitte Oppers-Walgreen, Liduine van den Bersselaar, Leo A. B. Joosten, PhD, Wim B. van den Berg, PhD: University Medical Center Nijmegen, Nijmegen, The Netherlands; 2Christina J. J. Coenen-de Roo, MSc: NV Organon, Oss, The Netherlands. Address correspondence and reprint requests to Erik Lubberts, University Medical Center Nijmegen, Nijmegen Center for Molecular Life Sciences, Rheumatology and Advanced Therapeutics, 189, 6500 HB Nijmegen, The Netherlands. E-mail: E.Lubberts@reuma.umcn.nl. Submitted for publication February 26, 2003; accepted in revised form October 21, 2003. Interleukin-17 (IL-17) is a T cell–derived cytokine produced by activated T cells, predominantly activated CD4⫹,CD45RO⫹ memory T cells (1,2). This cytokine may play a role in T cell–triggered inflammation by stimulating stromal cells to secrete various cytokines and growth factors associated with inflammation (1–4). A pathogenic role for IL-17 was found in organ allograft rejection (5), and increased IL-17 expression was detected in several diseases, such as systemic sclerosis (6), nephrotic syndrome (7), systemic lupus 650 ANTI–IL-17 THERAPY AFTER ONSET OF CIA erythematosus (8), and rheumatoid arthritis (RA) (9,10). In contrast with the restricted expression of IL-17, the IL-17 receptor is ubiquitously expressed in virtually all cells and tissues. It is a type I transmembrane protein that has no sequence similarity with any other known cytokine receptor (3). Binding of IL-17 to its unique receptor results in activation of the adaptor molecule tumor necrosis factor (TNF) receptor– associated factor 6, which is required for IL-17 signaling (11). RA is considered a systemic Th1-associated inflammatory joint disease that is characterized by chronic synovitis and destruction of cartilage and bone. T cells represent a large proportion of the inflammatory cells invading the synovial tissue. Since the etiology of RA is still unknown, regulating the cytokine imbalance might represent an effective way to control this disease. The proinflammatory cytokines TNF␣ and IL-1␤ play a crucial role in the pathology of arthritis, driving enhanced production of cytokines, chemokines, and degradative enzymes (12). In vivo studies have shown that neutralizing TNF␣ or IL-1␤ controls chronic inflammation and cartilage degradation, respectively (13–15). Consistent with this, clinical studies revealed efficacy after blocking of TNF␣ or IL-1␤. However, a subset of patients did not respond to these inhibitors, and none of the treatments cured the disease. Therefore, it is tempting to speculate that cytokines or factors other than IL-1␤ and TNF␣ also participate in the proinflammatory cytokine cascade. T cell cytokine IL-17 is spontaneously produced by RA synovial membrane cultures (9), and high levels have been detected in the synovial fluid of patients with RA (9,10). IL-17 can stimulate the production of IL-1␤ and TNF␣ from macrophages (4) and triggers human synoviocytes to produce IL-6, IL-8, granulocyte– macrophage colony-stimulating factor, and prostaglandin E2 (2,16), suggesting that IL-17 could be an upstream mediator in the pathogenesis of arthritis. Early neutralization of endogenous IL-17 prior to the development of arthritis in the experimental arthritis model suppresses the onset of disease (17,18). Furthermore, IL-17 may be involved in tissue destruction. IL-17 has biologic activities similar to those of IL-1␤, and additive/ synergistic effects with IL-1␤ and TNF␣ have been reported (19). In vitro, IL-17 suppresses matrix synthesis by articular chondrocytes through enhancement of nitric oxide (NO) production (20,21). In addition, in vitro studies suggested a role for IL-17 in bone erosion by induction of receptor activator of NF-B ligand (RANKL) expression (22). Recently, we showed that 651 IL-17 promotes bone erosion in murine collageninduced arthritis (CIA) through loss of the RANKL/ osteoprotegerin (OPG) balance (23). These observations indicate that IL-17 may promote joint inflammation as well as tissue destruction during the initial phase of arthritis. However, the role of T cell IL-17 during the effector phase of arthritis has still not been identified. In the present study, we demonstrated the therapeutic effect of anti–IL-17 antibody treatment in CIA, implying that the T cell cytokine IL-17 not only plays a role in the early stage of arthritis, but also has a function in propagating and prolonging the arthritis. Furthermore, fewer synovial IL-1␤–positive and RANKLpositive cells were found after treatment with neutralizing endogenous IL-17. This suggests that IL-17 might be a novel target for the treatment of destructive arthritis and implies that neutralization of this T cell factor during the effector phase of arthritis has therapeutic potential. Our data suggest that anti–IL-17 cytokine therapy is an interesting new approach that may contribute to the prevention of joint destruction and could provide an important additional strategy to the current anti-TNF and anti–IL-1␤ therapy for RA. MATERIALS AND METHODS Animals. Male DBA-1/Bom mice were purchased from Bomholtgård (Ry, Denmark). The mice were housed in filtertop cages. Arthritis was induced in mice between 10 and 12 weeks of age. Water and food were provided ad libitum. Animal studies were approved by the Institutional Review Board. Induction of CIA. Bovine type II collagen (CII) was prepared as previously described (24) and diluted in 0.05M acetic acid to a concentration of 2 mg/ml. This was emulsified in equal volumes of Freund’s complete adjuvant (CFA) (2 mg/ml Mycobacterium tuberculosis, strain H37Ra; Difco, Detroit, MI). DBA-1/Bom mice were immunized at the base of the tail with 100 g of bovine CII. On day 21, mice received an intraperitoneal (IP) booster injection of 100 g of CII dissolved in phosphate buffered saline (PBS), and the onset of arthritis usually occurred a few days after the booster injection. Assessment of arthritis. Mice were considered to have arthritis when significant changes in redness and/or swelling were noted in the digits or in other parts of the paws. Knee joint inflammation was scored visually after skin dissection, using a scale of 0–2 where 0 ⫽ uninflamed, 1 ⫽ mild, 1.5 ⫽ marked, and 2 ⫽ severe. Scoring was done by 2 independent observers (EL, MIK) without knowledge of the experimental groups. Rabbit anti-murine IL-17 antisera. Polyclonal rabbit antibodies were raised against recombinant murine IL-17 (mIL-17; R&D Systems, Minneapolis, MN) in our laboratory by immunization with CFA, and repeated subcutaneous injec- 652 tion of IL-17 mixed with Alum (Pierce, Rockford, IL). Anti– IL-17 titers were determined by a specific mIL-17 enzymelinked immunosorbent assay (ELISA; R&D Systems). All sera were complement deactivated. Anti–IL-17–positive sera blocked the stimulatory capacity of recombinant mIL-17 to induce NO production in the murine chondrocyte cell line H4 (25). Using this in vitro system, no cross-reactivity with IL-1␤, IL-1␣, or TNF␣ (R&D Systems) was observed. Study protocol. CIA was induced in male DBA-1/Bom mice as described above. After the first clinical signs of arthritis were observed (clinical arthritis score between 0.25 and 0.75), a single injection with the polyclonal mIL-17 antiserum (200 l/mouse) was given. Furthermore, mice with a higher CIA score (clinical arthritis score between 1.0 and 1.5) were selected and treated with a single IP injection of the polyclonal anti–mIL-17 serum (200 l/mouse). As a control, the same amount of normal rabbit serum was injected. The appearance of arthritis in the joints was assessed and the severity score was recorded as previously described (24). Thereafter, knee and ankle joints were isolated and processed by light microscopy. Assessment of the specificity of anti–IL-17 antibody in vitro. The H4 chondrocyte cell line was used to examine the specificity of the anti–IL-17 antibody. H4 cells (105) were incubated with IL-17 (25 ng/ml), IL-1␣ (10 ng/ml), IL-1␤ (1 ng/ml), or TNF␣ (10 ng/ml) with or without anti–IL-17 antibody (1:400) using RPMI 1640 tissue culture medium supplemented with 0.1% bovine serum albumin (BSA). After 24 hours, 100 l of conditioned medium was mixed with 100 l of Griess reagent (0.1% N-[1-naphthyl]ethylenediamine dihydrochloride [Sigma, St. Louis, MO] in 5% H3PO4) in a flatbottomed microtiter plate (Costar, Cambridge, MA), and the optical density at 545 nm (OD545) was measured using an ELISA plate reader (Titertek Multiscan MCC 340; Labsystems, Helsinki, Finland). Histologic analysis. Mice were killed by cervical dislocation. Thereafter, whole knee and/or ankle joints were removed and fixed for 4 days in 10% formalin. After decalcification in 5% formic acid, the specimens were processed for paraffin embedding (26). Tissue sections (7 m) were stained with hematoxylin and eosin or Safranin O. Histopathologic changes were scored using the following parameters. Infiltration of cells was scored on a scale of 0–3, depending on the amount of inflammatory cells in the synovial cavity (exudate) and synovial tissue (infiltrate). Proteoglycan depletion was determined using Safranin O staining. The loss of proteoglycans was scored on a scale of 0–3, ranging from fully stained cartilage to destained cartilage or complete loss of articular cartilage. A characteristic parameter in CIA is the progressive loss of articular cartilage. This destruction was graded separately on a scale of 0–3, ranging from the appearance of dead chondrocytes (empty lacunae) to the complete loss of articular cartilage (cartilage surface erosion). The degree of chondrocyte death was scored on a scale of 0–3, ranging from no empty lacunae to complete loss of chondrocytes in the cartilage layer. Cartilage surface erosion was scored on a scale of 0–3, ranging from no cartilage loss to complete loss of articular cartilage. Bone destruction was graded on a scale of 0–5, ranging from no damage to the complete loss of bone structure. Histopathologic changes in the ankle joints were scored on 5 semiserial sections of the joint, spaced 70 m apart. Two observers (EL, LUBBERTS ET AL MIK) without knowledge of the experimental group, as described earlier (24), performed scoring. Immunohistochemistry for RANKL and IL-1␤. Whole ankle joints were fixed, decalcified, and embedded in paraffin, as described above. Tissue sections (7 M) were treated with 3% H2O2 for 10 minutes at room temperature. Sections were incubated for 2 hours with 10 mM citrate (pH 6.0), and thereafter were incubated for 1 hour with the primary antibody directed against RANKL (rabbit polyclonal antibody raised against the epitope corresponding to amino acids 46–317 of RANKL of human origin [FL-317]) or IL-1␤ (rabbit antimouse IL-1␤ antibody [H153]) (Santa Cruz Biotechnology, Santa Cruz, CA) (27). Rabbit IgG antibody (X0936; Dako, Carpinteria, CA) was used as a control. After rinsing, sections were incubated for 30 minutes with biotinylated horseradish peroxidase–conjugated goat anti-rabbit IgG (Dako P0448). Development of the peroxidase staining was done with diaminobenzidine (Sigma). Counterstaining was done with Mayer’s hematoxylin. Sections were coded and randomly analyzed by 2 independent observers (EL, BO-W). Staining of IL-1␤ and RANKL was semiquantitatively scored on a 5-point scale (scores 0–4) at 200⫻ magnification; a score of 0 represented no staining and a score of 4 represented staining of a high number of inflammatory cells. IL-1␤–positive and RANKLpositive cells were counted manually in 5 random high-power fields, and then averaged and scored on a scale of 0–4 as follows: 0 (no staining), 1 (1–5 positive cells), 2 (6–10 positive cells), 3 (11–15 positive cells), and 4 (⬎20 positive cells). Radiologic assessment. At the end of the experiment, knee and/or ankle joints were isolated and used for radiographic analysis as a marker for joint destruction. Radiographs were carefully examined using a stereomicroscope, and joint destruction was scored on a scale of 0–5, ranging from no damage to complete destruction of the joint (24). Determination of IL-6 protein. IL-6 levels in sera were measured by a specific ELISA. Anti-murine IL-6 antibodies were from BioSource International (Camarillo, CA) (capture antibody rat anti-mouse IL-6 monoclonal antibody [mAb] [MP5-20F3], detection antibody rat anti-mouse IL-6 mAb biotin-labeled [MP5-32CK]). No cross-reactivity with the cytokines IL-1␤, IL-10, and TNF␣ was found. Briefly, ELISA plates were coated with the capture antibody (3 g/ml) by overnight incubation at 4°C in carbonate buffer (pH 9.6). Nonspecific binding sites were blocked by incubation for 1 hour at 37°C with 1% BSA in PBS/Tween. The sera from mice of different experimental groups were tested by incubation for 2 hours at room temperature. The plates were then incubated for 2 hours at room temperature with the biotinylated second antibody, followed by a 30-minute incubation at 37°C with streptavidin–polyperoxidase conjugate. Bound complexes were detected by reaction with orthophenylenediamine and H2O2. Absorbance was measured at 492 nm by an ELISA plate reader. The cytokine concentration in the samples was calculated as pg/ml using recombinant murine IL-6 (BioSource International) as a standard. The sensitivity of the IL-6 ELISA was 32 pg/ml. Statistical analysis. Differences between experimental groups were tested using the Mann-Whitney U test, unless stated otherwise. The data are expressed as the mean ⫾ SEM. ANTI–IL-17 THERAPY AFTER ONSET OF CIA 653 Figure 1. Specificity of the anti–interleukin-17 (anti–IL-17) antibody in vitro. H4 cells were incubated with IL-17, IL-1␣, IL-1␤, or tumor necrosis factor ␣ (TNF␣) with or without anti–IL-17 antibody, in triplicate for 24 hours. RPMI 1640 tissue culture medium was used as a control. Thereafter, 100 l of cell culture medium was mixed with 100 l of Griess reagent. The optical density (OD) was measured by an enzyme-linked immunosorbent assay plate reader. Data are expressed as OD545 and are the mean and SEM of 2 separate experiments. RESULTS Effects of neutralizing endogenous IL-17 using anti–IL-17 antibody treatment. Polyclonal rabbit antimurine IL-17 antibody–positive serum was generated in our laboratory. To examine the neutralizing capacity of the anti–IL-17 antibody, H4 chondrocytes were stimulated with IL-17 with or without the anti–IL-17 antibody for 24 hours. IL-17 stimulated H4 chondrocytes to produce NO in the supernatant, which was measured as OD545. H4 cells incubated with IL-17 resulted in an increase in OD545 (Figure 1). Coincubation with the anti–IL-17 antibody prevented the IL-17–induced increase, showing an IL-17 neutralizing capacity of the anti–IL-17 antibody (Figure 1). To further investigate the specificity of the anti–IL-17 antibody, similar studies were performed in which H4 cells were incubated with IL-1␣, IL-1␤, or TNF␣ cytokines. Coincubation with the anti–IL-17 antibody had no effect on the increase in OD induced by these proinflammatory cytokines (Figure 1). This indicates that the anti–IL-17 antibody had no cross-reactivity with these important cytokines in the pathogenesis of arthritis. Thereafter, arthritic mice were systemically treated with the anti–IL-17 antibody immediately after the first signs of CIA. As shown in Figure 2A, a single injection with the polyclonal anti–IL-17 antibody after the onset of CIA significantly suppressed the macro- Figure 2. Effect of anti–interleukin-17 (anti–IL-17) antibody treatment in collagen-induced arthritis. Immunized DBA-1/Bom mice received 1 intraperitoneal injection of anti–IL-17 antibody–positive serum after the first signs of arthritis (clinical score between 0.25 and 0.5). As a control, the same amount of normal rabbit serum was injected. The appearance of arthritis was assessed, and it was scored for severity in the fore and hind paws (A). At the end of the experiment, mice were killed by cervical dislocation, after which the hind knee (B) and ankle (C) joints were analyzed for joint damage by radiography. Data are the mean and SEM of 3 separate experiments with at least 30 mice per group. ⴱ ⫽ P ⫽ 0.01; ⴱⴱ ⫽ P ⬍ 0.005; ⴱⴱⴱ ⫽ P ⬍ 0.0005 versus control group, by MannWhitney U test. 654 LUBBERTS ET AL control group (P ⫽ 0.04) (Figure 3A). In the control arthritic joints, numerous granulocytes and mononuclear cells were present and several granulocytes adhered to cartilage. Less influx of granulocytes and mononuclear cells was noted after anti–IL-17 antibody treatment (Figure 4). In addition, histologic analysis revealed a significant reduction of focal bone erosion after blocking of IL-17 (P ⬍ 0.05) (Figures 3B and 4). Multinucleated cells were detected at sites of focal bone erosion in the control group (Figure 4C). However, anti–IL-17 treatment greatly decreased the number of multinucleated cells in the joint (Figure 4D). Prevention of chondrocyte death and cartilage surface erosion with anti–IL-17 antibody treatment. Joint sections were stained with Safranin O to investigate proteoglycan content in the articular cartilage. Furthermore, semiserial sections were scored for the degree of chondrocyte death and cartilage surface erosion. As shown in Figure 3C, marked proteoglycan depletion was observed in the control group, with significantly less after blocking of endogenous IL-17 (P ⫽ 0.04). In addition to the analysis of reversible proteoglycan loss, joint sections were scored for the degree of Figure 3. Histologic analysis of joint inflammation, bone erosion, and cartilage destruction after anti–interleukin-17 (anti–IL-17) cytokine therapy. Immunized DBA-1/Bom mice received 1 intraperitoneal injection of anti–IL-17 antibody–positive serum or normal rabbit serum (control) after the first signs of arthritis. Ten days later, mice were killed by cervical dislocation and the ankle joints were obtained for histologic analysis. Synovial inflammation (A), proteoglycan depletion (C), chondrocyte death (D), and cartilage surface erosion (E) were scored on a scale of 0–3. Focal bone erosion (B) was scored on a scale of 0–5. Data are the mean and SEM of 2 separate experiments with at least 9 mice per group. ⴱ ⫽ P ⬍ 0.05 versus control group, by Mann-Whitney U test. scopic arthritis score compared with the control group. Radiographic analysis revealed significantly less joint destruction in the knee (P ⫽ 0.01) and ankle (P ⬍ 0.0005) after anti–IL-17 therapy compared with the control group (Figures 2B and C). Reduction of synovitis and prevention of focal bone erosion with anti–IL-17 treatment. Histologic analysis revealed significant reduction of cell influx in the joint after blocking of endogenous IL-17 activity using the polyclonal anti–IL-17 antibody compared with the Figure 4. Effects of anti–interleukin-17 (anti–IL-17) cytokine therapy on synovial inflammation and joint destruction. A and C, Sections of an ankle joint of a mouse 10 days after a single systemic injection with normal rabbit control serum, showing pronounced inflammation, cartilage destruction (A) (arrowheads), and bone erosion (C) (arrows). B and D, Sections of an ankle joint of a mouse 10 days after a single systemic injection of anti–IL-17 antibody–positive serum. Note the decreased synovial inflammation, cartilage destruction, and bone erosion. BM ⫽ bone marrow; B ⫽ bone; C ⫽ cartilage; S ⫽ synovitis. (Original magnification ⫻ 200.) ANTI–IL-17 THERAPY AFTER ONSET OF CIA 655 Figure 5. Suppression of systemic interleukin-6 (IL-6) protein level by systemic anti–IL-17 cytokine therapy. Immunized DBA-1/Bom mice received 1 intraperitoneal injection of anti–IL-17 antibody–positive serum or normal rabbit serum (control) after the first signs of arthritis. Ten days later, sera were collected and IL-6 levels were measured by a specific enzyme-linked immunosorbent assay, as described in Materials and Methods. Data are the mean and SEM of 10 mice per group. ⴱ ⫽ P ⬍ 0.05 versus control group, by Mann-Whitney U test. irreversible cartilage damage. Anti–IL-17 antibody therapy resulted in a significant reduction of chondrocyte death (P ⬍ 0.05) and cartilage surface erosion (P ⫽ 0.02) (Figures 3D and E and Figure 4). This indicates that neutralizing IL-17 reduced the degree of cartilage destruction. Anti–IL-17 antibody treatment suppresses serum IL-6 levels. To gain insight into the mechanism of action during IL-17 neutralization, serum levels of IL-6 were measured in the treated animals at the time they were killed. Ten days after the onset of arthritis, anti–IL-17 antibody treatment significantly reduced the serum levels of IL-6 (74% lower [P ⫽ 0.02] compared with the control group) (Figure 5). Reduced number of synovial RANKL- and IL1␤–positive cells after anti–IL-17 antibody treatment. Specific immunohistochemistry revealed fewer IL-1␤– positive and RANKL-positive cells in the synovium of mice treated with the polyclonal anti–IL-17 antibody compared with the control group (P ⬍ 0.05) (Figure 6). This indicates that the joint-protective effect after neutralizing endogenous IL-17 might be mediated by suppression of synovial IL-1␤ and RANKL production. Role of T cell IL-17 in prolongation of CIA. The role of T cell IL-17 during joint inflammation and tissue destruction at a later stage of CIA is unknown and may be limited and overruled by monocyte/macrophage ac- Figure 6. Effects of anti–interleukin-17 (anti–IL-17) treatment on IL-1␤ and receptor activator of NF-B ligand (RANKL) expression in the synovium. Specific immunohistochemistry revealed A, IL-1␤ and B, RANKL-positive cells in the synovium of the control group. Neutralizing IL-17 resulted in a significant reduction of synovial IL-1␤–positive and RANKL-positive cells. Data are the mean and SEM of 2 separate experiments with at least 9 mice per group. See Figure 2 for more detail on the experimental protocol. No staining was observed in serial sections of the same area using the rabbit IgG control antibody (results not shown). ⴱ ⫽ P ⬍ 0.05 versus control group, by Mann-Whitney U test. tivity. Therefore, we examined whether T cell IL-17 plays a role in the prolongation of CIA. Anti–IL-17 antibody therapy was started during a later stage of CIA using mice with a clinical CIA score in the ankle joints between 1 and 1.5. As shown in Figure 7, a single systemic injection with the polyclonal anti–IL-17 antibody significantly slowed the progression of the disease in the ankle and knee joints, indicating that T cell IL-17 plays a role in the prolongation of CIA. 656 LUBBERTS ET AL Figure 7. Role of T cell interleukin-17 (IL-17) in prolongation of collagen-induced arthritis. Arthritic mice with a clinical score in the ankle joints between 1 and 1.5 received 1 intraperitoneal injection of anti–IL-17 antibody–positive serum. As a control, the same amount of normal rabbit serum was injected. The appearance of arthritis was assessed, and it was scored for severity in the fore and hind paws (A) and hind knee joints (B). There was significant suppression of the disease progression in the fore and hind paws and knee joints after neutralizing endogenous IL-17. Data are the mean and SEM of at least 10 mice per group. ⴱ ⫽ P ⫽ 0.03; ⴱⴱ ⫽ P ⫽ 0.009 versus control group, by Mann-Whitney U test. DISCUSSION This is the first study to demonstrate that neutralizing T cell IL-17 during the effector phase of CIA has therapeutic potential. Radiologic and histologic analyses revealed significant protection from joint damage when endogenous IL-17 was neutralized after arthritis expression. This protective effect was associated with down-regulation of IL-1␤ and RANKL in the synovium. Furthermore, neutralizing endogenous IL-17 at a later stage of CIA still slowed the progression of the disease. IL-17 shares many properties with IL-1␤ and TNF␣, and this T cell–derived cytokine has been shown to be present in the synovium of RA patients (9,10). IL-17 induces the production of proinflammatory mediators, such as IL-1␤ and TNF␣, from several joint cells including synovial fibroblasts, macrophages, and chondrocytes. In addition, IL-17 induces RANKL expression (22,23), which is a novel cytokine crucial for osteoclastogenesis (28). Moreover, IL-17, IL-1␤, TNF␣, and RANKL activate the common transcription factor NF-B in a variety of cells. IL-17 can synergize with these cytokines (IL-1␤, TNF␣, and RANKL), but probably has direct activity as well (17,29). Since a subset of RA patients does not respond to neutralizing IL-1␤ or TNF␣, other factors, such as IL-17, may participate in the proinflammatory cytokine cascade. In the present study, we showed the involvement of IL-17 in joint inflammation and prolongation of arthritis. IL-17 has dual effects on cartilage. In vitro, it inhibits chondrocyte metabolism in intact articular cartilage of mice and results in proteoglycan breakdown (19,20,30,31). Furthermore, in vitro studies show the induction of metalloproteinases by IL-17 in synoviocytes and chondrocytes (32–34). Interestingly, the effects of IL-17 on matrix degradation and synthesis were not dependent on IL-1␤ production by chondrocytes, and IL-1 receptor antagonist did not block IL-17–induced matrix release nor did it prevent the inhibition of matrix synthesis in vitro using porcine articular cartilage explants (31). Moreover, we recently identified an IL-1␤– independent role of IL-17 in the pathogenesis of arthritis in vivo (17). The downstream signaling pathways for IL-17 and IL-1␤ seem to be distinct, and differential activation of activator protein 1 members by IL-17 and IL-1␤ has been described (33). IL-1␤ is by far the more catabolic cytokine in experimental arthritis compared with TNF␣; however, IL-17 synergizes with TNF␣ to induce cartilage destruction in vitro (35). This underscores the potential of IL-17 to act additively or even synergistically with IL-1␤/TNF, but IL-17 may have direct catabolic effects as well. In the present study, we found a clear reduction of synovial IL-1␤ expression after neutralizing endogenous IL-17 in CIA, indicating that IL-17 is an upstream mediator of IL-1␤. Furthermore, we showed IL-17 to be a novel target for the treatment of cartilage destruction in experimental arthritis. IL-17 seems to be a potent stimulator of osteoclastogenesis (22,23). In the present study, we found ANTI–IL-17 THERAPY AFTER ONSET OF CIA reduced multinucleated cells after neutralizing endogenous IL-17, indicating that anti–IL-17 antibody treatment prevents the formation of osteoclast-like cells. Recently, it was shown that overexpression of IL-17 in the knee joints of CIA mice results in elevated expression of RANKL in the synovium and loss of the RANKL/OPG balance, leading to enhanced bone resorption (23). Of interest, systemic OPG treatment inhibits the local IL-17–induced bone resorption in the knee joints of CIA mice, suggesting that IL-17–induced bone erosion is at least partly mediated by RANKL (23). The observation that neutralizing IL-17 results in fewer RANKL-positive cells in the synovium further implies a relation between IL-17 and RANKL expression. Furthermore, it underscores the role of IL-17 in enhancement of the joint destruction process and makes IL-17 an attractive target for the treatment of destructive arthritis. Osteoclasts are potent bone-resorbing cells and play a crucial role in joint destruction (36). RANKL and TNF␣ contribute to osteoclast formation, while several other cytokines are responsible for osteoclast survival and/or activation. Neutralizing the RANKL/RANK pathway by administration of OPG prevents bone destruction (37–39). However, this kind of treatment is not antiinflammatory or chondroprotective, as shown in the present study with anti–IL-17, suggesting anti–IL-17 as a more appropriate therapy for destructive arthritis. In the present study, we used an anti–IL-17 antibody to neutralize endogenous IL-17 directly after onset and during a later stage of CIA. A single injection with anti–IL-17 antibody seems to be much more efficient than treatment with the soluble IL-17 receptor (sIL-17R):Fc fusion protein, as previously described (17,18). Since the treatment protocols are not identical between our previous study and the present study, we also treated CIA mice with the sIL-17R:Fc fusion protein starting after the onset of CIA (4 injections on alternative days using the same dose as described in ref. 17). This treatment did not result in suppression of the arthritis score; however, radiographic analysis revealed a significant reduction of the degree of joint destruction. In addition, semiquantitative analysis of messenger RNA (mRNA) expression for IL-1␤ and RANKL using reverse transcriptase–polymerase chain reaction showed down-regulation of IL-1␤ and RANKL mRNA expression in the synovium after sIL-17R:Fc treatment (Lubberts E, et al: unpublished observations). It is known that despite the ability of IL-17 to signal at low concentrations, it shows a low affinity to its receptor, with Ka values between 2.107 and 2.108 M⫺1 (40). Therefore, we speculate that the difference in affinity for IL-17 be- 657 tween the IL-17R:Fc and the anti-murine IL-17 antibodies may be an important reason for the higher efficacy using the anti–IL-17 antibody. Affinity studies must be performed to prove this hypothesis, and this is currently under investigation. The role of T cell cytokines such as IL-17 in propagating and prolonging arthritis must be identified. T cells and their cytokines may play an important role in initiating the arthritis and during an early phase. However, during the later stage of the arthritis, T cell cytokines may be overruled by mediators produced by activated macrophages. It has been shown that IL-17 plays an inflammatory role in the initial phase of experimental arthritis (17,18). The present study makes it clear that after the first clinical signs of arthritis, neutralizing endogenous IL-17 is still of therapeutic value. Systemic IL-6 levels were reduced and fewer synovial IL-1␤–positive and RANKL-positive cells were detected, suggesting both IL-1␤–dependent and IL-1␤– independent mechanisms of action. Furthermore, even at a later stage of CIA, T cell IL-17 contributes to prolongation of the arthritis, since blocking endogenous IL-17 in this phase of CIA slowed the progression of the disease. This suggests that despite the abundant expression of macrophage mediators, which are partly produced independently of IL-17, T cell IL-17 plays a role in maintaining the inflammation. We speculate that neutralizing IL-17 during this later stage of CIA has a suppressive effect on proinflammatory cytokine production. In addition, fewer additive/ synergistic effects between IL-17 and other proinflammatory cytokines such as TNF␣, IL-1␤, and IL-6 can be expected. Previous blocking studies with anti–IL-1␤ and anti-TNF␣ performed in our laboratory have shown that TNF␣ plays an important role in early CIA, and IL-1␤ is important in early and established CIA (15). IL-17 is a potent inducer of IL-1␤ (17), and we hypothesize that neutralizing IL-17 results in a reduction of IL-1␤ expression in the synovium during this later stage of CIA. Since IL-1␤ expression is not completely dependent on IL-17, not all IL-1 ␤ will be blocked, and this IL-17– independent IL-1␤ production will contribute to the progression of CIA. Fewer additive/synergistic effects between IL-17 and IL-1␤ may also play a role in slowing the progression of the disease. TNF␣ is hardly detectable in later stages of CIA; however, synergistic effects between TNF␣ and IL-17 have been documented (35). Blocking of IL-17 using the soluble receptor for IL-17 further improves the neutralizing effect of TNF␣ blocking after the onset of CIA (Lubberts E, et al: unpublished observations). 658 LUBBERTS ET AL We hypothesize that the mechanisms responsible for slowing the progression of the disease after neutralizing IL-17 during the later stage of CIA are suppression of proinflammatory cytokines such as IL-1␤, TNF␣, and IL-6, and elimination or reduction of the additive/ synergistic effects between IL-17 and these proinflammatory cytokines. Studies are currently being done in our laboratory to further prove this hypothesis. In summary, we have demonstrated the therapeutic potential of neutralizing T cell IL-17 during the effector phase of CIA. 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