alpha(2)-Macroglobulin from rheumatoid arthritis synovial fluid: functional analysis defines a role for oxidation in inflammation.

A hallmark of inflammation is the release of oxidants, proteinases, and cytokines, all important mediators of the inflammatory cascade. alpha(2)-Macroglobulin (alpha(2)M) is a high-affinity, broad-specificity proteinase inhibitor that also binds and regulates the biological activities of a number of cytokines. We demonstrated recently that hypochlorite-oxidized alpha(2)M has decreased ability to inhibit proteinases and regulate cytokines in vitro. The role of oxidation in regulating alpha(2)M functions in vivo is largely unknown. To determine the extent and biological consequence of in vivo alpha(2)M oxidation, we measured the degree of oxidative alpha(2)M modification from rheumatoid arthritis (RA) synovial fluid and compared this with osteoarthritis (OA) as noninflammatory controls. We found that RA synovial fluid alpha(2)M is significantly more oxidized than that from OA. RA synovial fluid also contains a twofold higher median alpha(2)M level than OA, while having only half the alpha(2)M-proteinase inhibitory activity. Detailed biochemical analysis demonstrates proteolytically degraded alpha(2)M in RA greater than in OA synovial fluid. Additionally, the hypochlorite-mediated oxidation product, chlorotyrosine, is present in RA more than in OA or plasma alpha(2)M samples. Taken together, these findings confirm a role for oxidative regulation of inflammation by altering the functions of extracellular mediators such as alpha(2)M.     

Further studies on the activation of procollagenase, the latent precursor of bone collagenase. Effects of lysosomal cathepsin B, plasmin and kallikrein, and spontaneous activation.

1. Cathepsin B, a tissue (lysosomal) proteinase, and two humoral proteinases, plasmin and kallikrein, activate the latent collagenase ('procollagenase') which is released by mouse bone explants in culture. Other lysosomal proteinases (carboxypeptidase B, cathepsin C and D) and thrombin did not activate the procollagenase. Dialysis of the culture fluids against 3M-NaSCN at 4 degrees C and, for some culture fluids, prolonged preincubation at 25 degrees C also caused the activation of procollagenase. 2. In all these cases, activation of procollagenase involved at least two successive steps: the activation of an endogenous latent activator present in the culture fluids and the activation of procollagenase itself. 3. An assay method was developed for the endogenous activator. Human serum, bovine serum albumin, casein and cysteine inhibited the endogenous activator at concentrations that did not influence the collagenase activity. N-Ethylmaleimide and 4-hydroxy-mercuribenzoate stimulated the endogenous activator, but iodoacetate had no effect. 4. It is proposed that cathepsin B, kallikrein and plasmin may play a role in the physiological activation of latent collagenase and thus initiate degradation of collagen in vivo. This may occur whatever the molecular nature of procollagenase (zymogen or enzyme-inhibitor complex) might be.     

Enzymatic alteration of C1q, the collagen-like subcomponent of the first component of complement, leads to cross-reactivity with type II collagen

Native serum C1q, the collagenous-like subcomponent of the first component of complement, is not recognized by polyclonal anti-collagen type II antibodies. However, when purified C1q was subjected to limited proteolysis by collagenase it showed antigenic cross-reactivity with collagen type II. The same cross-reactivity was observed with hemolytically active C1q in synovial fluids of patients with rheumatoid arthritis (RA), whereas C1q from synovial fluids of patients with osteoarthritis (OA), villo-nodular synovitis and ankylosing spondylitis was not recognized by this antibody. However, incubation of synovial fluid C1q of OA patients with synovial fluid leucocytes from RA patients led to an alteration of OA-C1q which was now recognized by the anti-collagen type II antibody.     

Increased adiponectin is negatively linked to the local inflammatory process in patients with rheumatoid arthritis

Adiponectin has been shown to exert insulin-sensitizing, anti-atherogenic, and anti-inflammatory properties in metabolic diseases. It has been suggested that adiponectin may play a role in rheumatoid arthritis (RA). To assess adiponectin in serum and synovial fluid from patients with RA and osteoarthritis (OA), and in serum from healthy controls. Adiponectin and CRP levels were analyzed by ELISA. The clinical activity of RA patients was assessed according to the 28 joint count Disease Activity Score. Synovial fluid adiponectin was significantly higher in RA than in OA patients (p<0.001). Adiponectin was negatively associated with the leukocyte count in RA synovial fluid (r=-0.45, p<0.05). Serum adiponectin was higher in RA compared to healthy controls (p<0.02), however comparable to OA patients. Serum adiponectin was higher than in synovial fluid in both diseases (p<0.001). In general, women had higher adiponectin levels than men. Adiponectin was not related to age, disease duration, body mass index, or disease activity of RA patients. Adiponectin is decreased in synovial fluid compared to serum indicating that peripheral fat stores are major producers of adiponectin into the blood stream. However, increased synovial fluid adiponectin in RA patients may counterpart the local inflammatory process.     

Activities of dipeptidyl peptidase II, dipeptidyl peptidase IV, prolyl endopeptidase, and collagenase-like peptidase in synovial membrane from patients with rheumatoid arthritis and osteoarthritis.

We examined the activities of peptidases in the synovial membrane from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Dipeptidyl peptidase II (DPP II), prolyl endopeptidase (PEP), and collagenase-like peptidase (CLP) activities were higher in knee joint synovial membrane from patients with RA than in that from patients with OA. DPP II and PEP activities in knee joint synovial membrane of patients with RA increased in parallel with the increase in joint fluid volume, whereas DPP IV activity decreased in parallel with the increase in joint fluid volume. These results suggest that these peptidases in the synovial membrane may play some role in immunological disturbances in the joints of patients with RA. Measurement of these peptidases in synovial membrane may be useful in the diagnosis of the severity of local joint inflammation.     

The migratory and phagocytic activity of polymorphonuclear leukocytes in rheumatoid arthritis and osteoarthritis patients

Polymorphonuclear neutrophils (PMN) play a central role in the elimination of most extracellular pathogenic microorganisms and any impairment of their functions therefore predisposes to defect immune defence. We investigated the migratory and phagocytic functions of the PMNs isolated from peripheral blood and synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The results suggest that in RA the number and the migratory but not phagocytic capacity of synovial fluid (SF) neutrophils were enhanced, while in OA they were significantly decreased in synovial fluid cells comparatively with peripheral blood (PB). The migratory function of both PB and SF cells from RA patients was increased comparatively with that of the cells from OA patients. We found the different abnormal functions in synovial fluid neutrophils from RA and OA patients. These results may help to elucidate the underlying mechanism which leads to severe joint destruction and different susceptibility to infectious diseases in patients with rheumatic disorders.     

Measurement of sulfidopeptide leukotrienes and their metabolism in human synovial fluid of patients with rheumatoid arthritis

Leukotriene (LT)C4 in the synovial fluid of patients with osteoarthritis deformans (OA) and rheumatoid arthritis (RA) was measured by radioimmunoassay (RIA) after extraction with Sep-Pak C18 cartridge. The amounts of immunoreactive LTC4 (i-LTC4) in samples from patients with OA and RA were not significantly different, being 0.198 +/- 0.018 pmol/ml (n = 11) and 0.179 +/- 0.016 pmol/ml (n = 12), respectively. After separation by high performance liquid chromatography (HPLC) and measurement by RIA, the levels of other sulfidopeptide LTs, such as LTD4 and LTE4, in synovial fluid from patients with RA were found to be significantly higher than those in fluid from patients with OA. The leukocyte number in synovial fluids did not correlate with the i-LTC4 level. The metabolic activities of these synovial fluids were determined by incubating them with 3H-LTC4 and then separating sulfidopeptide LTs by HPLC. The conversion of LTC4 to LTD4 in synovial fluids of patients with OA and RA were similar, but the dipeptidase activity converting LTD4 to LTE4 was higher in fluid from patients with RA. It is suggested that a high level of LTE4 may contribute to exudation of synovial fluid, since LTE4 increases vascular permeability.     

Pyroptosis by NLRP3/caspase-1/gasdermin-D pathway in synovial tissues of rheumatoid arthritis patients

We investigated the potential involvement of pyroptosis, a proinflammatory form of regulated cell death, in rheumatoid arthritis (RA). Synovial fluid, synovial tissues and/or serum were compared among 32 patients with RA, 46 patients with osteoarthritis (OA) and 30 healthy controls. Samples were assayed for interleukin (IL)-1β, IL-18 and lactate hydrogenase (LDH). Synovial expression of NLRP3, caspase-1 and cleaved gasdermin D (GSDMD) was assayed using immunohistochemistry and multiplex immunohistochemistry. Patients with RA showed significantly higher levels of IL-1β and IL-18 in synovial fluid than patients with OA, and significantly higher levels of both cytokines in serum than healthy controls. RA was associated with higher levels of LDH in synovial fluid than OA. Among patients with RA, levels of IL-1β, IL-18 and LDH were significantly higher in synovial fluid than in serum, and the levels in synovial fluid positively correlated with disease activity and inflammation. Synovial cells, particularly macrophages, showed upregulation of NLRP3, caspase-1 and cleaved GSDMD in RA compared to OA. Our results implicate pyroptosis in the pathogenesis of RA, perhaps as a driver of local inflammation in joints.     

Characterisation of the cannabinoid receptor system in synovial tissue and fluid in patients with osteoarthritis and rheumatoid arthritis

Introduction

Cannabis-based medicines have a number of therapeutic indications, including anti-inflammatory and analgesic effects. The endocannabinoid receptor system, including the cannabinoid receptor 1 (CB₁) and receptor 2 (CB₂) and the endocannabinoids, are implicated in a wide range of physiological and pathophysiological processes. Pre-clinical and clinical studies have demonstrated that cannabis-based drugs have therapeutic potential in inflammatory diseases, including rheumatoid arthritis (RA) and multiple sclerosis. The aim of this study was to determine whether the key elements of the endocannabinoid signalling system, which produces immunosuppression and analgesia, are expressed in the synovia of patients with osteoarthritis (OA) or RA.

Methods

Thirty-two OA and 13 RA patients undergoing total knee arthroplasty were included in this study. Clinical staging was conducted from x-rays scored according to Kellgren-Lawrence and Larsen scales, and synovitis of synovial biopsies was graded. Endocannabinoid levels were quantified in synovial fluid by liquid chromatography-mass spectrometry. The expression of CB₁ and CB₂ protein and RNA in synovial biopsies was investigated. Functional activity of these receptors was determined with mitogen-activated protein kinase assays. To assess the impact of OA and RA on this receptor system, levels of endocannabinoids in the synovial fluid of patients and non-inflamed healthy volunteers were compared. The activity of fatty acid amide hydrolase (FAAH), the predominant catabolic endocannabinoid enzyme, was measured in synovium.

Results

CB₁ and CB₂ protein and RNA were present in the synovia of OA and RA patients. Cannabinoid receptor stimulation of fibroblast-like cells from OA and RA patients produced a time-dependent phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 which was significantly blocked by the CB₁ antagonist SR141716A. The endocannabinoids anandamide (AEA) and 2-arachidonyl glycerol (2-AG) were identified in the synovial fluid of OA and RA patients. However, neither AEA nor 2-AG was detected in synovial fluid from normal volunteers. FAAH was active in the synovia of OA and RA patients and was sensitive to inhibition by URB597 (3'-(aminocarbonyl) [1,1'-biphenyl]-3-yl)-cyclohexylcarbamate).

Conclusion

Our data predict that the cannabinoid receptor system present in the synovium may be an important therapeutic target for the treatment of pain and inflammation associated with OA and RA.     

Apolipoprotein-A1 as a Damage-Associated Molecular Patterns Protein in Osteoarthritis: Ex Vivo and In Vitro Pro-Inflammatory Properties

Osteoarthritis (OA) is associated with a local inflammatory process. Dyslipidemia is known to be an underlying cause for the development of OA. Therefore, lipid and inflammatory levels were quantified ex vivo in blood and synovial fluid of OA patients (n=29) and compared to those of rheumatoid arthritis (RA) patients (n=27) or healthy volunteers (HV) (n=35). The role of apolipoprotein A-I (ApoA1) was investigated in vitro on inflammatory parameters using human joint cells isolated from cartilage and synovial membrane obtained from OA patients after joint replacement. Cells were stimulated with ApoA1 in the presence or not of serum amyloid A (SAA) protein and/or lipoproteins (LDL and HDL) at physiological concentration observed in OA synovial fluid. In our ex vivo study, ApoA1, LDL-C and total cholesterol levels were strongly correlated to each other inside the OA joint cavity whereas same levels were not or weakly correlated to their corresponding serum levels. In OA synovial fluid, ApoA1 was not as strongly correlated to HDL as observed in OA serum or in RA synovial fluid, suggesting a dissociative level between ApoA1 and HDL in OA synovial fluid. In vitro, ApoA1 induced IL-6, MMP-1 and MMP-3 expression by primary chondrocytes and fibroblast-like synoviocytes through TLR4 receptor. HDL and LDL attenuated joint inflammatory response induced by ApoA1 and SAA in a ratio dependent manner. In conclusion, a dysregulated lipidic profile in the synovial fluid of OA patients was observed and was correlated with inflammatory parameters in the OA joint cavity. Pro-inflammatory properties of ApoA1 were confirmed in vitro.     

Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin)

The mechanism of activation of tissue procollagenase by matrix metalloproteinase 3 (MMP-3)/stromelysin was investigated by kinetic and sequence analyses. MMP-3 slowly activated procollagenase by cleavage of the Gln80-Phe81 bond to generate a fully active collagenase of Mr = 41,000. The specific collagenolytic activity of this species was 27,000 units/mg (1 unit = 1 microgram of collagen digested in 1 min at 37 degrees C). Treatment of procollagenase with plasmin or plasma kallikrein gave intermediates of Mr = 46,000. These intermediates underwent rapid autolytic activation, via cleaving the Thr64-Leu65 bond, to give a collagenase species of Mr = 43,000 that exhibited only about 15% of the maximal specific activity. Similarly, (4-aminophenyl)mercuric acetate (APMA) activated procollagenase by intramolecular cleavage of the Val67-Met68 bond to generate a collagenase species of Mr = 43,000, but with only about 25% of the maximal specific activity. Subsequent incubation of the 43,000-Mr species with MMP-3 resulted in rapid, full activation and generated the 41,000-Mr collagenase by cleaving the Gln80-Phe81 bond. In the case of the proteinase-generated 43,000-Mr species, the action of MMP-3 was approximately 24,000 times faster than that on the native procollagenase. This indicates that the removal of a portion of the propeptide of procollagenase induces conformational changes around the Gln80-Phe81 bond, rendering it readily susceptible to MMP-3 activation. Prolonged treatment of procollagenase with APMA in the absence of MMP-3 also generated a 41,000-Mr collagenase, but this species had only 40% of the full activity and contained Val82 and Leu83 as NH2 termini. Thus, cleavage of the Gln80-Phe81 bond by MMP-3 is crucial for the expression of full collagenase activity. These results suggest that the activation of procollagenase by MMP-3 is regulated by two pathways: one with direct, slow activation by MMP-3 and the other with rapid activation in conjunction with tissue and/or plasma proteinases. The latter event may explain an accelerated degradation of collagens under certain physiological and pathological conditions.     

Expression of MDC/CCL22 and its receptor CCR4 in rheumatoid arthritis,psoriatic arthritis and osteoarthritis

The pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA) involves an abnormal chemokine regulation. The chemokine receptor CCR4 is necessary for T cell migration to the skin. We, therefore, studied if CCR4 and its ligand macrophage-derived chemokine (MDC/CCL22) could participate in spreading the disease between skin and joints by examining RA, PsA and osteoarthritis (OA) patients. In synovial fluid from RA and PsA patients we observed a significantly higher MDC/CCL22 level compared to OA patients. Additionally, the MDC/CCL22 protein was found to be elevated in RA and PsA plasma compared to OA and healthy volunteers. Flow cytometry revealed that most CD4⁺CCR4⁺ lymphocytes also co-expressed CD45RO. Neither the MDC/CCL22 level nor the expression of CCR4 correlated to CRP. Immunohistochemistry of the RA and OA synovial membrane demonstrated CCR4 to be expressed by mononuclear cells and endothelial cells. Our results show that MDC/CCL22 is present within the synovial membrane of RA and OA patients and in high amount in the synovial fluid of patients with RA and PsA. This will enable migration of CCR4 expressing memory cells supporting that MDC/CCR4 could play a role in attracting skin specific memory T cells to the joints.     

A critical role for allograft inflammatory factor-1 in the pathogenesis of rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by massive synovial proliferation, angiogenesis, subintimal infiltration of inflammatory cells and the production of cytokines such as TNF-alpha and IL-6. Allograft inflammatory factor-1 (AIF-1) has been identified in chronic rejection of rat cardiac allografts as well as tissue inflammation in various autoimmune diseases. AIF-1 is thought to play an important role in chronic immune inflammatory processes, especially those involving macrophages. In the current work, we examined the expression of AIF-1 in synovial tissues and measured AIF-1 in synovial fluid (SF) derived from patients with either RA or osteoarthritis (OA). We also examined the proliferation of synovial cells and induction of IL-6 following AIF-1 stimulation. Immunohistochemical staining showed that AIF-1 was strongly expressed in infiltrating mononuclear cells and synovial fibroblasts in RA compared with OA. Western blot analysis and semiquantitative RT-PCR analysis demonstrated that synovial expression of AIF-1 in RA was significantly greater than the expression in OA. AIF-1 induced the proliferation of cultured synovial cells in a dose-dependent manner and increased the IL-6 production of synovial fibroblasts and PBMC. The levels of AIF-1 protein were higher in synovial fluid from patients with RA compared with patients with OA (p < 0.05). Furthermore, the concentration of AIF-1 significantly correlated with the IL-6 concentration (r = 0.618, p < 0.01). These findings suggest that AIF-1 is closely associated with the pathogenesis of RA and is a novel member of the cytokine network involved in the immunological processes underlying RA.     

The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis

S100A8 and S100A9, two Ca²⁺-binding proteins of the S100 family, are secreted as a heterodimeric complex (S100A8/A9) from neutrophils and monocytes/macrophages. Serum and synovial fluid levels of S100A8, S100A9, and S100A8/A9 were all higher in patients with rheumatoid arthritis (RA) than in patients with osteoarthritis (OA), with the S100A8/A9 heterodimer being prevalent. By two-color immunofluorescence labeling, S100A8/A9 antigens were found to be expressed mainly by infiltrating CD68⁺ macrophages in RA synovial tissue (ST). Isolated ST cells from patients with RA spontaneously released larger amounts of S100A8/A9 protein than did the cells from patients with OA. S100A8/A9 complexes, as well as S100A9 homodimers, stimulated the production of proinflammatory cytokines, such as tumor necrosis factor alpha, by purified monocytes and in vitro-differentiated macrophages. S100A8/A9-mediated cytokine production was suppressed significantly by p38 mitogen-activated protein kinase (MAPK) inhibitors and almost completely by nuclear factor kappa B (NF-κB) inhibitors. NF-κB activation was induced in S100A8/A9-stimulated monocytes, but this activity was not inhibited by p38 MAPK inhibitors. These results indicate that the S100A8/A9 heterodimer, secreted extracellularly from activated tissue macrophages, may amplify proinflammatory cytokine responses through activation of NF-κB and p38 MAPK pathways in RA.     

Synovial fluid and plasma selenium, copper, zinc, and iron concentrations in patients with rheumatoid arthritis and osteoarthritis

In recent years, a great number of studies have investigated the possible role of trace elements in the etiology and pathogenesis of rheumatoid arthritis (RA) and osteoartritis (OA). We studied synovial fluid and plasma concentrations of selenium (Se), zinc (Zn), copper (Cu), and iron (Fe) in patients with RA and OA and compared them with sex- and age-matched healthy subjects. Plasma albumin levels were measured as an index of nutritional status. Plasma Se, Cu, and Zn concentrations were determined by atomic absorption spectrophotometry and Fe concentrations were determined by the colorimetric method. Although plasma and synovial fluid Se concentration were found to be significantly lower (p<0.05, and p<0.05, respectively), Cu concentrations were significantly higher in patients with RA than those of healthy subjects and OA (p<0.05 and p<0.05, respectively). There were no significant differences in plasma and synovial fluid Zn concentrations and albumin levels among three groups (p>0.05). On the other hand, synovial fluid Cu and Fe concentrations were significantly higher in patients with OA than those of healthy subjects (p<0.05). There was a significantly positive correlation between synovial fluid Se−Cu values and Zn−Fe values in patients with RA. Our results showed that synovial fluid and plasma trace element concentrations, excluding Zn, change in inflammatory RA, but not in OA. These alterations in trace element concentrations in inflammatory Ra might be a result on the changes of the immunoregulatory cytokines.     

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

Introduction

TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro.

Methods

TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry.

Results

TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38⁺ plasma cells and with CD22⁺ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22⁺ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1⁺ osteoblasts.

Conclusions

The expression of TWEAK by CD22⁺ B cells and CD38⁺ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.     

Expression of matrix metalloproteinase-9 (gelatinase B) in gouty arthritis and stimulation of MMP-9 by urate crystals in macrophages

To investigate the relevance of gelatinase-B (matrix metalloproteinase 9, MMP-9) in gouty arthritis (GA), we tested the occurrence of MMP-9 in GA patients and cell culture system. Gelatinolytic activity in the synovial fluid (SF) of patients with different kinds of arthritis was assessed by gelatin zymography. A predominant 92-kDa MMP-9 gelatinolytic activity was evident in rheumatoid arthritis (RA) and GA samples, but no activity was observed in osteoarthritis (OA) samples. Among the 53 SF samples (9 RA, 24 GA, and 20 OA) analyzed for MMP-9 and tissue inhibitor of metalloproteinase (TIMP-1) antigen levels by ELISA, MMP-9 antigen levels were elevated tenfold in GA SF compared with OA SF. In addition, GA synovial tissue extracts revealed elevated levels of MMP-9 expression as compared to OA tissue extracts by Western blot and RT-PCR analysis. Immunohistochemical studies demonstrated that MMP-9 immunoreactivity was more intense in GA than in OA synovial tissues. Furthermore, macrophages activation by gouty crystals in vitro was examined. Crystals stimulated MMP-9 gene expression in macrophage cell line and such stimulation was suppressed by PD98059. These findings suggest that the abnormal production of MMP-9 by macrophages is a reflection of the pathological conditions in joints of patients with GA, and that the activation of MMP-9 in the joint is known to play an important role in joint disease.