Streptococcus pyogenes Infection Induces Septic Arthritis with Increased Production of the Receptor Activator of the NF-{kappa}B Ligand

Bacterial arthritis is a rapidly progressive and highly destructivejoint disease in humans, with Staphylococcus aureus and Neisseriagonorrhoeae the major causative agents, although beta-hemolyticstreptococci as well often induce the disease. We demonstratehere that intravenous inoculation of CD-1 mice with the groupA streptococcus (GAS) species Streptococcus pyogenes resultedin a high incidence of septic arthritis. Signs of arthritisemerged within the first few days after injection, and bacterialexaminations revealed that colonization of the inoculated GASin the arthritic joints persisted for 21 days. Induction ofpersistent septic arthritis was dependent on the number of microorganismsinoculated. Immunohistochemical staining of GAS with anti-GASantibodies revealed colonization in the joints of infected mice.Cytokine levels were quantified in the joints and sera of infectedmice by using an enzyme-linked immunosorbent assay. High levelsof interleukin-1ß (IL-1ß) and IL-6 weredetected in the joints from 3 to 20 days after infection. Wenoted that an increase in the amount of receptor activator ofNF- ${kappa}$ B ligand (RANKL), which is a key cytokine in osteoclastogenesis,was also evident in the joints of the infected mice. RANKL wasnot detected in sera, indicating local production of RANKL inthe infected joints. Blocking of RANKL by osteoprotegerin, adecoy receptor of RANKL, prevented bone destruction in the infectedjoints. These results suggest that GAS can colonize in the jointsand induce bacterial arthritis. Local RANKL production in theinfected joints may be involved in bone destruction.

INTRODUCTION

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Introduction

The group A streptococcus (GAS) Streptococcus pyogenes is agram-positive bacterial pathogen that causes infections suchas pharyngitis, bacteremia, and necrotizing fasciitis, as wellas postinfectious sequelae, such as acute rheumatic fever (7).Further streptococcal infections can induce acute rheumaticfever and poststreptococcal reactive arthritis (3, 20, 27).This type of reactive arthritis has been suggested to be dueto the production of antibodies to the infecting agent, whichcross-react with joint synovial tissue or cartilage (3). Inaddition to reactive arthritis, infectious septic arthritishas been reported as a clinical manifestation of GAS infection(10, 27).

Bacterial infections can be localized in joints, causing septicarthritis, the most rapidly progressing joint disease, afterwhich permanent joint damage develops in most cases, and mortalitycontinues to be high (10). Staphylococcus aureus and Neisseriagonorrhoeae are the most frequently isolated bacterial speciesassociated with septic arthritis; however, GAS is the next mostcommon bacterium isolated from septic joints of patients andaccounts for 8 to 16% of cases of septic arthritis (10, 27).Therefore, GAS induces several types of arthritis, includingpoststreptococcal reactive arthritis, rheumatic fever, and septicarthritis.

To study the pathogenesis of septic arthritis, a murine modelof the disease has been developed using S. aureus (5) and thegroup B streptococcus (GBS) Streptococcus agalactiae (24, 34).Certain bacterial virulence factors such as superantigens andexotoxins have been implicated in S. aureus arthritis (33),and host immune responses were reported to be important in theinduction and progression of this disease (12, 32, 35). Inflammatorycytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosisfactor alpha (TNF- ${alpha}$ ) participate in the pathogenesis of septicarthritis (4). Regarding GAS, it has been established that injectionof cell wall or peptidoglycan-polysaccharide fractions fromthe organism produces acute joint lesions in rats and mice (6,15), and such streptococcal cell wall arthritis has been usedas a model of rheumatoid arthritis. However, investigationsof septic arthritis induced by S. aureus or GBS have revealedthat septic arthritis is not induced by inactivated cells orsonicated cellular extracts containing bacterial peptidoglycan,polysaccharides, and proteins (5, 34); thus, the relationshipbetween septic arthritis and streptococcal cell wall arthritisis not well known.

Pathogenic bone destruction is associated with the up-regulationof osteoclastic bone resorption. Until recently, the molecularmechanisms of osteoclast differentiation and up-regulation ofbone resorption have been poorly understood. However, recentinvestigations have identified members of the TNF family ofligands and their receptors as critical regulators of osteoclastogenesis.The cytokine receptor activator of NF- ${kappa}$ B ligand (RANKL) is nowrecognized as a critical cytokine in osteoclast differentiation(2, 11, 30). RANKL induces osteoclast differentiation from osteoclastprecursor cells in the presence of macrophage colony-stimulatingfactor (11, 30), and RANKL-deficient mice show severe osteopetrosis,revealing the importance of the cytokine in osteoclastogenesis(8, 17). Although recent reports have suggested the involvementof RANKL in rheumatoid arthritis (18, 25), its role in septicarthritis has not been studied.

Osteoclastogenesis is blocked in the presence of osteoprotegerin(OPG) (11, 28, 30, 37). OPG is a soluble decoy receptor thatinhibits osteoclast formation, function, and survival by preventingthe binding of RANKL to its receptor, which is present on osteoclastprecursors and mature osteoclasts (11, 30, 37). Mice with nullmutations of the OPG gene exhibit severe osteoporosis (28),while mice with ablated RANKL genes exhibit osteopetrosis (11,17, 30).

In the present study, we found that systemic challenge of micewith GAS resulted in chronic infection with a high incidenceof septic arthritis. High levels of proinflammatory cytokinesincluding RANKL were detected in the arthritic joints of theinfected mice. In addition, administration of OPG inhibitedthe bone destruction induced by GAS infection.

MATERIALS AND METHODS

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Materials and Methods

Mice and reagents. Female outbred CD-1 mice, 6 weeks old, were obtained from CharlesRiver Japan (Yokohama, Japan) and used throughout the experiments.Recombinant mouse OPG (a recombinant OPG fused to human immunoglobulinG [IgG] Fc) was purchased from R&D Systems (Minneapolis,Minn.).

Administration of bacteria. This study was reviewed and approved by the Animal Care Committeeof Osaka University. GAS strain JRS4 (serotype M6; streptomycinresistant) was provided by E. Hanski (The Hebrew UniversityHadassah Medical School, Jerusalem, Israel) (13). Strains SSI-1(M3) and SSI-9 (M1) were clinical isolates from patients withtoxic shock-like syndrome as described previously (14, 21).The microorganisms were grown at 37°C in Todd-Hewitt brothmedium (BD Bioscience, Sparks, Md.) supplemented with 0.2% yeastextract (BD Bioscience) (THY) until the mid-log phase. The bacterialcells were washed and diluted in serum-free Dulbecco's modifiedEagle's medium (DMEM) (Sigma, St. Louis, Mo.). The GAS inoculumsize was determined turbidimetrically at 600 nm, and the numberof live bacterial cells was confirmed by enumeration of theCFU on THY agar plates. On day 0, the desired number of GASorganisms was inoculated intravenously (i.v.) via a tail veinin a 0.5-ml volume of DMEM. Control mice were injected in thesame way with 0.5 ml of DMEM. In order to demonstrate the involvementof RANKL in bone destruction induced by the GAS infection, recombinantOPG (1 mg/kg of body weight/day) was administered subcutaneouslydaily from day 3 to day 9.

Clinical evaluation of arthritis. Mice infected with GAS strain JRS4 were examined 1 day afterinfection and then every other day for 3 weeks to evaluate theclinical features of the disease—in particular, the presenceof joint arthritis. The degree of erythema or swelling was evaluatedfor each hind paw and recorded as follows: 0 points, no swellingor erythema; 1 point, mild erythema; 2 points, mild swellingand moderate erythema; and 3 points, severe swelling and erythema.The points for each hind paw were added, and the sum was designatedas the arthritis index. A single mouse could have a maximumarthritis index of 6. To evaluate the extent of the functionaldisorder and/or ankylosis that occurred in the hind paws, eachjoint was scored from 0 (no functional change) to 3 points (severefunctional disorder and/or ankylosis). Similar to the articularindex, the points of each hind paw were added, and the sum wasdesignated as the joint dysfunction index. The body weightsof the infected and control mice were also monitored at regularintervals.

Bacterial growth in organs of infected mice. Organ infections in mice administered GAS JRS4 were determinedby CFU evaluation 21 days after i.v. inoculation. The heart,lungs, liver, and knee joints of the hind paws were removedfrom mice with arthritis (arthritis index of >2) and placedin a tissue homogenizer with 1 ml of sterile phosphate-bufferedsaline (PBS). After the organ samples were homogenized, variousdilutions were made in PBS and plated on THY agar plates containing1 mg of streptomycin per ml. CFU were enumerated, and the resultswere expressed as number of CFU per whole organ.

Cytokine assays. Blood samples from mice infected with GAS JRS4 and from uninfectedcontrol mice were obtained before euthanasia on days 1, 3, 5,10, and 20 after inoculation. Samples were incubated at 37°Cfor 1 h and then centrifuged, and the supernatant sera werestored at -80°C until use. Knee joints were obtained frommice infected or uninfected with GAS. Adjacent soft tissueswere removed as much as possible and then homogenized in 1 mlof PBS containing 20 µl of protease inhibitor cocktailper ml (Sigma). The homogenized tissues were then centrifugedat 3,000 x g for 10 min, and the supernatants were stored at-80°C until analysis (35). TNF- ${alpha}$ , IL-1ß, and IL-6levels in sera and joints were measured with a commercial enzyme-linkedimmunosorbent assay (ELISA) kit (Genzyme, Minneapolis, Minn.)according to the manufacturer's recommendations. The RANKL concentrationsin sera and joints were also measured with an ELISA kit (R&DSystems). The results are expressed as picograms per milliliterof serum or of joint homogenates. The detection limits of theassays were 3 pg/ml (each) for IL-1ß and IL-6 and5 pg/ml for TNF- ${alpha}$ .

Serum sialic acid concentration. Serum samples were obtained from infected and uninfected mice21 days after infection. The sialic acid concentration in serumwas measured with a commercial kit (Kyokuto Pharmaceutical,Tokyo, Japan) (32).

Histology. Infected and uninfected control mice were sacrificed 21 daysafter inoculation, and histological studies were performed.Hind paw knee joints were removed aseptically, fixed in 4% paraformaldehydefor 4 days, decalcified in 7.5% EDTA for 3 weeks, dehydrated,embedded in paraffin, and sectioned at 5 to 6 µm. To evaluatethe extent of bone destruction, sections were stained with hematoxylinand eosin. Scores for bone destruction were acquired accordingto the following criteria: 0 points, no destruction; 1 point,mild loss of cortical or trabecular bone at a few sites; 2 points,moderate loss of bone at many sites; and 3 points, marked lossof bone at many sites. The total points for each knee jointwere designated as the bone destruction index.

Immunohistochemistry. Immunohistochemical staining for GAS JRS4 was performed withanti-GAS whole-cell antibodies. An immunofluorescence stainingmethod was applied to the sections as previously described (21).The sections were preincubated with 5% skim milk for 30 minand then incubated with rabbit anti-GAS antibodies (1:200) overnightat room temperature. The sections were then treated with AlexaFluor 568-conjugated anti-rabbit IgG (1:500; Molecular Probes,Eugene, Oreg.). The sections were observed with a confocal microscopesystem (model LSM510; Carl Zeiss, Oberkochen, Germany) (21).

Statistical analysis. A Mann-Whitney U test and Sheffe's test were performed withSTATVIEW software (SAS Institute, Cary, N.C.). Values are expressedas the mean ± standard error, with P < 0.05 consideredsignificant.

RESULTS

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Results

Clinical course of arthritis. The capacity of GAS strain JRS4 to produce a lethal infectionwas examined by i.v. inoculation of CD-1 mice with differentnumbers of bacteria. Table 1 shows that an inoculation of lessthan 2 x 10⁸ CFU of GAS JRS4 did not cause death, while themortality rate increased to 80% with an inoculum size of 4 x10⁸ CFU per mouse. It was also noted that mice given 2 x 10⁸CFU showed severe joint inflammation (Fig. 1B). The clinicalsigns of joint swelling were observed as early as 1 day afterinjection of 2 x 10⁸ CFU of GAS JRS4. The incidence of arthritiswas greater than 80% on day 5, and the most commonly involvedjoints were the ankles and wrists. Joint dysfunction was alsoevident in mice infected with 2 x 10⁸ CFU. At later time points,ankylosis and depositions of fibrous exudate in the articularcavities were observed. Furthermore, marked pus formation aroundthe infected joints was seen (Fig. 1C). Inoculation with 5 x10⁷ CFU of strain JRS4 did not cause death of mice, and DMEM-injectedcontrol mice did not exhibit arthritis or any extra-articularmanifestations. In this regard, we found that GAS strain SSI-1induced septic arthritis in CD-1 mice when more than 2 x 10⁶CFU was inoculated, and infection with more than 1 x 10⁷ CFUresulted in death of mice within 7 days. However, infectionwith SSI-9 required more than 2 x 10⁷ CFU organisms to developseptic arthritis in CD-1 mice (data not shown).

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TABLE 1. Effect of S. pyogenes JRS4 inoculum size on arthritis in and mortality of CD-1 mice

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FIG. 1. Characteristics of appearance of hind paws of female CD-1 mice infected with GAS strain JRS4. (A) Control hind paw (arthritis index = 0). (B) Swollen joint (arthritis index = 3) of a mouse on day 7 after i.v. infection of 2 x 10⁸ CFU of GAS strain JRS4. (C) Appearance of arthritic joint on day 21. Severe abscesses are shown around the infected joint (arrowheads).

The induction of joint arthritis was dependent on the numberof GAS JRS4 CFU inoculated (Table 1). Mice inoculated with 5x 10⁷ CFU showed mild articular swelling and erythema, and theirarthritis index never exceeded a mean value of 1.5 throughoutthe observation period (Fig. 2B). In contrast, the arthritisindex of mice inoculated with 2 x 10⁸ CFU reached 3 on day 11(Fig. 2B). Inoculation with 2 x 10⁸ CFU resulted in a decreasein body weight (Fig. 2A), and severe ankylosis or joint dysfunctionwas also observed in those mice (Fig. 2C). Increasing the infectiondose resulted in increased arthritis incidence, severity, andjoint dysfunction.

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FIG. 2. Time course of arthritis in CD-1 mice after i.v. inoculation of GAS. Mice were injected with 2 x 10⁸ CFU ( ${circ}$ ), 5 x 10⁷ CFU ( ${square}$ ), or 1 x 10⁷ CFU (•) suspended in serum-free DMEM or DMEM alone ( ${blacksquare}$ ). (A) Influence of GAS JRS4 infection on weight development. (B) Scoring of severity of erythema and swelling in hind paws of infected mice (arthritis index). (C) Scoring of functional disorder and ankylosis of hind paws of infected mice (dysfunction index). Results are expressed as the mean ± standard deviation. In each group, 10 mice were examined. *, P < 0.05 versus uninfected control mice.

Histopathology and immunohistochemical staining. The appearance of joints with an arthritis index of 3 on day21 is shown in Fig. 1C. Histopathological analyses of knee jointsof mice with an arthritis index of >2 showed synovial proliferation,marked infiltration of mononuclear cells, and polymorphonuclearleukocytes in the soft tissues (Fig. 3B and E). Destructionof both cartilage and subchondral bone was also found in scatteredareas of the joints, and the articular cavities were filledwith purulent exudate (Fig. 3B). Immunohistochemical stainingusing anti-GAS antibodies revealed colonization of GAS strainJRS4 in the joints of infected mice. Bacteria were detectedin both the articular cavities and bone marrow cavities. Asshown in Fig. 3D, most of the organisms were colonized alongthe bone trabecula. In contrast, the joints of uninfected controlmice were not stained by the anti-GAS antibodies (Fig. 3C).

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FIG. 3. Histological and immunohistochemical staining. Uninfected control and infected (2 x 10⁸ CFU of GAS strain JRS4) mice were sacrificed at 21 days after inoculation. Control and infected joints were fixed, decalcified, and embedded in paraffin. Sections were stained with hematoxylin and eosin (A, B, and E) or stained immunohistochemically with rabbit anti-GAS antibodies and Alexa Fluor-conjugated anti-rabbit IgG (C and D). (A and C) Uninfected control joints. (B, D, and E) Infected arthritic joints. Margins of the bone body are indicated by arrows, and accumulation of inflammatory cells is indicated by an arrowhead. (E) Increased magnification of the arrowhead portion in Fig. 3B (bar, 50 µm).

Colonization of GAS strain JRS4 in various organs was also investigatedon day 21 (Table 2). While numerous bacteria (10⁷ CFU/joints)were found in the joints of infected mice with arthritis (arthritisindex of >2), the number of GAS organisms in the heart, lungs,and liver was less than 10⁴ CFU per organ.

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TABLE 2. Growth of S. pyogenes JRS4 in various organs of infected mice

Kinetics of cytokine appearance. Figure 4 shows that IL-1ß concentrations in jointsbegan to increase 3 days after injection of GAS strain JRS4and reached a level of approximately 350 pg/ml. The concentrationsof both of IL-1ß and IL-6 in joints were higher thanthose of controls throughout the experimental periods. However,the cytokine concentrations in sera reached the maximal valueon day 5, and a progressive decrease was observed from day 10after injection (Fig. 4). TNF- ${alpha}$ levels never exceeded 50 pg/mlin the joints (Fig. 4C) and remained lower than 10 pg/ml inserum. RANKL concentrations in both the joints and serum ofinfected mice were also assayed with an ELISA kit. The concentrationsof RANKL in the joints were comparable to those of IL-6 andreached 200 pg (Fig. 5), while the concentrations in sera werebelow the detection limit. A progressive decrease in RANKL concentrationin the joints was observed on day 21 after infection. We alsomeasured serum sialic acid concentrations on day 21 and founda significant increase in infected mice (data not shown).

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FIG. 4. IL-1ß, IL-6, and TNF- ${alpha}$ concentrations in sera and joints of CD-1 mice. Mice were injected with 2 x 10⁸ CFU of GAS strain JRS4 in DMEM ( ${blacksquare}$ ) or with DMEM alone (). Samples were obtained 1, 3, 5, 10, and 20 days after infection. Results are expressed as the mean ± standard error of three experiments. *, P < 0.05 versus uninfected control mice.

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FIG. 5. RANKL concentrations in sera and joints of GAS-infected CD-1 mice. Mice were injected with 2 x 10⁸ CFU of GAS strain JRS4 in DMEM ( ${blacksquare}$ ) or with DMEM alone (). Samples were obtained at 1, 3, 5, 10, and 20 days after injection. Results are expressed as the mean ± standard error of three experiments. *, P < 0.05 versus uninfected control mice.

Effect of OPG on bone destruction in septic arthritis. Since a significant increase in RANKL in the infected jointswas observed, we investigated the effect of OPG, a decoy receptorof RANKL, on GAS-induced bone destruction (Fig. 6). At the onsetof arthritis, GAS-infected mice were administered OPG (1 mg/kgof body weight/day) subcutaneously daily from day 3 to day 9.On day 21, the mice were sacrificed, and the hind paws wereexamined for bone destruction. Inhibition of RANKL by OPG hadno effect on the severity of inflammation (data not shown).However, OPG-treated mice exhibited a reduction in loss of corticaland trabecular bone (Fig. 6C), whereas untreated infected micedeveloped severe bone lesions, characterized by partial to completedestruction of cortical and trabecular bone and erosion of thearticular cartilages (Fig. 6B). Table 3 summarizes the effectof OPG treatment on GAS-induced arthritis. OPG-treated miceshowed significant reduction of bone destruction.

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FIG. 6. Effect of OPG treatment on GAS-induced bone destruction. Recombinant OPG (1 mg/kg/day) was administered subcutaneously daily from day 3 to day 9 after infection with GAS strain JRS4 (2 x 10⁸ CFU). Hind paw joints were obtained from uninfected control (A), infected (2 x 10⁸ CFU of strain JRS4) control (B), and OPG-treated infected (C) mice 21 days after inoculation. The joints were fixed, decalcified, and embedded in paraffin, followed by staining with hematoxylin and eosin.

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TABLE 3. Effect of OPG treatment on severity of joint destruction

DISCUSSION

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Discussion

We found in this study that infection with live GAS inducedseptic arthritis in mice. Most mice inoculated with 2 x 10⁸CFU of GAS strain JRS4 suffered septic arthritis. This may bea good experimental model of septic arthritis induced by GAS.To study the pathogenesis of septic arthritis, a similar murinemodel was developed using S. aureus (5, 33) and GBS (24, 34).Certain virulence factors such as superantigens and exotoxinshave been implicated in S. aureus-induced arthritis (33). SinceGAS produces several kinds of virulence factors, such as superantigensand exotoxins, these factors may contribute to the pathogenesisof GAS arthritis. It was known that cell wall components ofGAS induced experimental arthritis in strains of rats (6). However,induction of this type of streptococcal cell wall arthritisis difficult in experimental mice, and i.v. injection of GAScell wall components induced acute arthritis in BALB/c and DBA/1Jmice (6, 15). Acute swelling and erythema of the ankles andwrists reached maximum severity within a few days after injection;however, histological studies showed that there was no pannusformation or destruction of cartilage and subchondral bone inthe mice (15). These pathological features are quite differentfrom those of the septic arthritis shown in this study.

It was reported that synovial fluid cultures were bacteriallypositive in 90% of nongonococcal bacterial arthritis cases (10).Recent studies have shown that GAS can attach to and invadeseveral types of eukaryotic cells, such as epithelial cellsand keratinocytes (1, 7, 13, 14, 19, 21). Furthermore, entryof GAS into various host cells has been shown to occur in vivoand may contribute to bacterial persistence despite antibiotictherapy (22). Several investigators have suggested the needfor chronic penicillin prophylaxis in patients with poststreptococcalreactive arthritis (20), and it was also reported that >50%of patients suffering from such arthritis were positive forGAS in throat cultures (3). Therefore, GAS can invade jointtissues, and its persistence may induce not only septic arthritisbut also poststreptococcal reactive arthritis. In fact, therewas chronic colonization of GAS in the joints of infected micein the present study. GAS colonization in the joints may directlytrigger tissue damage by producing several tissue-damaging enzymes,such as proteases (7), as well as superantigens, such as SpeA,which eventually stimulate host immune systems. The presenthistological examinations showed that infiltration of mononuclearcells in the infected joints occurred in GAS-induced arthritis(Fig. 3). These cells produce inflammatory cytokines, such asIL-1 and TNF- ${alpha}$ , which in turn also induce the destruction ofbone and cartilage in joint tissues.

Cytokines play pivotal roles in inflammation and immune responses.We found high levels of proinflammatory cytokines, includingIL-1ß, IL-6, and TNF- ${alpha}$ , in the joints of mice infectedwith GAS. Tissi et al. (35) also showed an increase in the levelsof IL-1, IL-6, and TNF- ${alpha}$ in serum as well as locally in GBS-inducedseptic arthritis, and they revealed that systemic inoculationwith pentoxifylline, an inhibitor of IL-1ß and IL-6production, resulted in a decrease or abolition of cytokineproduction, along with a consequent reduction in both the incidenceand severity of GBS arthritis. Regarding mouse septic arthritiscaused by S. aureus, TNF and lymphotoxin double-mutant miceare resistant (12). These results suggest the participationof proinflammatory cytokines in septic arthritis caused by GAS,GBS, and S. aureus.

RANKL is a newly discovered key mediator of osteoclast differentiationand bone resorption (11, 30). Several investigations have revealedthe involvement of RANKL in the bone destruction in rheumatoidarthritis. Kotake et al. (18) first reported that increasedconcentrations of RANKL were detected in synovial fluid frompatients with rheumatoid arthritis. Others also reported thatsynovial tissues from patients from rheumatoid arthrtis expressedincreased levels of RANKL (31). These studies together withour results suggest that RANKL in inflammatory joint tissuesis involved in bone destruction in septic arthritis as wellas rheumatoid arthritis.

OPG, a decoy receptor of RANKL, inhibits the differentiation,activation, and accumulation of osteoclasts (11, 16, 26, 28,30). Blockade of RANKL by administration of exogenous OPG suppressedthe development of joint erosion in experimental models of adjuvantarthritis in rats (16). We found that injections of recombinantOPG significantly reduced bone destruction in GAS-induced septicarthritis, suggesting that RANKL plays an essential role inbone destruction in bacterial arthritis. In this regard, ourprevious study demonstrated that in vitro infection of GAS inducesRANKL expression in mouse osteoblasts (23), suggesting thatthe colonization of GAS in joints induces local RANKL productionfrom osteoblasts.

Several investigations have shown a possible link between thepathogenesis of rheumatoid arthritis and bacterial components.Both bacterial DNA and peptidoglycan have been detected in thejoints of some patients with rheumatoid arthritis (9, 36). Herewe observed an increase in serum sialic acid, a marker of rheumatoidarthritis (29), in GAS-infected mice. Experimental rats withadjuvant arthritis showed a similar increase in serum sialicacid (32). Therefore, some common mechanisms may underlie bothseptic arthritis and rheumatoid arthritis.

In summary, we have demonstrated that i.v. inoculation of micewith GAS induces experimental septic arthritis and local productionof RANKL is involved in bone destruction in the infected joints.

ACKNOWLEDGMENTS

We thank T. Onishi and M. Miura for technical advice regardingthe histological and immunohistochemical analyses.

This work was supported in part by a grant-in-aid from the JapanSociety for the Promotion of Science (A14207074); a Health SciencesResearch grant (H12-shinko-27) from the Ministry of Health,Labour and Welfare; and a grant from the Japan Science and TechnologyCorporation.

FOOTNOTES

* Corresponding author. Mailing address: Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, 1-8, Yamadaoka, Suita-Osaka 565-0871, Japan. Phone: 81-6-6879-2976. Fax: 81-6-6879-2976. E-mail: okahashi{at}dent.osaka-u.ac.jp.

Editor: J. N. Weiser

REFERENCES

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