ARTHRITIS & RHEUMATISM Vol. 50, No. 6, June 2004, pp 2005–2013 DOI 10.1002/art.20014 © 2004, American College of Rheumatology Role of Interleukin-18 in Experimental Group B Streptococcal Arthritis Luciana Tissi,1 Bradford McRae,2 Tariq Ghayur,2 Christina von Hunolstein,3 Graziella Orefici,3 Francesco Bistoni,1 and Manuela Puliti1 Objective. To assess the role of interleukin-18 (IL-18) in the evolution of septic arthritis induced by group B streptococci (GBS) in mice. Methods. CD1 mice were inoculated intravenously with 8 ⴛ 106 colony-forming units (CFU) of type IV GBS (strain 1/82), and administered intraperitoneally 1 hour before infection with anti–IL-18 monoclonal antibodies (0.25 mg/mouse). In a subsequent set of experiments, mice infected with a suboptimal arthritogenic dose of GBS (4 ⴛ 106 CFU/mouse) were administered different doses of recombinant IL-18 for 4 days, starting 1 hour after infection. Mortality, evolution of arthritis, bacterial clearance, joint histopathology, and cytokine production were examined in infected mice that did or did not receive treatment with anti–IL-18 antibodies or IL-18. Results. IL-18 was produced during GBS infection. Neutralization of IL-18 resulted in a decrease in mortality rates, and in the incidence and severity of arthritis. Amelioration of arthritis was accompanied by a dramatic reduction in local IL-1␤, IL-6, macrophage inflammatory protein 1␣ (MIP-1␣) and MIP-2 production, and reduced bacterial burden. Administration of exogenous IL-18 resulted in increased mortality rates and increased incidence and severity of GBS arthritis, concomitant with a higher number of GBS and increased levels of IL-6, IL-1␤, MIP-1␤, and MIP-2 production in the joints. Conclusion. The present study indicated some involvement of IL-18 in the pathogenesis of GBSinduced arthritis. The role of IL-18 in joint pathology is shown by a regulatory effect on inflammatory mediator levels and local cell influx. Thus, IL-18 should be regarded as a potential therapeutic target in GBS infection and arthritis. Group B streptococci (GBS) are a leading cause of life-threatening infections in neonates and infants (1). Invasive neonatal GBS infection has either an early (usually the first 24 hours after birth) or late (7 days after birth) onset. Common manifestations of GBS disease in neonates include pneumonia, septicemia, meningitis, bacteremia, and bone or joint infections (1). Invasive disease caused by GBS has also been recognized in adults (2,3). Septic arthritis is one of the clinical manifestations of late-onset GBS infection in neonates (1) and requires prolonged antibiotic treatment to ensure an uncomplicated outcome. In adults, GBS septic arthritis is often associated with age and severe underlying diseases (4–7). GBS arthritis is usually hematogenously acquired, and the most frequently affected joints are the hip, ankle, and wrist (1). In our mouse model of hematogenously induced GBS arthritis, mice inoculated with the reference serotype IV GBS strain manifested clinical signs of arthritis characterized by early onset, with evolution from an acute exudative synovitis to permanent lesions with irreversible joint damage and/or ankylosis (8). This laboratory mouse model offers outstanding potential for GBS arthritis, in that bacteremia persists for ⬎10 days after GBS infection and localization of articular lesions is similar to that in humans. We initially demonstrated that induction of GBS arthritis depends on the viability and number of microorganisms injected (8), the presence and amount of bacterial capsule, and Supported by a grant (2001061479-003) from the Ministero dell’Università e della Ricerca Scientifica 2001–2002, Italy. 1 Luciana Tissi, PhD, Francesco Bistoni, MD, Manuela Puliti, PhD: University of Perugia, Perugia, Italy; 2Bradford McRae, PhD, Tariq Ghayur, PhD: Abbott Bioresearch Center, Worcester, Massachusetts; 3Christina von Hunolstein, PhD, Graziella Orefici, MD: Istituto Superiore di Sanità, Rome, Italy. Address correspondence and reprint requests to Luciana Tissi, PhD, Department of Experimental Medicine and Biochemical Sciences, Microbiology Section, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. E-mail: [email protected] Submitted for publication October 7, 2003; accepted in revised form February 23, 2004. 2005 2006 TISSI ET AL sialic acid in the capsular polysaccharide (9). In this model, high-level systemic and local production of interleukin-6 (IL-6) and IL-1␤ and scant production of tumor necrosis factor ␣ (TNF␣) were observed in response to GBS infection (10). Direct correlation between the severity of arthritis and joint concentrations of IL-6 and IL-1␤, but not TNF␣, was observed (10). IL-18 is a novel cytokine that exhibits proinflammatory features and is a member of the IL-1 family of proteins, originally identified as the interferon-␥ (IFN␥)–inducing factor (11). IL-18 is structurally related to IL-1␤; both cytokines require IL-1␤–converting enzyme for cleavage of the precursor to release the bioactive molecules for IL-1␤ and IL-18 (12,13). Pro–IL-18 expression has been detected in antigen-presenting cells such as activated macrophages, Kupffer cells (11), and dendritic cells (14), as well as articular chondrocytes (15) and osteoblasts (16). IL-18 induces the production of proinflammatory cytokines such as TNF␣ and IL-1 (17). Furthermore, IL-18 stimulates the proliferation of activated T cells and inhibits the formation of osteoclast-like cells (16). In several diseases, including rheumatoid arthritis (RA), IL-18 is considered a proinflammatory cytokine (18). IL-18 is expressed in human RA synovium, and enhanced levels of IL-18 have been found in the sera of RA patients (19). Furthermore, IL-18 plays a role in the induction of RA synovial fibroblast expression of CXC chemokines through NF-B (20). IL-18 induces chondrocyte proliferation, up-regulates inducible nitric oxide synthase, stromelysin, IL-6, and cyclooxygenase 2 expression, and increases glycosaminoglycan release (15). A central role of IL-18 has also been shown in acute streptococcal cell wall (SCW)–induced joint inflammation, where neutralization of endogenous IL-18 suppresses joint swelling by reducing local TNF␣ and IL-1␤ levels (21). The aim of the present study was to investigate the role of IL-18 in our experimental model of GBSinduced septic arthritis. Endogenous IL-18 was neutralized before arthritis induction and the effect on joint pathology was determined. The impact of exogenous IL-18 administration on the severity of articular lesions using a suboptimal arthritogenic dose of GBS was also examined. MATERIALS AND METHODS Mice. Sex-matched, 8-week-old male or female outbred CD1 mice were obtained from Charles River (Calco, Italy). Microorganism. Type IV GBS, reference strain 1/82, were grown overnight at 37°C in Todd-Hewitt broth (Oxoid, Basingstoke, UK), washed, and diluted in RPMI 1640 medium (Gibco Life Technologies, Milan, Italy). The inoculum size was estimated turbidimetrically, and viability counts were performed by plating on tryptic soy agar–5% sheep blood agar (blood agar), and incubating overnight at 37°C under anaerobic conditions. Mice were inoculated intravenously via the tail vein with 8 ⫻ 106 or 4 ⫻ 106 GBS in a volume of 0.5 ml. Control mice were injected in the same manner with 0.5 ml of RPMI 1640 medium. Cytokines and antibodies. Recombinant murine IL-18 (6.7 ⫻ 106 units/mg) was purchased from R&D Systems (Minneapolis, MN) and diluted according to the manufacturer’s recommendations in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). IL-18 was injected intraperitoneally in a 0.2-ml volume at different doses (ranging from 0.01 to 0.1 g per mouse) once a day for 4 days starting 1 hour before GBS infection. As controls, infected mice received PBS plus BSA according to the same experimental schedule, and uninfected mice received IL-18. Mouse anti-mouse IL-18 monoclonal antibody (mAb; clone 1C5, isotype IgG1) was generated at Abbott Bioresearch Center (Worcester, MA). Normal mouse IgG was purchased from Sigma (Milan, Italy). The antibodies (0.25 mg/mouse) were injected intraperitoneally 1 hour before GBS infection. The dose was chosen on the basis of preliminary titration experiments showing complete neutralization of in vivo IL-18 serum levels as determined by enzyme-linked immunosorbent assay (ELISA). Control mice received PBS according to the same experimental schedule. Clinical evaluation of arthritis and mortality. Mice that were injected with GBS and that did or did not receive treatment with mAb or IL-18 (as described above) were evaluated for signs of arthritis and mortality. Mortality was recorded at 24-hour intervals for 30 days. After introduction of GBS, mice were examined daily by 2 observers (LT and MP) for 1 month to evaluate the presence of joint inflammation, and scores for arthritis severity (macroscopic score) were given as previously described (10,22). Arthritis was defined as visible erythema and/or swelling of at least 1 joint. Clinical severity of arthritis was graded on a scale of 0–3 for each paw, according to changes in erythema and swelling (0 ⫽ no change, 1 ⫽ mild swelling and/or erythema, 2 ⫽ moderate swelling and erythema, and 3 ⫽ marked swelling, erythema, and/or ankylosis). Thus, a mouse could have a maximum score of 12. The arthritis index (mean ⫾ SD) was constructed by dividing the total score (cumulative value of all paws) by the number of animals used in each experimental group. Histologic assessment. Groups of mice that were infected with GBS and that did or did not receive treatment with anti–IL-18 mAb or IL-18 were examined 7 days after infection for histopathologic features of arthritis. Mice were euthanized and arthritic hind paws (1 per mouse) were removed aseptically, fixed in formalin (10% volume/volume) for 24 hours and then decalcified in trichloroacetic acid (5% v/v) for 7 days, dehydrated, embedded in paraffin, sectioned at 3–4 m, and stained with hematoxylin and eosin (H&E). Samples were examined under blinded conditions. Tibiotarsal, tarsometatarsal, and metatarsophalangeal joints were examined, and a histologic score was assigned to each joint based on the extent IL-18 IN GBS ARTHRITIS of infiltrate (presence of inflammatory cells in the subcutaneous and/or periarticular tissues), exudate (presence of inflammatory cells in the articular cavity), cartilage damage, bone erosion, and loss of joint architecture. Arthritis severity was classified as mild (minimal infiltrate), moderate (presence of infiltrate, minimal exudate, integrity of joint architecture), or severe (presence of massive infiltrate/exudate, cartilage and bone erosion, and disrupted joint architecture). GBS growth in blood, kidneys, and joints. Blood, kidney, and joint infections in GBS-infected mice that did or did not receive treatment with anti–IL-18 mAb or IL-18 were determined by evaluation of colony-forming units (CFU) at different times after inoculation. Blood samples were obtained by retroorbital sinus bleeding before the mice were killed. Ten-fold dilutions were prepared in RPMI 1640 medium, and 0.1 ml of each dilution was plated in triplicate on blood agar and incubated under anaerobic conditions for 24 hours. The number of CFU was determined and the results were expressed as the number of CFU per milliliter of blood. Kidneys were removed aseptically and placed in a tissue homogenizer with 3 ml of sterile RPMI 1640 medium. All wrist and ankle joints from each mouse were removed, weighed, and ground in a mortar in sterile RPMI 1640 medium (1 ml/100 mg joint weight). After homogenization, all tissue samples were diluted and plated in triplicate on blood agar, and the results were expressed as the number of CFU per whole organ or per milliliter of joint homogenate. Sample preparation for cytokine assessment. Blood samples from the different experimental groups were obtained by retroorbital sinus bleeding at different times after infection before the mice were killed. Sera were stored at ⫺80°C until analyzed. Joint tissues were prepared as previously described (10). Briefly, all wrist and ankle joints from each mouse were removed and then homogenized in toto in 1 ml/100 mg joint weight of lysis medium (RPMI 1640 containing 2 mM phenylmethylsulfonyl fluoride and 1 g/ml final concentration of aprotinin, leupeptin, and pepstatin A). The homogenized tissues were then centrifuged at 2,000g for 10 minutes, and supernatants were sterilized using a Millipore filter (0.45 m) and stored at ⫺80°C until analyzed. Cytokine assays. IL-18, IL-6, IL-1␤, macrophage inflammatory protein 1␣ (MIP-1␣), MIP-2, IFN␥, and TNF␣ concentrations in the biologic samples were measured with commercial ELISA kits (IL-6, IL-1␤; Amersham Pharmacia Biotech, Little Chalfont, UK, and IFN␥, TNF␣, IL-18, MIP1␣, and MIP-2; R&D Systems), according to the manufacturers’ recommendations. Results were expressed as picograms per milliliter of serum or supernatant from joint homogenates. The detection limits of the assays were 25 pg/ml for IL-18, 7 pg/ml for IL-6, 3 pg/ml for IL-1␤, 1.5 pg/ml for MIP-1␣, 1.5 pg/ml for MIP-2, 15 pg/ml for IFN␥, and 5.1 pg/ml for TNF␣. Statistical analysis. Differences in the arthritis index, number of CFU, and cytokine concentrations between the groups of mice were analyzed by Student’s unpaired t-test. Between-group differences in survival data were analyzed by the Mann-Whitney U test, and incidence of arthritis and histologic data were analyzed by the chi-square test. Each experiment was repeated 3 times. Results are expressed as the mean ⫾ SD. P values less than 0.05 were considered significant. 2007 Figure 1. Interleukin-18 (IL-18) levels in supernatants from joint homogenates and sera from mice infected with 8 ⫻ 106 colony-forming units/mouse of group B streptococcus (GBS) and from uninfected controls. Supernatants from joint homogenates and sera were collected as described in Materials and Methods. IL-18 levels in the biologic samples were determined by enzyme-linked immunosorbent assay. Values are the mean ⫾ SD of 3 separate experiments. In each experiment, 3 mice per group were killed at each time point. ⴱ ⫽ P ⬍ 0.01 versus uninfected controls. RESULTS IL-18 production during GBS infection. IL-18 production was assessed in sera and supernatants from the joints at different times after infection with 8 ⫻ 106 CFU/mouse of GBS. As shown in Figure 1, joint levels of IL-18 were significantly higher (P ⬍ 0.01) in GBSinfected mice compared with naive mice (mean ⫾ SD 60.1 ⫾ 9.8 versus 25.8 ⫾ 4.9 pg/ml of joint homogenate, day 1 after infection). Maximum values were reached on day 7 (578 ⫾ 70.2 pg/ml of joint supernatant versus 20.6 ⫾ 5.5 pg/ml in naive animals). Circulating levels of IL-18 were also augmented during infection and peaked on day 7, although systemic production was lower and later than that observed locally. Effect of IL-18 blockade on clinical course of arthritis. Clinical signs of joint swelling were observed in 30% of the mice as early as 24 hours after infection with 8 ⫻ 106 CFU of GBS. The incidence of arthritis increased to 70% by day 5, and the maximum prevalence was observed on day 7 after inoculation, when 80% of the mice manifested clinical signs of arthritis (Figure 2A). Similarly, the arthritis index progressively increased and reached maximum value 10 days after GBS introduction (mean ⫾ SD 3.0 ⫾ 0.5) (Figure 2B); most of the animals had articular lesions in both the hind paws and fore paws. Forty percent of mice died during the course of infection (Figure 2C). Neutralization of endogenous IL-18 was performed by administering anti–IL-18 mAb (0.25 mg/ mouse) 1 hour before infection. Efficacy of anti–IL-18 2008 Figure 2. Effect of endogenous IL-18 neutralization on mortality rates and on the incidence and severity of arthritis in mice infected with GBS (8 ⫻ 106 colony-forming units/mouse). Monoclonal antibodies (0.25 mg/mouse) were injected intraperitoneally 1 hour before GBS infection. Control mice received phosphate buffered saline (PBS) according to the same protocol. Ten mice were used in each experimental group. A, Incidence of arthritis (percentage of mice with visible arthritis). B, Arthritis index (clinical severity of arthritis, evaluated as described in Materials and Methods). For A and B, values are the mean ⫾ SD of 3 separate experiments. C, Survival curves. Mortality was recorded at 24-hour intervals for 30 days. Data represent the cumulative results of 3 separate experiments. D, Histopathologic severity of arthritis in joints from hind paw sections, assessed 7 days after infection. Arthritis was scored as mild, moderate, or severe, as described in Materials and Methods. For anti–IL-18 treatment, 10 paws and 24 joints were assessed; for PBS treatment 10 paws and 28 joints were assessed. ⴱ ⫽ P ⬍ 0.01 versus controls; F ⫽ P ⬍ 0.05 versus controls. See Figure 1 for other definitions. mAb treatment was assessed by measuring IL-18 levels in serum and joints after antibody injection (days 1, 2, 3, and 5). Total abrogation of systemic free IL-18 production and a significant (P ⬍ 0.01) decrease in local cytokine levels were observed at all time points checked (data not shown). Anti–IL-18 mAb treatment resulted in a reduced number of animals showing articular lesions compared with controls, although the differences between the 2 experimental groups were not significant (Figure 2A). The arthritis index in mice treated with anti–IL-18 mAb was significantly lower than that in controls, reaching a maximum value of 1.8 ⫾ 0.4, compared with 3.0 ⫾ 0.5, on day 10 after infection (Figure 2B). Differences in the arthritis index between the 2 experimental groups were still significant at the end of the observation period (data not shown). There were significant differences in mortality rates between mice treated with anti–IL-18 mAb and control mice (P ⫽ 0.042). Only 10% of animals treated with anti–IL-18 mAb died, versus 40% of controls (Figure 2C). Irrele- TISSI ET AL vant antibodies did not affect mortality rates, or the incidence or severity of arthritis (data not shown). Seven days after infection, mice were killed and the most frequently affected paw (hind paw) was removed for histologic examination. Microscopic analysis of H&E-stained sections was performed. In control animals, 57.1% of the examined joints were classified as severely affected, with massive infiltrate/exudate, cartilage and bone erosion, and loss of joint integrity, and 35.7% were classified as moderately affected; only 7.2% of the joints were classified as mildly affected (Figure 2D). In contrast, most (58.3%) of the examined joints from mice treated with anti–IL-18 mAb were classified as moderately affected, and 33.4% of the joints were classified as mildly affected; only 8.3% of the joints in this group were severely affected (Figure 2D). Quantitative monitoring of bacteremia and bacterial growth in the kidneys and joints of mice that did or did not receive treatment with anti–IL-18 mAb was performed. A significantly lower (P ⬍ 0.01) number of microorganisms was recovered from the joints of mice treated with anti–IL-18 mAb compared with controls 3 days after infection (2.4 ⫻ 105 ⫾ 0.4 ⫻ 105 versus 8.4 ⫻ 106 ⫾ 0.7 ⫻ 106, respectively). Such differences were also found in subsequent days (data not shown). A similar trend was observed in GBS growth rates in the kidneys, while no significant differences were observed between the experimental groups in the blood (data not shown). Effect of IL-18 blockade on cytokine production. Since IL-6 and IL-1␤ play a major role in the pathogenesis of GBS arthritis (10), the effect of IL-18 blockade on these cytokine levels was assayed. Based on results from other experimental models indicating an involvement of chemokines in the development of arthritis (23–25), MIP-1␣ (a CC chemokine) and MIP-2 (a CXC chemokine) concentrations were also determined. Plasma and joint specimens were collected daily from day 0 to day 3, and then on days 5 and 10 after GBS infection. As previously described (10) and as shown in Figure 3, a rapid increase in IL-6 and IL-1␤ production was observed in the joints and sera of GBS-treated mice. Sustained levels of both cytokines were still present 10 days after infection. A time-dependent increase in chemokine concentrations was also observed in the joints. In particular, MIP-2 levels peaked on day 3 after bacterial inoculation (681 ⫾ 98 pg/ml), while the MIP-1␣ concentration was 256 ⫾ 47 pg/ml. In the serum, MIP-1␣ concentrations slightly increased, whereas more sustained levels were reached by MIP-2. IL-18 neutralization resulted in an early (24 hours) decrease in IL-6 joint IL-18 IN GBS ARTHRITIS 2009 in IL-18–treated animals than in controls, with a marked worsening of articular lesions. A similar negative effect of IL-18 administration upon survival was observed. In fact, with an inoculum size of 4 ⫻ 106 GBS/mouse only 10% of the control mice had died at the end of the observation period, while mortality rates in mice treated with 0.05 or 0.1 g/mouse of IL-18 were 40% and 50%, respectively (Figure 4C). No effects were observed in terms of mortality rates and the incidence and severity of arthritis when mice were injected with 0.01 g/mouse Figure 3. Effect of anti–IL-18 monoclonal antibody (mAb) administration on IL-6, IL-1␤, macrophage inflammatory protein 1␣ (MIP1␣), and MIP-2 production in the sera and joints of mice infected with GBS (8 ⫻ 106 colony-forming units/mouse). Anti–IL-18 mAb (0.25 mg/mouse) or phosphate buffered saline (PBS) was injected intraperitoneally 1 hour before infection. Uninfected mice (day 0) received PBS at the time of mAb administration. Blood samples and supernatants from joint homogenates were collected at the indicated times after treatment (see Materials and Methods). Levels of IL-6, IL-1␤, MIP1␣, and MIP-2 were determined by enzyme-linked immunosorbent assay. Values are the mean ⫾ SD of 3 separate experiments. In each experiment, 3 mice per group were killed at each time point. ⴱ ⫽ P ⬍ 0.01 versus control mice. See Figure 1 for other definitions. concentrations. IL-6 levels in the joints of mice treated with anti–IL-18 mAb remained significantly lower (P ⬍ 0.01) than those in the joints of control mice at all time points assessed. In addition, anti–IL-18 treatment resulted in lower levels of IL-1␤, MIP-1␣, and MIP-2 production. However, in this case, the phenomenon was evident starting 2 days after mAb administration, and at a later time point (day 10), all cytokine levels in treated and control mice were comparable. In the first days (days 0–3) after infection, anti–IL-18 treatment resulted in a strong decrease in systemic levels of IL-6 and MIP-2, but not IL-1␤. Irrelevant antibodies did not affect cytokine production (data not shown). Effect of IL-18 administration on mortality and arthritis. To further define the role of IL-18 in GBSinduced articular pathology, mice were injected with a suboptimal arthritogenic dose of GBS (4 ⫻ 106 CFU/ mouse) and treated with different doses of IL-18 (ranging from 0.01 to 0.1 g/mouse) for 4 days, starting 1 hour before infection. Control infected mice received PBS plus BSA (vehicle) following the same treatment schedule. As shown in Figures 4A and B, IL-18 treatment dramatically influenced the clinical course of GBS arthritis. The frequency of arthritis was more pronounced Figure 4. Effect of exogenous IL-18 administration on survival rates and on the incidence and severity of GBS arthritis in CD1 mice infected intravenously with GBS (4 ⫻ 106 colony-forming units/ mouse). Murine recombinant IL-18 (0.1 or 0.05 g/mouse) or phosphate buffered saline plus 0.1% bovine serum albumin (vehicle) was administered intraperitoneally for 4 days starting 1 hour before infection. Ten mice were used in each experimental group. A, Incidence of arthritis (percentage of mice with visible arthritis). B, Arthritis index (clinical severity of arthritis, evaluated as described in Materials and Methods). C, Survival curves. Mortality was recorded at 24-hour intervals for 30 days. Data represent cumulative results of 3 separate experiments. For A and B, values are the mean ⫾ SD of 3 separate experiments. ⴱ ⫽ P ⬍ 0.01; F ⫽ P ⬍ 0.05 versus vehicletreated mice. See Figure 1 for definitions. 2010 Figure 5. Effect of exogenous IL-18 administration on IL-6, IL-1␤, macrophage inflammatory protein 1␣ (MIP-1␣), and MIP-2 production in the sera and joints of mice infected with GBS (4 ⫻ 106 colony-forming units/mouse). IL-18 (0.05 g/mouse) or phosphate buffered saline plus 0.1% bovine serum albumin (vehicle) was injected intraperitoneally for 4 days starting 1 hour before infection. Control uninfected mice (day 0) received vehicle according to the same protocol. Blood samples and supernatants from joint homogenates were collected at the indicated times after treatment (see Materials and Methods). Levels of IL-6, IL-1␤, MIP-1␣, and MIP-2 were determined by enzyme-linked immunosorbent assay. Values are the mean ⫾ SD of 3 separate experiments. In each experiment, 3 mice per group were killed at each time point. ⴱ ⫽ P ⬍ 0.01 versus vehicletreated mice. See Figure 1 for other definitions. of IL-18 (data not shown). Histologic findings confirmed the clinical observations (data not shown). Effect of IL-18 administration on GBS growth and cytokine production. In vivo GBS growth was assessed in blood, kidneys, and joints of mice treated with IL-18 (0.05 g/mouse for 4 days) or vehicle. There were no significant differences in the number of GBS recovered from the bloodstream between the experimental groups (data not shown). In contrast, higher GBS titers were observed in the joints and kidneys of IL-18– treated mice compared with controls from day 2 after infection on (data not shown). Ten days after injection, 7.6 ⫻ 107 ⫾ 0.2 ⫻ 107 GBS were recovered in the joints and 1.2 ⫻ 108 ⫾ 0.2 ⫻ 108 in the kidneys of IL-18– treated mice, compared with 1.0 ⫻ 106 ⫾ 0.3 ⫻ 106 and 9.8 ⫻ 106 ⫾ 1.0 ⫻ 106 in controls, respectively. Animals infected with GBS and treated with IL-18 or vehicle were monitored for systemic and local production of proinflammatory cytokines and chemokines. As shown in Figure 5, administration of a subarthritogenic dose of GBS resulted in moderate local production of IL-6, IL-1␤, MIP-1␣, and MIP-2. A significantly dramatic increase (P ⬍ 0.01) in cytokine and chemokine production was evident upon treatment with TISSI ET AL IL-18. This effect was not limited to the period of IL-18 administration, since higher levels of all the secreted proteins examined were still found in the joints of IL-18–treated animals compared with controls at the end of the observation period. Systemic production of IL-6 and MIP-2 was also increased by IL-18 treatment, while no effect was evident on IL-1␤ and MIP-1␣ secretion. As expected, IL-18 administration resulted in rapid (within 4 hours after infection) IFN␥ and TNF␣ production, particularly sustained at the systemic level (Figure 6). Subsequently, IFN␥ levels remained significantly higher (P ⬍ 0.01) in treated mice compared with controls until day 4 after infection, while no significant differences between experimental groups were found for TNF␣ levels. Treatment with IL-18 alone induced only a weak systemic production of IL-6, MIP-2, IFN␥, and TNF␣, which was limited to the period of cytokine administration (data not shown). Figure 6. Effect of exogenous IL-18 administration on interferon-␥ (IFN␥) and tumor necrosis factor ␣ (TNF␣) in sera and joints of mice infected with GBS (4 ⫻ 106 colony-forming units/mouse). IL-18 (0.05 g/mouse) or phosphate buffered saline plus 0.1% bovine serum albumin (vehicle) was injected intraperitoneally for 4 days starting 1 hour before infection. Control uninfected mice (day 0) received vehicle according to the same protocol. Blood samples and supernatants from joint homogenates were collected at the indicated times after treatment (see Materials and Methods). Levels of IFN␥ and TNF␣ were determined by enzyme-linked immunosorbent assay. Values are the mean ⫾ SD of 3 separate experiments. In each experiment, 3 mice per group were killed at each time point. ⴱ ⫽ P ⬍ 0.01; F ⫽ P ⬍ 0.05 versus vehicle-treated mice. See Figure 1 for other definitions. IL-18 IN GBS ARTHRITIS DISCUSSION The present study assessed the role of IL-18 in murine GBS arthritis by investigating the effect of its blockade or supplementation in an experimental model of GBS infection. The murine model of GBS infection has been beneficial in elucidating bacterial and host factors responsible for GBS arthritis (8–10,26,27). In particular, a strong involvement of IL-6 and IL-1␤, but not TNF␣, in the pathogenesis of GBS arthritis has been established (10). Significantly high levels of IL-18 were evident upon GBS infection, particularly in the joints. Endogenous IL-18 plays an important role in GBS arthritis, since neutralization resulted in a decrease in the severity of arthritis. Similarly, improved outcome was achieved after administration of neutralizing anti– IL-18 antibodies or IL-18 binding protein in collageninduced arthritis (CIA) (28,29), and in a mouse model of SCW arthritis (23). In these experimental models, attenuation of the disease was associated with a marked reduction of local proinflammatory cytokines responsible for articular damage. In the present study, an early marked decrease in IL-6 and IL-1␤ production was observed upon IL-18 neutralization. Both cytokines are known to contribute directly to articular damage. In fact, IL-1␤, together with TNF␣, induces the release of tissue-damaging enzymes from synovial cells and articular chondrocytes and activates osteoclasts (30,31). IL-6 participates together with IL-1 in the catabolism of connective tissue components at inflammation sites (32,33), and activates osteoclasts, resulting in joint destruction (34). It is likely that a decrease in IL-6 and IL-1␤ is one of the factors involved in the amelioration of articular lesions upon anti–IL-18 treatment in our experimental model. There appears to be a connection between inflammatory cell accumulation and joint destruction in septic arthritis (35–37). In fact, invading macrophages and granulocytes produce cytokines and proteolytic enzymes that contribute to cartilage and bone destruction (38–40). In addition to proinflammatory cytokine production, in our experimental model the extent of articular inflammatory infiltrate and exudate and the number of microorganisms that reach the joints also dictate the severity of GBS arthritis (41). Selective recruitment of activated leukocytes into a site of inflammation is mediated by many factors, including chemokines (42). These low molecular weight proteins, divided into 2 distinct groups, CXC and CC, based on the position of the first 2 cysteine amino acid residues (42), are produced by leukocytes, endothelial cells, chondrocytes, osteoblasts, 2011 and other cell types in response to antigens, microbial products, and endogenous cytokines, and are detected in various inflammatory diseases (42,43). As expected, MIP-1␣ and MIP-2 production was observed during GBS infection, particularly at the joint level, and treatment with anti–IL-18 antibodies significantly impaired chemokine production. IL-18 induces CXC chemokine production from synovial fibroblasts in RA patients (22). It is likely that, in our experimental model, blockade of IL-18 acts directly on the production of MIP-2 (a CXC chemokine), thus lowering polymorphonuclear cell influx into the joints. Since there is evidence that polymorphonuclear cells from the synovial fluid of RA patients produce high levels of MIP-1␣ (44), the observed decrease in MIP-1␣ concentrations in the joints of mice treated with anti–IL-18 may be due to a small number of polymorphonuclear cells that are locally recruited. In fact, histopathologic analysis revealed reduced infiltrate/exudate in the joints of mice treated with anti–IL-18. It should be noted that GBS persist in macrophages for up to 24–48 hours (45), and macrophages may carry GBS to different body sites, such as the joints, thereby disseminating infection. Thus, it is likely that the small number of GBS recovered from the joints of mice treated with anti–IL-18 is due to the low number of locally recruited inflammatory cells. Administration of exogenous IL-18 to mice injected with GBS led to a worsening of articular lesions, as observed in a model of CIA (46,47). In the latter case, IL-18 likely mediated inflammatory arthritis not only by enhancing Th1 activity, but also by directly inducing the production of proinflammatory cytokines (IL-6, TNF␣, IFN␥) from different cell types of the innate immune system. In our experimental model, infection with a suboptimal arthritogenic dose resulted in mild articular lesions in a few animals. Low levels of proinflammatory cytokines and chemokines, together with a low number of GBS, were evident in the joints. Upon treatment with recombinant IL-18, a dramatic increase in both cytokines and chemokines was observed. IL-18 may exacerbate arthritis by multiple mechanisms. It might directly act on synovial macrophages and articular chondrocytes. In fact, in vitro experiments have demonstrated that IL-18 induces the release of proinflammatory cytokines from macrophages and the release of matrix metalloproteinases and glycosaminoglycans by articular cartilage, supporting a possible direct contribution of IL-18 in joint destruction (17). But, by inducing MIP-2 production, IL-18 might enhance the local influx of polymorphonuclear cells, which, in turn, secrete MIP-1␣ with consequent recruitment of 2012 TISSI ET AL mononuclear cells. As stated above, all these inflammatory cells not only contribute to articular damage by cytokine and proteolytic enzyme production (35), but by carrying microorganisms (45) to the different body sites, also augment local bacterial load, thus amplifying the inflammatory response. 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