Enzymatic activity and distribution of phospholipase A2 in human cartilage

Extracellular phospholipase A2 (PLA2) with proinflammatory activity has recently been discovered in synovial fluids in inflammatory arthritides. In the search for the sources of synovial fluid PLA2, human synovium and articular cartilage were found to contain large quantities of the enzyme. In rheumatoid arthritis (RA), PLA2 activity in synovium, superficial and deep layers of articular cartilage was 20 +/- 14 (SEM), 168 +/- 62 and 533 +/- 176 nmol/min/mg protein respectively. Corresponding values in osteoarthritis (OA) were 49 +/- 11, 569 +/- 109 and 1709 +/- 243 nmol/min/mg protein, all significantly higher (p less than .01) than in RA. Nasal septal cartilage contained much less PLA2, 19 +/- 5.6. PLA2 in human articular and nasal cartilage has sn-2 specificity, a neutral pH optimum and absolute calcium dependence. High PLA2 concentration in articular cartilage may imply that, at least in part, cartilage is the source of PLA2 in the joint space. Since RA cartilage and synovium have less PLA2 activity than the corresponding OA tissues, additional sources of PLA2 in RA synovial fluids are implicated.     

Characterization of extracellular phospholipase A2 in rheumatoid synovial fluid

Phospholipase A2 (PLA2) activity has now been identified in rheumatoid synovial fluids. This PLA2 is a calcium-requiring protein of MW 11,000 with a neutral pH optimum. Its activity was inhibited by high concentrations of Mg2+, and by the active site-directed histidine reagent p-bromophenacyl bromide. Ionic and nonionic detergents, or the sulfhydryl reagent dithiothreitol caused loss of enzyme activity. Synovial fluid PLA2 did not interact with sulphated mucopolysaccharides such as heparin or chondroitin sulphate. Release and sequestration of PLA2 in the joint space may contribute to the characteristic rheumatoid inflammatory changes.     

Suppression of murine endotoxic shock by sPLA2 inhibitor, indoxam, through group IIA sPLA2-independent mechanisms.

Endotoxic shock is a systemic inflammatory process, involving a variety of proinflammatory mediators. Two types of secretory phospholipase A2 (sPLA2) have been implicated in this process. Group IB sPLA2 (PLA2-IB) binds to the PLA2 receptor (PLA2R), and PLA2R-deficient mice exhibit resistance to endotoxin-induced lethality with reduced plasma levels of proinflammatory cytokines, such as TNF-alpha. Group IIA sPLA2 (PLA2-IIA) is found in many tissues and cell types, and local and systemic levels are elevated under numerous inflammatory conditions including sepsis. In this study, we investigated the effect of a specific sPLA2 inhibitor, indoxam, on murine endotoxic shock. Indoxam suppressed the elevation of plasma TNF-alpha with a similar potency in PLA2-IIA-expressing and PLA2-IIA-deficient mice after LPS challenge. In PLA2-IIA-deficient mice, indoxam also suppressed the elevation of plasma IL-1beta, IL-6 and NO, and prolonged survival after LPS challenge. Indoxam was found to block the PLA2-IB binding to murine PLA2R with a high potency (Ki=30 nM). The inhibitory effects of indoxam on the LPS-induced elevation of plasma TNF-alpha levels could not be observed in mice deficient in PLA2R. These findings suggest that indoxam blocks the production of proinflammatory cytokines during endotoxemia through PLA2-IIA-independent mechanisms, possibly via blockade of the PLA2R function.     

Regulation of synovial cell growth: basic fibroblast growth factor synergizes with interleukin 1 beta stimulating phospholipase A2 enzyme activity, phospholipase A2 activating protein production and release of prostaglandin E2 by rheumatoid arthritis synovial cells in culture.

Cytokines have been implicated in the regulation of eicosanoid synthesis and synovial cell proliferation. To further define these mechanisms, we have compared the effects of basic fibroblast growth factor and platelet-derived growth factor on cell growth, prostaglandin E2 (PGE2) production and phospholipase A2 enzyme activity in long-term cultures of synovial cells from rheumatoid arthritis (RA) patients capable of proliferating in serum-free medium. Compared with serum-free medium alone, RA synovial cell growth was significantly enhanced by adding either basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF) to the culture medium. Growing RA synovial cells for 14 days in serum-free medium plus bFGF caused them to spontaneously release significant amounts of PGE2, an effect not seen if cells were grown in serum-free medium alone, or serum-free medium plus PDGF. Enhanced release of PGE2 occurred when arachidonic acid was added to bFGF but not PDGF-treated RA synovial cells, suggesting that bFGF increased cyclooxygenase enzyme activity in these cells. Moreover, phospholipase A2 (PLA2) enzyme activity was found to be significantly greater in RA synovial cells grown for 14 days in serum-free medium containing bFGF alone, or bFGF plus interleukin 1 beta (IL-1 beta) compared with cells grown in either serum-free medium alone, or serum-free medium plus PDGF. Similarly, bFGF plus IL-1 beta-stimulated release of PLA2 activating protein, a novel mammalian phospholipase stimulator found in high concentrations in RA synovial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant human secretory phospholipase A2: purification and characterization of the enzyme for active site studies.

A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.     

Transglutaminase 2 and phospholipase A2 interactions in the inflammatory response in human Thp-1 monocytes

Several experimental approaches have demonstrated that transglutaminase 2 (TG2) increased activity is involved in monocyte activation and inflammatory response. Preliminary results also demonstrate a TG-mediated post-translational modification of phospholipase A₂ (PLA2), which catalyzes the release of arachidonic acid from its lipid storage sites. The control of PLA2-mediated production of eicosanoids has been found to be of great benefit for inflammatory disease treatment. However, the identification of the mechanisms of PLA2 activation is a very complex issue, because of the presence of multiple PLA2 forms. The aim of this study was to characterize the interactions between TG2 and sPLA2 in LPS-stimulated THP-1 cells, which were treated with TPA to induce early differentiated macrophage-type model. We demonstrated that increases in TG2 enzyme activity and protein expression may be considered an early event in monocyte/macrophage activation by LPS. Under these conditions, TG2 protein was co-immunoprecipitated with PLA2 by monoclonal antibody directed against the secretory form of the enzyme (sPLA2-V). Concomitantly, the PLA2 enzyme activity increased in TPA-treated cells exposed to LPS; these high levels of enzyme activity were significant reduced by R283, a site-specific inhibitor of TG2. Moreover, confocal laser scanning microscopy analysis of double-immunostained cytochemical specimens confirmed a co-localization of BAPA-labeled proteins and sPLA2-V in LPS-treated cells. These findings give evidence of a complex TG2/sPLA2-V, suggesting the possibility that sPLA2-V is a substrate for TG2. These results demonstrated that TG2 increases produced a sustained activation of PLA2 activity, suggesting a functional interaction between these enzymes in the regulation of inflammatory response.     

α-Enolase expressed on the surfaces of monocytes and macrophages induces robust synovial inflammation in rheumatoid arthritis

α-Enolase (ENO1) is a multifunctional glycolytic enzyme expressed abundantly in the cytosol. It has been implicated in autoimmune and inflammatory diseases. Serum Abs against ENO1 were reported in rheumatoid arthritis (RA). Cell-surface expression of ENO1 has been found to be increased rapidly in response to inflammatory stimuli, but its expression and function has not been reported in RA. In this study, we show that cell-surface expression of ENO1 is increased on monocytes and macrophages isolated from RA patients but not on those from osteoarthritis patients, and Ab against ENO1 can stimulate these cells to produce higher amounts of proinflammatory mediators, such as TNF-α, IL-1 α/β, IFN-γ, and PGE(2) via p38 MAPK and NF-κB pathway. The frequency of ENO1-positive cells in synovial fluid mononuclear cells was higher than PBMCs. ENO1-positive cells were also found in the inflamed synovium from RA patients and arthritic ankle tissues of mice with collagen-induced arthritis. Taken together, these findings suggest that Abs against ENO1 present in RA sera may stimulate monocytes and macrophages expressing cell-surface ENO1 and contribute to production of proinflammatory mediators during the effector phase of synovial inflammation.     

Cells of the synovium in rheumatoid arthritis. Synovial fibroblasts

For some time synovial fibroblasts have been regarded simply as innocent synovial cells, mainly responsible for synovial homeostasis. During the past decade, however, a body of evidence has accumulated illustrating that rheumatoid arthritis synovial fibroblasts (RASFs) are active drivers of joint destruction in rheumatoid arthritis. Details regarding the intracellular signalling cascades that result in long-term activation and synthesis of proinflammatory molecules and matrix-degrading enzymes by RASFs have been analyzed. Molecular, cellular and animal studies have identified various interactions with other synovial and inflammatory cells. This expanded knowledge of the distinct role played by RASFs in the pathophysiology of rheumatoid arthritis has moved these fascinating cells to the fore, and work to identify targeted therapies to inhibit their joint destructive potential is underway.     

The emerging role of Interleukin 27 in inflammatory arthritis and bone destruction

Although the causes of inflammatory arthritis elude us, aberrant cytokine expression has been linked to joint pathology. Consequently, several approaches in the clinic and/or in clinical trials are targeting cytokines, e.g. tumor necrosis factor (TNF), Interleukin 23 (IL-23) and Interleukin 17 (IL-17), with the goal of antagonizing their respective biologic activity through therapeutic neutralizing antibodies. Such, cytokine signaling-dependent molecular networks orchestrate synovial inflammation on multiple levels including differentiation of myeloid cells to osteoclasts, the central cellular players in arthritis-associated pathologic bone resorption. Hence, understanding of the cellular and molecular mechanisms elicited by synovial cytokine networks that dictate recruitment, differentiation and activation of osteoclast precursors and osteoclasts, respectively, is central to shaping novel therapeutic options for inflammatory arthritis patients. In this article we are discussing the complex signaling interactions involved in the regulation of inflammatory arthritis and it's associated bone loss with a focus on Interleukin 27 (IL-27). The present review will discuss the primary bone-degrading cell, the osteoclast, and on how IL-27, directly or indirectly, modulates osteoclast activity in autoimmune-driven inflammatory joint diseases.     

Sera of patients suffering from inflammatory diseases contain group IIA but not group V phospholipase A(2)

During recent years, the high phospholipase A(2) (PLA(2)) concentrations at sites of inflammation and in circulation in several life-threatening diseases, such as sepsis, multi-organ dysfunction and acute respiratory distress syndrome, has generally been ascribed to the non-pancreatic group IIA PLA(2). Recently the family of secreted low molecular mass PLA(2) enzymes has rapidly expanded. In some cases, a newly described enzyme appeared to be cross-reactive with antibodies against the group IIA enzyme. For this reason, reports describing the expression of group IIA PLA(2) during inflammatory conditions need to be reevaluated. Here we describe the identification of the PLA(2) activity in sera of acute chest syndrome patients and in sera of trauma victims. In both cases, the PLA(2) activity was identified as group IIA. This classification was based upon cross-reactivity with monoclonal antibodies against group IIA PLA(2) which do not recognize the recombinant human group V enzyme. Moreover, purification of the enzymatic activity from the two sera followed by N-terminal amino acid sequence analyses revealed only the presence of group IIA enzyme.     

Enhanced tissue expression and elevated circulating level of phospholipase A(2) receptor during murine endotoxic shock

Phospholipase A(2) receptor (PLA(2)R) mediates a variety of biological responses elicited by mammalian secretory phospholipase A(2) (sPLA(2)). In mice, group IB sPLA(2) (sPLA(2)-IB) acts as an endogenous ligand of PLA(2)R, and analysis of PLA(2)R-deficient mice has demonstrated a critical role of the sPLA(2)-IB/PLA(2)R system in the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) in the development of endotoxic shock. Here, we generated specific antibodies against a recombinant soluble form of PLA(2)R and examined its expression in the lung and spleen where a remarkable elevation of TNF-alpha expression has been observed during endotoxemia. Immunohistochemical analysis revealed the expression of PLA(2)R in type II alveolar epithelial cells and a subset of splenic lymphocytes, and its expression levels were markedly enhanced at 1 h after endotoxin challenge. Analysis with a newly developed sandwich enzyme-linked immunosorbent assay system revealed the presence of a soluble form of PLA(2)R in plasma of wild-type mice compared with its absence in plasma of PLA(2)R-deficient mice. After exposure to endotoxin, its circulating level was significantly elevated to the maximum level at 2-3 h after the treatment. These results suggest that tissue expression and the circulating level of PLA(2)R are elevated during murine endotoxemia, which might be relevant to its potential roles in the production of proinflammatory mediators during the development of inflammatory conditions.     

Domain-induced activation of human phospholipase A2 type IIA: local versus global lipid composition

Secretory human phospholipase A2 type IIA (PLA2-IIA) catalyzes the hydrolysis of the sn-2 ester bond in glycerolipids to produce fatty acids and lysolipids. The enzyme is coupled to the inflammatory response, and its specificity toward anionic membrane interfaces suggests a role as a bactericidal agent. PLA2-IIA may also target perturbed native cell membranes that expose anionic lipids to the extracellular face. However, anionic lipid contents in native cells appear lower than the threshold levels necessary for activation. By using phosphatidylcholine/phosphatidylglycerol model systems, we show that local enrichment of anionic lipids into fluid domains triggers PLA2-IIA activity. In addition, the compositional range of enzyme activity is shown to be related to the underlying lipid phase diagram. A comparison is done between PLA2-IIA and snake venom PLA2, which in contrast to PLA2-IIA hydrolyzes both anionic and zwitterionic membranes. In general, this work shows that PLA2-IIA activation can be accomplished through local enrichment of anionic lipids into domains, indicating a mechanism for PLA2-IIA to target perturbed native membranes with low global anionic lipid contents. The results also show that the underlying lipid phase diagram, which determines the lipid composition at a local level, can be used to predict PLA2-IIA activity.     

Substrate specificities and properties of human phospholipases A2 in a mixed vesicle model.

Studies of the specificity of phospholipases A2 (PLA2s) for different substrates have usually been carried out in vesicles or mixed micelles, where differences in shape, size, or charge of vesicles formed with different phospholipids may give misleading results. Another factor is binding of the enzyme to the phospholipid surface, which has recently been addressed using vesicles of an anionic phospholipid, dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) to which some extracellular PLA2s were shown to bind with a very high affinity (Jain, M. K., and Berg, O. G. (1989) Biochem. Biophys. Acta 1002, 127-156). In the present report we have used a similar system to study the substrate preferences of two human PLA2s that are thought to be physiologically relevant in the metabolism of arachidonic acid: a recombinant form of the human synovial fluid (14 kDa) PLA2 and the cytosolic (85 kDa) PLA2 found in monocytic cells. It is shown that both human enzymes bind tightly to DMPM vesicles and follow the basic characteristics of processive hydrolysis in this model using analysis of progress curves and substrate competition experiments. Mixed vesicles containing DMPM with small amounts (3-5 mol%) of other phospholipids have been used to study the substrate selectivity of the two human isoenzymes. The synovial fluid PLA2 shows a clear preference (approximately 7-fold) for sn-glycero-3-phosphoethanolamine over sn-glycero-3-phosphocholine. Within glycerophosphocholines, this enzyme displays little preference for the sn-2 fatty acyl group, and a slight preference for phospholipids with sn-1-acyl versus sn-1-alkyl substituents. In contrast, the cytosolic PLA2 shows a marked selectivity for arachidonoyl in the sn-2 position and only minor differences in selectivity for the polar head group in the sn-3 position. This enzyme does not distinguish between sn-1-acyl and sn-1-alkyl subclasses of glycerophosphocholines.     

ATPase and Adpase activities in synovial membrane of equine metacarpophalangeal joint

ATPase and ADPase activities capable of hydrolyzing nucleoside di- and triphosphates in the presence of Ca2+ are present in synovial membrane of metacarpophalangeal joint mainly associated to membrane fractions. These hydrolytic activities have been considered involved in the inflammatory process where ATP and ADP are inflammatory mediators while adenosine counteracts this effect. Both, subcellular localization and kinetic properties of these nucleotidase activities, suggest that could correspond to single enzyme called ATP-diphosphohydrolase or apyrase. The comparison of the activity on ATP-Ca and ADP-Ca from normal and pathological equine synovial membrane did not show significant differences either in the subcellular fraction distribution or in the enrichment of each subcellular fraction. Neither differences on 5'-nucleotidase activity present in the microsomal fraction were observed.     

IL-6/IFN-beta 2 in synovial effusions of patients with rheumatoid arthritis and other arthritides. Identification of several isoforms and studies of cellular sources

We have looked for IL-6, a cytokine that has immunomodulating and inflammation-associated activities, in joint exudates (fluid and mononuclear cells) from patients with rheumatoid arthritis and other arthritides using both biologic and biochemical assays. IL-6 was assessed by its ability to stimulate alpha 1-antichymotrypsin secretion from the human hepatoma cell line Hep3B clone 2, an activity which is blocked by an antiserum to Escherichia coli derived IL-6, and by the growth of the IL-6-dependent murine hybridoma 7TD1 cell line. IL-6 isoforms in synovial fluid were characterized by immunoaffinity chromatography followed by Western blotting. The presence of IL-1 in synovial fluids and its production by synovial fluid mononuclear cells was monitored by Western blotting and indirect immunofluorescence with polyclonal anti-IL-1 beta antisera. In an analysis of 30 effusions from 27 rheumatoid patients with acutely inflamed joints, abundant quantities of IL-6 (greater than 2 ng/ml) were detected in 23 by the alpha 1-antichymotrypsin bioassay. Several rheumatoid synovial fluids also had elevated IL-6 levels in the 7TD1 bioassay. Seven of nine nonrheumatoid effusions also contained high levels of IL-6 (greater than 2 ng/ml). No IL-1 (less than 0.25 ng/ml) could be detected by Western blotting in 10 rheumatoid effusions even though eight of these contained high levels of IL-6. The IL-6 activity could be neutralized with a rabbit antiserum to rIL-6. Multiple IL-6 isoforms (25, 30, 45 kDa) were present in two rheumatoid and one traumatic effusion studied. Fresh mononuclear cells isolated from various synovial effusions did not appear to make IL-6 constitutively, as no IL-6 could be detected in the media of cells cultured for 12 to 18 h after isolation. Similarly, there was no constitutive production of IL-1 by these cells. However, synovial fluid mononuclear cells could be induced to secrete both IL-6 and IL-1 after stimulation with LPS. The LPS-responsive cells were monocytes and not lymphocytes or dendritic cells. These findings suggest that IL-6 is involved in inflammatory joint disease. However, the primary cells synthesizing it may be located in the synovial lining instead of the joint exudate.     

Eicosanoids in insect immunity: bacterial infection stimulates hemocytic phospholipase A2 activity in tobacco hornworms

Intracellular phospholipase A(2) (PLA(2)) is responsible for releasing arachidonic acid from cellular phospholipids, and is thought to be the first step in eicosanoid biosynthesis. Intracellular PLA(2)s have been characterized in fat body and hemocytes from tobacco hornworms, Manduca sexta. Here we show that bacterial challenge stimulated increased PLA(2) activity in isolated hemocyte preparations, relative to control hemocyte preparations that were challenged with water. The increased activity was detected as early as 15 s post-challenge and lasted for at least 1 h. The increased activity depended on a minimum bacterial challenge dose, and was inhibited in reactions conducted in the presence of oleyoxyethylphosphorylcholine, a site-specific PLA(2) inhibitor. In independent experiments with serum prepared from whole hemolymph, we found no PLA(2) activity was secreted into serum during the first 24 h following bacterial infection. We infer that a hemocytic intracellular PLA(2) activity is increased immediately an infection is detected. The significance of this enzyme lies in its role in launching the biosynthesis of eicosanoids, which mediate cellular immune reactions to bacterial infection.     

Inhibition of human sPLA2 and 5-lipoxygenase activities by two neo-clerodane diterpenoids.

The inhibitory effect of two neo-clerodane diterpenoids, E-isolinaridial (EI) and its methylketone derivative (EIM), isolated from Linaria saxatilis var. glutinosa, on PLA2 and other enzyme activities involved in the inflammatory process was studied. Both compounds inhibited human synovial sPLA2 in a concentration-dependent manner with IC50 values of 0.20 and 0.49 microM, respectively, similar to scalaradial. Besides, these compounds decreased the cell-free 5-lipoxygenase activity and A23187-induced neutrophil LTB4 biosynthesis. Another function of human neutrophils, such as receptor-mediated degranulation, was also significantly reduced. In contrast, none of the compounds affected superoxide generation in leukocytes, or cyclooxygenase-1, cyclooxygenase-2 and inducible nitric oxide synthase activities in cell-free assays.     

Synovial DKK1 expression is regulated by local glucocorticoid metabolism in inflammatory arthritis

Introduction

Inflammatory arthritis is associated with increased bone resorption and suppressed bone formation. The Wnt antagonist dickkopf-1 (DKK1) is secreted by synovial fibroblasts in response to inflammation and this protein has been proposed to be a master regulator of bone remodelling in inflammatory arthritis. Local glucocorticoid production is also significantly increased during joint inflammation. Therefore, we investigated how locally derived glucocorticoids and inflammatory cytokines regulate DKK1 synthesis in synovial fibroblasts during inflammatory arthritis.

Methods

We examined expression and regulation of DKK1 in primary cultures of human synovial fibroblasts isolated from patients with inflammatory arthritis. The effect of TNFα, IL-1β and glucocorticoids on DKK1 mRNA and protein expression was examined by real-time PCR and ELISA. The ability of inflammatory cytokine-induced expression of the glucocorticoid-activating enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to sensitise fibroblasts to endogenous glucocorticoids was explored. Global expression of Wnt signalling and target genes in response to TNFα and glucocorticoids was assessed using a custom array.

Results

DKK1 expression in human synovial fibroblasts was directly regulated by glucocorticoids but not proinflammatory cytokines. Glucocorticoids, but not TNFα, regulated expression of multiple Wnt agonists and antagonists in favour of inhibition of Wnt signalling. However, TNFα and IL-1β indirectly stimulated DKK1 production through increased expression of 11β-HSD1.

Conclusions

These results demonstrate that in rheumatoid arthritis synovial fibroblasts, DKK1 expression is directly regulated by glucocorticoids rather than TNFα. Consequently, the links between synovial inflammation, altered Wnt signalling and bone remodelling are not direct but are dependent on local activation of endogenous glucocorticoids.     

Toll-like receptor 2 pathway drives streptococcal cell wall-induced joint inflammation: critical role of myeloid differentiation factor 88

The IL-1R/Toll-like receptor (TLR) superfamily of receptors has a key role in innate immunity and inflammation. In this study, we report that streptococcal cell wall (SCW)-induced joint inflammation is predominantly dependent on TLR-2 signaling, since TLR-2-deficient mice were unable to develop either joint swelling or inhibition of cartilage matrix synthesis. Myeloid differentiation factor 88 (MyD88) is a Toll/IL-1R domain containing adaptor molecule known to have a central role in both IL-1R/IL-18R and TLR signaling. Mice deficient for MyD88 did not develop SCW-induced arthritis; both joint swelling and disturbance of cartilage chondrocyte anabolic function was completely abolished. Local levels of proinflammatory cytokines and chemokines in synovial tissue washouts were strongly reduced in MyD88-deficient mice. Histology confirmed the pivotal role of MyD88 in acute joint inflammation. TLR-2-deficient mice still allow influx of inflammatory cells into the joint cavity, although the number of cells was markedly reduced. No influx of inflammatory cells was seen in joints of MyD88-deficient mice. In addition, cartilage matrix proteoglycan loss was completely absent in MyD88 knockout mice. These findings clearly demonstrated that MyD88 is a key component in SCW-induced joint inflammation. Since agonists of the Toll-like pathway are abundantly involved in both septic and rheumatoid arthritis, targeting of MyD88 may be a novel therapy in inflammatory joint diseases.