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Synoviocytes
Kinds of Synoviocytes Selected AbstractsThrombin-activatable carboxypeptidase B cleavage of osteopontin regulates neutrophil survival and synoviocyte binding in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 10 2009Shadi A. Sharif Objective Osteopontin (OPN) is a proinflammatory cytokine that plays an important role in the pathogenesis of rheumatoid arthritis (RA). OPN can be cleaved by thrombin, resulting in OPN-R and exposing the cryptic C-terminal ,4,1 and ,9,1 integrin,binding motif (SVVYGLR). Thrombin-activatable carboxypeptidase B (CPB), also called thrombin-activatable fibrinolysis inhibitor, removes the C-terminal arginine from OPN-R, generating OPN-L and abrogating its enhanced cell binding. We undertook this study to investigate the roles of OPN-R and OPN-L in synoviocyte adhesion, which contributes to the formation of invasive pannus, and in neutrophil survival, which affects inflammatory infiltrates in RA. Methods Using specifically developed enzyme-linked immunosorbent assays, we tested the synovial fluid of patients with RA, osteoarthritis (OA), and psoriatic arthritis (PsA) to determine OPN-R, OPN-L, and full-length OPN (OPN-FL) levels. Results Elevated levels of OPN-R and OPN-L were found in synovial fluid samples from RA patients, but not in samples from OA or PsA patients. Increased levels of OPN-R and OPN-L correlated with increased levels of multiple inflammatory cytokines, including tumor necrosis factor , and interleukin-6. Immunohistochemical analyses revealed robust expression of OPN-FL, but only minimal expression of OPN-R, in RA synovium, suggesting that cleaved OPN is released into synovial fluid. In cellular assays, OPN-FL, and to a lesser extent OPN-R and OPN-L, had an antiapoptotic effect on neutrophils. OPN-R augmented RA fibroblast-like synoviocyte binding mediated by SVVYGLR binding to ,4,1, whereas OPN-L did not. Conclusion Thrombin activation of OPN (resulting in OPN-R) and its subsequent inactivation by thrombin-activatable CPB (generating OPN-L) occurs locally within inflamed joints in RA. Our data suggest that thrombin-activatable CPB plays a central homeostatic role in RA by regulating neutrophil viability and reducing synoviocyte adhesion. [source] Adenosine downregulates cytokine-induced expression of intercellular adhesion molecule-1 on rheumatoid synovial fibroblasts independently of adenosine receptor signalingDRUG DEVELOPMENT RESEARCH, Issue 4 2003Takashi Nakazawa Abstract Adhesion of fibroblast-like synoviocytes (FLSs) to T cells through the interaction of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). We therefore used flow cytometry and quantitative polymerase chain reaction (PCR) to examine the effect of adenosine and its derivatives on expression of ICAM-1 induced by tumor necrosis factor-alpha and interferon-gamma in primary rheumatoid FLSs (RA-FLSs) and E11 cells, an RA-FLS line. Exposing cells to adenosine (5,500 µM) for 24 h in the presence of coformycin, an adenosine deaminase inhibitor, concentration-dependently inhibited cytokine-induced transcription of ICAM-1 mRNA, as well as subsequent surface expression of the protein. Although transcription of all four adenosine receptor isoforms has been detected in FLSs, neither the A1 receptor agonist R-PIA, the A2A receptor agonist CGS21680 nor the A3 agonist Cl-IB-MECA had any effect on cytokine-induced ICAM-1 expression. Conversely, A1/A2 receptor antagonist xanthine amine congener and A2A antagonist ZM240385 both failed to suppress the effect of adenosine. Adenosine appears to inhibit cytokine-induced ICAM-1 expression in FLSs independently of adenosine receptor-mediated signaling. By contrast, the effect of adenosine was neutralized by nitrobenzylmercaptopurin, a nucleoside transporter inhibitor, or by ABT702, an adenosine kinase inhibitor. This suggests that adenosine taken up via the nucleoside transporter is phosphorylated by adenosine kinase, and the resultant phospho-adenosine interferes with the ICAM-1 transcription and cell surface expression. Downregulation of T cell,FLS interaction by adenosine may thus represent a novel approach to the treatment of RA. Drug Dev. Res. 58:368,376, 2003. © 2003 Wiley-Liss, Inc. [source] Inhibition of synoviocytes proliferation by two types of c-myc antisense oligodeoxynucleotidesINTERNATIONAL JOURNAL OF RHEUMATIC DISEASES, Issue 2 2004Xinxin ZHAO Abstract Aim:, The c-myc proto-oncogene is over-expressed in synoviocytes from patients with rheumatoid arthritis (RA). For improving the inhibition of c-myc antisense oligodeoxynucleotides (AS ODN) on RA synoviocytes proliferation, we used two antisense sequences: one (antimyc-AUG AS ODN) targeting the initiation codon (AUG) and the next four codons on c-myc mRNA; another (antimyc-CRD AS ODN) targeting the coding region determinant (CRD) on c-myc mRNA to investigate if there was a difference on inhibiting synoviocytes proliferation. Methods:, Cultured human synoviocytes from patients with RA. The sequences were modified by phosphorothioates. Lipofectin was used as carrier. MTT assay was used to examine the inhibition of cell proliferation. Results:, Antimyc-AUG AS ODN and antimyc-CRD AS ODN both can inhibit synoviocytes proliferation dose-dependently. The maximum decrement of cell number was 40% at 2.5 µM and 48 h, 41.4% at 5 µM and 48 h, respectively. The action time of antimyc-AUG AS ODN inhibiting synoviocytes proliferation was earlier than that of antimyc-CRD AS ODN. ODN at high levels had non-sequence-specific cytotoxicity. Conclusions:, Both c-myc AS ODN are useful in inhibiting synoviocytes proliferation. [source] Vitamin K and rheumatoid arthritisIUBMB LIFE, Issue 6 2008Hiroshi Okamoto Abstract Vitamin K2 [menaquinone-4 (MK-4)] has been reported to induce apoptosis in hepatocellular carcinoma, leukemia, and MDS cell lines. The effects of MK-4 on the development of arthritis have never been addressed so far. In this study, we investigated the effect of MK-4 upon the proliferation of rheumatoid synovial cells and the development of arthritis in collagen-induced arthritis (CIA). We analyzed the effect of MK-4 on the proliferation of fibroblast-like synoviocytes (FLSs) using the3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The proapoptotic effect of MK-4 upon FLS was investigated with annexin V staining and DNA fragmentation and caspase 3/7 assays. Moreover, we analyzed the effect of MK-4 on the development of CIA in female dark agouti rats. Our results indicated that MK-4 inhibited the proliferation of FLS and the development of CIA in a dose-dependent manner. We concluded that MK-4 may represent a new agent for the treatment of RA in the setting of combination therapy with other disease-modifying antirheumatic drugs. © 2008 IUBMB IUBMB Life, 60(6): 355,361, 2008 [source] Efficacy of ex vivo OPG gene therapy in preventing wear debris induced osteolysisJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2002J. Jeffrey Goater Aseptic loosening of prosthetic implants remains a serious orthopaedic problem and the greatest limitation to total joint arthroplasty. Central to the etiology of aseptic loosening is periprosthetic osteolysis at the bone-implant interface, which is caused by wear debris-induced inflammation. This inflammation produces the critical osteoclast differentiation factor RANKL, which directly stimulates osteoclastogenesis and osteoclastic bone resorption. A dominant factor known to counteract this process is the natural RANKL receptor antagonist protein OPG. Here we explore the potential of ex vivo OPG gene therapy for aseptic loosening by evaluating the efficacy of stably transfected fibroblast-like synoviocytes (FLS) expressing OPG in preventing wear debris-induced osteoclastogenesis, in a mouse calvaria model. Although the stably transfected fibroblasts produced small amounts of OPG (0.3 ng/ml/72 h/106 cells), this protein was very effective in preventing osteoclastic resorption as determined in a bone wafer assay. More importantly, implantation of 107 FLS,OPG, together with 30 mg of Ti wear debris, onto the calvaria of mice, completely inhibited osteoclastogenesis 3 days after surgery. Animals given FLS-LacZ control cells, which persisted for 3 days as determined by X-gal staining, together with the Ti particles, had a 6-fold increase in osteoclastogenesis compared to controls without Ti. This increased osteoclastogenesis was completely inhibited by the FLS-OPG, as osteoclast numbers in the calvaria of these animals were similar to that seen in the SHAM controls. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Effects of carprofen (R and S enantiomers and racemate) on the production of IL-1, IL-6 and TNF-, by equine chondrocytes and synoviocytesJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 2 2002S. ARMSTRONG Chondrocytes and synoviocytes harvested from the joints of healthy horses were maintained in tissue culture. Production of the cytokines interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour necrosis factor- , (TNF- ,) in response to lipopolysaccharide (LPS), and the effects of addition of carprofen (racemate and R and S enantiomers) were determined. Lipopolysaccharide failed to stimulate TNF- , activity in both cell types but concentrations of IL-1 and IL-6 were both increased in a concentration and time-related manner. Both carprofen enantiomers and the racemic mixture attenuated the increase in IL-6 induced by LPS in synoviocytes, and S carprofen exerted a similar effect on chondrocytes. Neither enantiomer nor the racemate of carprofen suppressed the increase in IL-1 release produced by LPS in chondrocytes and synoviocytes. An action of carprofen to suppress IL-6 release might contribute to the actions which occur in vivo. [source] Immunocytochemical Localization of Caveolin-3 in the Synoviocytes of the Rat Temporomandibular Joint During DevelopmentTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 3 2008Masahiro Niwano Abstract Caveolins,caveolin-1, -2, -3 (Cav1, 2, 3),are major components of caveolae, which have diverse functions. Our recent study on the temporomandibular joint (TMJ) revealed expressions of Cav1 and muscle-specific Cav3 in some synovial fibroblast-like type B cells with well-developed caveolae. However, the involvement of Cav3 expression in the differentiation and maturation of type B cells remains unclear. The present study therefore examined the chronological alterations in the localization of Cav3 in the synovial lining cells of the rat TMJ during postnatal development by immunocytochemical techniques. Observations showed immature type B cells possessed a few caveolae with Cav1 but lacked Cav3 protein at postnatal day 5 (P5). At P14, Cav3-immunopositive type B cells first appeared in the synovial lining layer. They increased in number and immunointensity from P14 to P21 as occlusion became active. In immunoelectron microscopy and double immunolabeling with heat shock protein 25 (Hsp25) and Cav3, coexpressed type B cells developed rough endoplasmic reticulum and numerous caveolae, while the Cav3-immunonegative type B cell with Hsp25 immunoreaction possessed few of these. Results suggest that Cav3 expression, which is closely related to added functional stimuli, reflects the differentiation of the type B synoviocytes. Anat Rec, 291:233,241, 2008. © 2008 Wiley-Liss, Inc. [source] Expression of caveolin-3 in the fibroblast-like type B synoviocytes in the rat temporomandibular jointTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 3 2007Kayoko Nozawa-Inoue Abstract The present study revealed that the fibroblast-like type B synoviocytes (covering the surface of the synovial membrane in the rat temporomandibular joint) had muscle-specific caveolin-3 protein in their caveolae. The existence of two kinds of type B synoviocytes (with and without caveolin-3-immunoreactions even in the synovial lining layer) might reflect the functional difference between them. Anat Rec, 2007. © 2007 Wiley-Liss, Inc. [source] Antimicrobial peptides are expressed and produced in healthy and inflamed human synovial membranesTHE JOURNAL OF PATHOLOGY, Issue 3 2002PD Dr Med Friedrich Paulsen Abstract The objective of this study was to determine the expression and production of antimicrobial peptides by healthy and inflamed human synovial membranes. Deposition of the antimicrobial peptides lysozyme, lactoferrin, secretory phospholipase A2 (sPA2), matrilysin (MMP7), human neutrophil alpha-defensins 1,3 (HNP 1,3), human beta-defensin 1 (HBD-1), and human beta-defensin 2 (HBD-2) was determined by immunohistochemistry. Expression of mRNA for the antimicrobial peptides bactericidal permeability-increasing protein (BPI), heparin binding protein (CAP37), human cationic antimicrobial protein (LL37), human alpha-defensin 5 (HD5), human alpha-defensin 6 (HD6), HBD-1, HBD-2, and human beta-defensin 3 (HBD-3) was analysed by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR revealed CAP37 and HBD-1 mRNA in samples of healthy synovial membrane. Additionally, HBD-3 and/or LL37 mRNA was detected in synovial membrane samples from patients with pyogenic arthritis (PA), osteoarthritis (OA) or rheumatoid arthritis (RA). BPI, HD5, HD6, and HBD-2 mRNAs were absent from all samples investigated. Immunohistochemistry identified lysozyme, lactoferrin, sPA2, and MMP7 in type A synoviocytes of all samples. HBD-1 was only present in type B synoviocytes of some of the samples. Immunoreactive HBD-2 peptide was only visible in some inflamed samples. HNP1-3 was detected in both healthy and inflamed synovial membranes. The data suggest that human synovial membranes produce a broad spectrum of antimicrobial peptides. Under inflammatory conditions, the expression pattern changes, with induction of HBD-3 in PA (LL37 in RA; HBD-3 and LL37 in OA) as well as down-regulation of HBD-1. HBD-3 holds therapeutic potential in PA as it has a broad spectrum of antimicrobial activity and accelerates epithelial healing. However, caution is appropriate since defensins also promote fibrin formation and cell proliferation , key elements in joint infection. Clarification of the role of antimicrobial peptides in OA and RA will require further investigation. Copyright © 2002 John Wiley & Sons, Ltd. [source] Glucocorticoid-induced leucine zipper is an endogenous antiinflammatory mediator in arthritisARTHRITIS & RHEUMATISM, Issue 9 2010Elaine Beaulieu Objective Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid-induced protein, the reported molecular interactions of which suggest that it functions to inhibit inflammation. However, the role of endogenous GILZ in the regulation of inflammation in vivo has not been established. This study was undertaken to examine the expression and function of GILZ in vivo in collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis (RA), and in RA synoviocytes. Methods GILZ expression was detected in mouse and human synovium by immunohistochemistry and in cultured cells by real-time polymerase chain reaction and permeabilization flow cytometry. GILZ function was assessed in vivo by small interfering RNA (siRNA) silencing using cationic liposome,encapsulated GILZ or control nontargeting siRNA and was assessed in vitro using transient overexpression. Results GILZ was readily detectable in the synovium of mice with CIA and was up-regulated by therapeutic doses of glucocorticoids. Depleting GILZ expression in vivo increased the clinical and histologic severity of CIA and increased synovial expression of tumor necrosis factor and interleukin-1 (IL-1), without affecting the levels of circulating cytokines or anticollagen antibodies. GILZ was highly expressed in the synovium of patients with active RA and in cultured RA synovial fibroblasts, and GILZ overexpression in synovial fibroblasts inhibited IL-6 and IL-8 release. Conclusion Our findings indicate that GILZ functions as an endogenous inhibitor of chronic inflammation via effects on cytokine expression and suggest that local modulation of GILZ expression could be a beneficial therapeutic strategy. [source] A20 suppresses inflammatory responses and bone destruction in human fibroblast-like synoviocytes and in mice with collagen-induced arthritisARTHRITIS & RHEUMATISM, Issue 8 2010Young-Sool Hah Objective Nuclear factor-,B (NF-,B) has been implicated as a therapeutic target for the treatment of rheumatoid arthritis (RA). The purpose of this study was to determine whether A20, a universal inhibitor of NF-,B, might have antiarthritic effects. Methods An adenovirus containing A20 complementary DNA (AdA20) was used to deliver A20 to human rheumatoid fibroblast-like synoviocytes (FLS) in vitro as well as to mice with collagen-induced arthritis (CIA) in vivo via intraarticular injection into the ankle joints bilaterally. Results In vitro experiments demonstrated that AdA20 suppressed NF-,B activation, chemokine production, and matrix metalloproteinase secretion induced by tumor necrosis factor , in FLS. Mice with CIA that were treated with AdA20 had a lower cumulative disease incidence and severity of arthritis, based on hind paw thickness, radiologic and histopathologic findings, and inflammatory cytokine levels, than did control virus,injected mice. The protective effects of AdA20 were mediated by the inhibition of the NF-,B signaling pathway. The severity of arthritis was also significantly decreased in the untreated front paws, indicating a beneficial systemic effect of local suppression of NF-,B. Surprisingly, mice treated with AdA20 after the onset of CIA had significantly decreased arthritis severity from the onset of clinical signs to the end of the study. Conclusion These results suggest that using A20 to block the NF-,B pathway in rheumatoid joints reduces both the inflammatory response and the tissue destruction. The development of an immunoregulatory strategy based on A20 may therefore have therapeutic potential in the treatment of RA. [source] Prevention of cartilage degeneration and restoration of chondroprotection by lubricin tribosupplementation in the rat following anterior cruciate ligament transectionARTHRITIS & RHEUMATISM, Issue 8 2010Gregory D. Jay Objective To investigate whether cartilage degeneration is prevented or minimized following intraarticular injections of lubricin derived from human synoviocytes in culture, recombinant human PRG4 (rhPRG4), or human synovial fluid (SF) in a rat model of anterior cruciate ligament (ACL) injury. Methods Unilateral ACL transection (ACLT) was performed in Lewis rats (n = 45). Nine animals were left untreated. The remaining rats were given intraarticular injections (50 ,l/injection) of either phosphate buffered saline (PBS) (n = 9), human synoviocyte lubricin (200 ,g/ml; n = 9), rhPRG4 (200 ,g/ml; n = 9), or human SF lubricin (200 ,g/ml; n = 9) twice weekly beginning on day 7 after injury. Joints were harvested on day 32 after injury. Histologic analysis was performed using Safranin O,fast green staining, and articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI),modified Mankin criteria. Histologic specimens were immunoprobed for lubricin and sulfated glycosaminoglycans. A 24-hour urine collection was performed on days 17 and 29 postinjury, and urinary C-terminal telopeptide of type II collagen (CTX-II) levels were measured. Results Treatment with human synoviocyte lubricin resulted in significantly lower OARSI scores for cartilage degeneration compared with no treatment or PBS treatment (P < 0.05). Increased immunostaining for lubricin in the superficial zone chondrocytes and on the surface of cartilage was observed in lubricin-treated, but not untreated or PBS-treated, joints. On day 17, urinary CTX-II levels in human synoviocyte lubricin, and human SF lubricin,treated animals were significantly lower than those in untreated animals (P = 0.005 and P = 0.002, respectively) and in PBS-treated animals (P = 0.002 and P < 0.001, respectively). Conclusion After treatment with any of the 3 types of lubricin evaluated in this study, a reduction in cartilage damage following ACLT was evident, combined with a reduction in type II collagen degradation. Our findings indicate that intraarticular lubricin injection following an ACL injury may be beneficial in retarding the degeneration of cartilage and the development of posttraumatic OA. [source] Follistatin-like protein 1 is a mesenchyme-derived inflammatory protein and may represent a biomarker for systemic-onset juvenile rheumatoid arthritis,ARTHRITIS & RHEUMATISM, Issue 8 2010David C. Wilson Objective To examine both the source of follistatin-like protein 1 (FSTL-1) and the factors that induce its expression in arthritis, and to determine whether juvenile rheumatoid arthritis (JRA) is characterized by overexpression of FSTL-1. Methods FSTL-1 expression patterns were analyzed by immunohistochemical staining of joint tissue derived from mice with collagen-induced arthritis. Induction of FSTL-1 secretion was assessed in osteoblasts, adipocytes, and human fibroblast-like synoviocytes in response to transforming growth factor , (TGF,), interleukin-1, (IL-1,), tumor necrosis factor , (TNF,), and IL-6. In addition, sera and synovial fluid from children with oligoarticular, polyarticular, or systemic-onset JRA were assayed for FSTL-1 using a custom enzyme-linked immunosorbent assay. FSTL-1 concentrations in these patients were assessed for correlations with the erythrocyte sedimentation rate (ESR) and platelet count. Results Immunohistochemical staining of murine joint sections demonstrated expression of FSTL-1 in all cell types of the mesenchymal lineage, including osteocytes, chondrocytes, adipocytes, and fibroblasts. FSTL-1 could be induced in osteoblasts, adipocytes, and human fibroblast-like synoviocytes by TGF,, IL-1,, TNF,, and IL-6. The IL-1, response was significantly greater than the TNF, response (P < 0.05). In human serum and synovial fluid, only those samples from children with the systemic-onset JRA subtype had elevated concentrations of FSTL-1. The synovial fluid concentrations of FSTL-1 were 2,3-fold higher than the serum concentrations. The elevation in serum FSTL-1 concentrations seen in children with systemic-onset JRA correlated closely with elevations in the ESR and platelet count. Conclusion These findings demonstrate that the arthritic joint matrix is a major source of FSTL-1 and that IL-1, is a central mediator of FSTL-1 secretion. Furthermore, FSTL-1 may represent a useful biomarker of disease activity in systemic-onset JRA. [source] Deletion of either CD55 or CD97 ameliorates arthritis in mouse modelsARTHRITIS & RHEUMATISM, Issue 4 2010Robert M. Hoek Objective CD55 (decay-accelerating factor) is best known for its role in the negative regulation of the complement system. Indeed, lack of this molecule leads to disease aggravation in many autoimmune disease models. However, CD55 is abundantly present on fibroblast-like synoviocytes and is also a ligand of the adhesion-class heptahelical receptor CD97, which is expressed by infiltrating macrophages. Treatment with antibodies to CD97 ameliorates the collagen-induced model of rheumatoid arthritis (RA) in DBA/1 mice, but the net contribution of CD55 is unknown. This study was undertaken to investigate the role of CD55 in experimental RA. Methods Arthritis was induced in wild-type, CD55,/,, and CD97,/, mice using collagen-induced and K/BxN serum,transfer models. Incidence of arthritis was monitored over time, and disease activity was assessed by clinical and immunohistochemical evaluation. Results In contrast to observations in many inflammatory disease models, lack of CD55 resulted in decreased arthritis in experimental models of RA. Consistent with the previously reported effects of anti-CD97 antibody treatment, CD97,/, mice had reduced arthritis activity compared with wild-type controls. Conclusion Our findings indicate that the lack of CD55 or CD97 in 2 different models of arthritis increases resistance to the disease. These findings provide insight into a role for CD55 interaction with CD97 in the pathogenesis of RA and suggest that therapeutic strategies that disrupt CD55/CD97 may be clinically beneficial. [source] Synovial fibroblasts self-direct multicellular lining architecture and synthetic function in three-dimensional organ cultureARTHRITIS & RHEUMATISM, Issue 3 2010Hans P. Kiener Objective To define the intrinsic capacity of fibroblast-like synoviocytes (FLS) to establish a 3-dimensional (3-D) complex synovial lining architecture characterized by the multicellular organization of the compacted synovial lining and the elaboration of synovial fluid constituents. Methods FLS were cultured in spherical extracellular matrix (ECM) micromasses for 3 weeks. The FLS micromass architecture was assessed histologically and compared with that of dermal fibroblast controls. Lubricin synthesis was measured via immunodetection. Basement membrane matrix and reticular fiber stains were performed to examine ECM organization. Primary human and mouse monocytes were prepared and cocultured with FLS in micromass to investigate cocompaction in the lining architecture. Cytokine stimuli were applied to determine the capacity for inflammatory architecture rearrangement. Results FLS, but not dermal fibroblasts, spontaneously formed a compacted lining architecture over 3 weeks in the 3-D ECM micromass organ cultures. These lining cells produced lubricin. FLS rearranged their surrounding ECM into a complex architecture resembling the synovial lining and supported the survival and cocompaction of monocyte/macrophages in the neo,lining structure. Furthermore, when stimulated by cytokines, FLS lining structures displayed features of the hyperplastic rheumatoid arthritis synovial lining. Conclusion This 3-D micromass organ culture method demonstrates that many of the phenotypic characteristics of the normal and the hyperplastic synovial lining in vivo are intrinsic functions of FLS. Moreover, FLS promote survival and cocompaction of primary monocytes in a manner remarkably similar to that of synovial lining macrophages. These findings provide new insight into inherent functions of the FLS lineage and establish a powerful in vitro method for further investigation of this lineage. [source] Effect of interleukin-32, on differentiation of osteoclasts from CD14+ monocytesARTHRITIS & RHEUMATISM, Issue 2 2010Yong-Gil Kim Objective Interleukin-32 (IL-32) induces various inflammatory molecules in human monocytes and differentiation of monocytes into macrophage-like cells. This study was undertaken to evaluate the effects of IL-32,, the most biologically active isoform, on the differentiation and activation of osteoclasts. Methods CD14+ monocytes were obtained from healthy volunteers, and samples of synovial tissue and synovial fluid were obtained from patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). The concentration and expression levels of IL-32, in RA and OA samples were evaluated by enzyme-linked immunosorbent assay and immunoblotting, respectively. To examine the osteoclastogenic effects and functional activities, isolated monocytes were treated with either IL-32, or IL-17 in the presence or absence of soluble RANKL (sRANKL) on a culture system and on Osteologic disks. The expression of RANKL and osteoprotegerin (OPG) messenger RNA (mRNA) in RA fibroblast-like synoviocytes (FLS) was measured using reverse transcription,polymerase chain reaction (PCR) and real-time PCR. Results The concentration and expression levels of IL-32, were higher in the RA samples than in the OA samples. Upon costimulation with sRANKL, the osteoclast count and resorbed area increased more significantly in the IL-32,,stimulated cultures than in those stimulated with IL-17. In the IL-32,,treated group without sRANKL stimulation, osteoclasts were differentiated, but the cells displayed low resorption activity. In RA FLS, RANKL mRNA expression increased in the presence of both IL-32, and IL-17. However, transcription of OPG decreased following IL-32, stimulation, resulting in a significant increase in the RANKL:OPG ratio. Conclusion Our results suggest that IL-32, is a potent mediator of active osteoclast generation in the presence of sRANKL. Moreover, this novel cytokine creates more favorable conditions for osteoclastogenesis in the RA joint by increasing the RANKL:OPG ratio in FLS. [source] Inhibition of NF-,B signaling by fasudil as a potential therapeutic strategy for rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 1 2010Hiroshi Okamoto Objective Rheumatoid arthritis (RA) is the most common systemic autoimmune disease and is characterized mainly by symmetric polyarticular joint disorders. The pathologic processes are mediated by a number of cytokines, chemokines, cell adhesion molecules, and matrix metalloproteinases. The expression of most of these molecules is controlled at the transcriptional level. In addition, activation of NF-,B is involved in RA pathogenesis. This study was performed to explore the role of a novel serine/threonine kinase inhibitor, fasudil, in the control of the NF-,B activation pathway and to investigate the therapeutic effects of fasudil on arthritis development in a rat model of RA. Methods Fibroblast-like synoviocytes (FLS) from RA patients and human endothelial cells (ECs) were established and maintained. To study the role of fasudil on cytokine expression, various cytokines expressed in the RA FLS and human ECs were measured by enzyme-linked immunosorbent assay following stimulation of the cells with interleukin-1, (IL-1,) in the presence of various concentrations of fasudil. The role of fasudil on NF-,B activation was studied using a reporter gene assay, Western blotting of I,B,, immunofluorescence analysis of the p65 subunit of NF-,B, and electrophoretic mobility shift assay. The in vivo effects of fasudil on arthritis were studied in a rat adjuvant-induced arthritis (AIA) model. Results Fasudil inhibited cytokine expression in RA FLS and human ECs and also inhibited the activation of ECs, in a dose-dependent manner. Fasudil inhibited IL-1,,induced activation of NF-,B independent of the inhibition of I,B, degradation and nuclear translocation of NF-,B, and inhibited IL-1,,induced DNA binding of NF-,B. Finally, in vivo, fasudil ameliorated arthritis in rats with AIA, without any adverse effects. Conclusion Serine/threonine kinase inhibitor fasudil inhibits the development of arthritis in a rat model of RA, and also inhibits the NF-,B signaling required for binding of NF-,B to specific DNA sequences through, for example, the phosphorylation of p65, suggesting that a specific target of fasudil might be a novel NF-,B kinase. Thus, fasudil serves as a novel strategy for the treatment of RA. [source] Anti,neuropilin-1 peptide inhibition of synoviocyte survival, angiogenesis, and experimental arthritisARTHRITIS & RHEUMATISM, Issue 1 2010Jin-Sun Kong Objective To delineate the role of neuropilin-1 (NP-1), a vascular endothelial growth factor receptor (VEGFR), in rheumatoid inflammation and to determine whether blockade of NP-1 could suppress synoviocyte survival and angiogenesis. Methods VEGF111,165 peptide, which encompasses the NP-1 binding domain of VEGF165, was generated by cleaving VEGF165 with plasmin. The effect of this peptide on the interaction between VEGF165 and its receptor was determined by 125I-VEGFR binding assay. Assays to determine synoviocyte apoptosis, adhesion, and migration were performed in the presence of VEGF165 and/or the peptide. VEGF165 -induced angiogenesis was assessed by measuring the proliferation, tube formation, and wounding migration of endothelial cells (ECs). Mice were immunized with type II collagen to induce experimental arthritis. Results VEGF111,165 peptide specifically inhibited the binding of 125I-VEGF165 to NP-1 on rheumatoid synoviocytes and ECs. The peptide eliminated the VEGF165 -mediated increase in synoviocyte survival and activation of p-ERK and Bcl-2. The peptide also completely inhibited a VEGF165 -induced increase in synoviocyte adhesion and migration. In addition, the anti,NP-1 peptide blocked VEGF165 -stimulated proliferation, capillary tube formation, and wounding migration of ECs in vitro. VEGF165 -induced neovascularization in a Matrigel plug in mice was also blocked by treatment with the peptide. Finally, subcutaneous injection of anti,NP-1 peptide suppressed arthritis severity and autoantibody formation in mice with experimental arthritis and inhibited synoviocyte hyperplasia and angiogenesis in arthritic joints. Conclusion Anti,NP-1 peptide suppressed VEGF165 -induced increases in synoviocyte survival and angiogenesis, and thereby blocked experimental arthritis. Our findings suggest that anti,NP-1 peptide could be useful in alleviating chronic arthritis. [source] Specifically modified osteopontin in rheumatoid arthritis fibroblast-like synoviocytes supports interaction with B cells and enhances production of interleukin-6ARTHRITIS & RHEUMATISM, Issue 12 2009Yasuhiro Take Objective Osteopontin (OPN) is expressed by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA), but its pathologic role is still obscure. The present study was undertaken to analyze the role of OPN in RA by focusing on its effects on cell,cell interactions between FLS and B lymphocytes. Methods FLS obtained from 10 patients with RA and 10 non-RA subjects and a B lymphocyte cell line were studied. The characteristics of OPN expression by FLS were analyzed by Western blotting, immunoprecipitation, and immunofluorescence studies. In cocultures of FLS and B lymphocytes, the effects of OPN on adhesion of B lymphocytes to FLS and the consequent production of interleukin-6 (IL-6) were analyzed in experiments involving overexpression and knockdown of OPN and inhibitory studies with an OPN-blocking antibody. In vivo, the expression of OPN in RA synovium was examined by immunohistochemistry. Results A specifically modified 75-kd form of OPN was predominantly expressed in RA FLS, and this was associated with expression of >200-kd thrombin-cleaved OPN that was crosslinked with fibronectin and localized on the surface of the FLS. In FLS,B lymphocyte cocultures, 75-kd OPN,positive FLS produced a significantly higher amount of IL-6 than did 75-kd OPN,negative FLS. When the FLS were separated from B lymphocytes or cultured alone, the production of IL-6 was low and was not significantly different between these 2 culture conditions. Moreover, OPN overexpression enhanced production of IL-6 in 75-kd OPN,positive FLS,B lymphocyte cocultures. Addition of the OPN-blocking antibody inhibited the adhesion of B lymphocytes to FLS. Immunohistochemical analyses revealed that localization of IL-6,positive cells coincided with the sites at which OPN and B lymphocytes were colocalized. Conclusion Specifically modified 75-kd OPN was expressed by RA FLS. This form of OPN affected FLS,B lymphocyte interactions by supporting the adhesion of B lymphocytes to FLS and enhancing the production of IL-6. [source] A critical role of Cyr61 in interleukin-17,dependent proliferation of fibroblast-like synoviocytes in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 12 2009Qiuyu Zhang Objective Fibroblast-like synoviocytes (FLS) are a major component of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Cyr61 (CCN1) is a product of a growth factor,inducible immediate early gene and is involved in cell adhesion, proliferation, and differentiation. However, the role that Cyr61 plays in FLS proliferation has remained undetermined. The aim of this study was to identify the role of Cyr61 in regulating the proliferation of FLS derived from patients with RA. Methods Expression of Cyr61 in synovial tissue (ST) and in FLS was determined simultaneously using immunohistochemistry, real-time polymerase chain reaction, and Western blotting. Cyr61 levels in synovial fluid (SF) were determined by enzyme-linked immunosorbent assay. FLS proliferation stimulated by SF, Cyr61, and interleukin-17 (IL-17) was measured by thymidine incorporation. Activation of signal transduction pathways was determined by Western blotting and confocal microscopy. Results Cyr61 was overexpressed in ST, FLS, and SF samples from RA patients as compared with samples from normal controls. Elevated levels of Cyr61 in RA SF promoted the proliferation of FLS, an effect that was abrogated by a neutralizing monoclonal antibody against human Cyr61. Furthermore, in samples from RA patients, Cyr61 was found to protect FLS from apoptosis and to sustain the expression of Bcl-2 in FLS. Most importantly, the expression of Cyr61 in FLS was regulated by IL-17 mainly via the p38 MAPK and NF-,B signaling pathways. Knockdown of expression of the Cyr61 gene inhibited IL-17,stimulated FLS proliferation. Conclusion Our findings indicate that Cyr61 plays a critical role in IL-17,mediated proliferation of FLS in RA and likely contributes to hyperplasia of synovial lining cells and eventually to joint destruction in patients with RA. [source] Gadd45, deficiency in rheumatoid arthritis: Enhanced synovitis through JNK signalingARTHRITIS & RHEUMATISM, Issue 11 2009Camilla I. Svensson Objective JNK-mediated cell signaling plays a critical role in matrix metalloproteinase (MMP) expression and joint destruction in rheumatoid arthritis (RA). Gadd45,, which is an NF-,B,regulated gene, was recently identified as an endogenous negative regulator of the JNK pathway, since it could block the upstream kinase MKK-7. This study was carried out to evaluate whether low Gadd45, expression in RA enhances JNK activation and overproduction of MMPs in RA, and whether Gadd45, deficiency increases arthritis severity in passive K/BxN murine arthritis. Methods Activation of the NF-,B and JNK pathways and Gadd45, expression were analyzed in human synovium and fibroblast-like synoviocytes (FLS) using quantitative polymerase chain reaction, immunoblotting, immunohistochemistry, electrophoretic mobility shift assay, and luciferase reporter constructs. Gadd45,,/, and wild-type mice were evaluated in the K/BxN serum transfer model of inflammatory arthritis, and clinical signs of arthritis, osteoclast formation, and bone erosion were assessed. Results Expression levels of the Gadd45, gene and protein were unexpectedly low in human RA synovium despite abundant NF-,B activity. Forced Gadd45, expression in human FLS attenuated tumor necrosis factor,induced signaling through the JNK pathway, reduced the activation of activator protein 1, and decreased the expression of MMP genes. Furthermore, Gadd45, deficiency exacerbated K/BxN serum,induced arthritis in mice, dramatically increased signaling through the JNK pathway, elevated MMP3 and MMP13 gene expression in the mouse joints, and increased the synovial inflammation and number of osteoclasts. Conclusion Deficient Gadd45, expression in RA can contribute to activation of JNK, exacerbate clinical arthritis, and augment joint destruction. This process can be mitigated by enhancing Gadd45, expression or by inhibiting the activity of JNK or its upstream regulator, MKK-7. [source] Differential mechanism of NF-,B inhibition by two glucocorticoid receptor modulators in rheumatoid arthritis synovial fibroblastsARTHRITIS & RHEUMATISM, Issue 11 2009Valerie Gossye Objective To investigate and compare the molecular mechanisms by which 2 glucocorticoid receptor (GR),activating compounds, dexamethasone (DEX) and Compound A (CpdA), interfere with the NF-,B activation pathway in rheumatoid arthritis (RA) synovial cells. Methods Quantitative polymerase chain reaction was performed to detect the tumor necrosis factor , (TNF,),induced cytokine gene expression of interleukin-1, (IL-1,) and to investigate the effects of DEX and CpdA in RA fibroblast-like synoviocytes (FLS) transfected with small interfering RNA (siRNA) against GR (siGR) compared with nontransfected cells. Immunofluorescence analysis was used to detect the subcellular distribution of NF-,B (p65) under the various treatment conditions, and active DNA-bound p65 was measured using a TransAM assay and by chromatin immunoprecipitation analysis of IL-1,. Signaling pathways were studied via Western blotting of siGR-transfected cells, compared with nontransfected and nontargeting siRNA,transfected control cells, to detect the regulation of phospho-IKK, I,B,, phospho-p38, phospho-ERK, and phospho-JNK. Results Both DEX and CpdA efficiently inhibited IL-1, gene expression in a GR-dependent manner. In addition, CpdA attenuated the TNF,-induced nuclear translocation and DNA binding of p65 in RA FLS, via the attenuation of IKK phosphorylation and subsequent I,B, degradation. CpdA also displayed profound effects on TNF,-induced MAPK activation. The effects of CpdA on TNF,-induced kinase activities occurred independently of the presence of GR. In sharp contrast, DEX did not affect TNF,-induced IKK phosphorylation, I,B, degradation, p65 nuclear translocation, or MAPK activation in RA FLS. Conclusion DEX and CpdA display a dissimilar molecular mechanism of interaction with the NF-,B activation pathway ex vivo. A dual pathway, partially dependent and partially independent of GR (nongenomic), may explain the gene-inhibitory effects of CpdA in RA FLS. [source] The small ubiquitin-like modifier mediates the resistance of prosthesis-loosening fibroblast-like synoviocytes against fas-induced apoptosisARTHRITIS & RHEUMATISM, Issue 7 2009Ingmar Meinecke Objective To study the expression of small ubiquitin-like modifier 1 (SUMO-1) in aseptic loosening of prosthesis implants and to investigate its role in regulating the susceptibility of prosthesis-loosening fibroblast-like synoviocytes (FLS) to Fas-induced apoptosis. Methods Specimens of aseptically loosened tissue were obtained at revision surgery, and the expression of SUMO-1 was analyzed by in situ hybridization. SUMO-1 levels in FLS were determined by quantitative polymerase chain reaction and Western blot analysis. Immunohistochemistry and confocal microscopy were used to study the subcellular localization of SUMO-1. The functional role of SUMO-1 in Fas-induced apoptosis of prosthesis-loosening FLS was investigated by small interfering RNA,mediated knockdown of SUMO-1 and by gene transfer of the nuclear SUMO-specific protease SENP1. Results SUMO-1 was expressed strongly in aseptically loosened tissue and was found prominently at sites adjacent to bone. Prosthesis-loosening FLS expressed levels of SUMO-1 similar to the levels expressed by rheumatoid arthritis (RA) FLS, with SUMO-1 being found mainly in promyelocytic leukemia protein nuclear bodies. Knockdown of SUMO-1 had no effect on spontaneous apoptosis but significantly increased the susceptibility of prosthesis-loosening FLS to Fas-induced apoptosis. Gene transfer of the nuclear SUMO-specific protease SENP1 reverted the apoptosis-inhibiting effects of SUMO-1. Conclusion These data suggest that SUMO-1 is involved in the activation of both RA FLS and prosthesis-loosening FLS by preventing these cells from undergoing apoptosis. Modification of nuclear proteins by SUMO-1 contributes to the antiapoptotic effects of SUMO-1 in prosthesis-loosening FLS, providing evidence for the specific activation of sumoylation during their differentiation. Therefore, SUMO-1 may be an interesting target for novel strategies to prevent aseptic prosthesis loosening. [source] Transmembrane BAFF from rheumatoid synoviocytes requires interleukin-6 to induce the expression of recombination-activating gene in B lymphocytesARTHRITIS & RHEUMATISM, Issue 5 2009Caroline Rochas Objective B cells that accumulate in the synovial tissue of rheumatoid arthritis (RA) patients revise their receptors due to coordinate expression of recombination-activating gene 1 (RAG-1) and RAG-2 genes. The aim of this study was to determine the mechanisms that control this re-expression. Methods B cells from healthy control subjects were cocultured with fibroblast-like synoviocytes (FLS) from patients with RA and osteoarthritis (OA). Re-expression of RAG messenger RNA (mRNA) and proteins was analyzed by reverse transcription,polymerase chain reaction (RT-PCR) and indirect immunofluorescence. Activity of RAG enzymes was evaluated by flow cytometry to measure variations in immunoglobulin , and , light chain expression and by ligation-mediated,PCR to assess specific DNA breaks. Blocking antibodies, short hairpin RNA, and recombinant cytokine were used to identify the molecules involved in RAG re-expression. Results RA FLS, but not OA FLS, induced B cells to re-express RAG mRNA and proteins. Enzymes were functional, since the ,-to-, ratios decreased and specific DNA breaks were detectable after coculture with RA FLS. Transmembrane BAFF provided the first signal of RAG re-expression, since its down-regulation in RA FLS prevented RAG gene transcription in B cells. The failure of transmembrane BAFF from OA FLS to induce RAG suggests that a second signal was provided by RA FLS. Interleukin-6 (IL-6) is a candidate, since blockade of its receptors precluded transcription of RAG genes by RA FLS. Unless supplemented with IL-6, OA FLS were unable to induce RAG gene expression in normal B cells. Conclusion Two independent signals are required for the induction of RAG gene expression in B cells that infiltrate the synovium of patients with RA. [source] The ,7 nicotinic acetylcholine receptor on fibroblast-like synoviocytes and in synovial tissue from rheumatoid arthritis patients: A possible role for a key neurotransmitter in synovial inflammationARTHRITIS & RHEUMATISM, Issue 5 2009Marjolein A. Van Maanen Objective Recent studies have suggested an important role for neurotransmitters as modulators of inflammation. Therefore, we undertook this study to investigate the expression of the ,7 subunit of the nicotinic acetylcholine receptor (,7nAChR) and its function in rheumatoid arthritis (RA). Methods The potential role of the ,7nAChR in modulating proinflammatory cytokine expression in fibroblast-like synoviocytes (FLS) was identified by screening an adenoviral short hairpin RNA (Ad.shRNA) library. An ,7-specific antibody was used for immunohistochemistry, and fluorescein isothiocyanate,labeled ,-bungarotoxin, which binds specifically to the ,7nAChR, was used for immunofluorescence. Gene expression in FLS was determined by quantitative polymerase chain reaction with primers specific for the ,7nAChR. In addition, we analyzed messenger RNA (mRNA) expression of dup,7, a variant ,7 transcript. Next, we studied the functional role of the ,7nAChR in RA FLS by examining the effects of ,7-specific agonists on the production of interleukin-6 (IL-6) and IL-8 by activated FLS. Results A screen using an Ad.shRNA library against 807 transcripts revealed that a specific ,7nAChR shRNA potently modulated IL-8 and matrix metalloproteinase expression in FLS. The ,7nAChR was expressed in the inflamed synovium from RA patients, predominantly in the intimal lining layer. We found ,7nAChR expression at both the mRNA and protein level in cultured RA FLS. FLS also constitutively expressed dup,7 mRNA. Specific ,7nAChR agonists reduced tumor necrosis factor ,,induced IL-6 and IL-8 production by FLS. Conclusion The ,7nAChR and its dup,7 variant are expressed in RA synovium, where they may play a critical role in regulating inflammation. Targeting the ,7nAChR could provide a novel antiinflammatory approach to the treatment of RA. [source] MicroRNA-124a is a key regulator of proliferation and monocyte chemoattractant protein 1 secretion in fibroblast-like synoviocytes from patients with rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 5 2009Yuji Nakamachi Objective To elucidate the role of microRNA (miRNA) in the pathogenesis of rheumatoid arthritis (RA), we analyzed synoviocytes from RA patients for their miRNA expression. Methods Synoviocytes derived from surgical specimens obtained from RA patients were compared with those obtained from osteoarthritis (OA) patients for their expression of a panel of 156 miRNA with quantitative stem-loop reverse transcription,polymerase chain reaction. The miRNA whose expression decreased or increased in RA synoviocytes as compared with OA synoviocytes were identified, and their target genes were predicted by computer analysis. We used an in vitro system of enhancing the expression of specific miRNA by transfection of precursors into synoviocytes, and then we performed proliferation, cell cycle, and apoptosis assays, as well as enzyme-linked immunosorbent assays for cytokine production. The effects of transfection on predicted target protein and messenger RNA (mRNA) were then examined by Western blot analysis and luciferase reporter assay. Results We found that miR-124a levels significantly decreased in RA synoviocytes as compared with OA synoviocytes. Transfection of precursor miR-124a into RA synoviocytes significantly suppressed their proliferation and arrested the cell cycle at the G1 phase. We identified a putative consensus site for miR-124a binding in the 3,-untranslated region of cyclin-dependent kinase 2 (CDK-2) and monocyte chemoattractant protein 1 (MCP-1) mRNA. Induction of miR-124a in RA synoviocytes significantly suppressed the production of the CDK-2 and MCP-1 proteins. Luciferase reporter assay demonstrated that miR-124a specifically suppressed the reporter activity driven by the 3,-untranslated regions of CDK-2 and MCP-1 mRNA. Conclusion The results of this study suggest that miR-124a is a key miRNA in the posttranscriptional regulatory mechanisms of RA synoviocytes. [source] Cadherin 11 promotes invasive behavior of fibroblast-like synoviocytesARTHRITIS & RHEUMATISM, Issue 5 2009Hans P. Kiener Objective To define the expression pattern of cadherin 11 in the destructive pannus tissue of patients with rheumatoid arthritis, and to determine whether cadherin 11 expression in fibroblast-like synoviocytes controls their invasive capacity. Methods Cadherin 11 expression in rheumatoid synovial tissue was evaluated using immunohistochemistry. To examine the role of cadherin 11 in regulating the invasive behavior of fibroblast-like synoviocytes, we generated L cell clones expressing wild-type cadherin 11, mutant cadherin 11, and empty vector,transfected controls. The invasive capacity of L cell transfectants and cultured fibroblast-like synoviocytes treated with a blocking cadherin 11,Fc fusion protein or control immunoglobulin was determined in Matrigel invasion assays. Results Immunohistochemical analysis revealed that cadherin 11 is abundantly expressed in cells at the cartilage,pannus junction in rheumatoid synovitis. Assays to determine invasion demonstrated a 2-fold increased invasive capacity of cadherin 11,transfected L cells compared with L cells transfected with E-cadherin or control vector. The invasive behavior of L cells stably transfected with a cadherin 11 construct that lacked the juxtamembrane cytoplasmic domain was diminished to the level of vector control L cells. Furthermore, treatment with the cadherin 11,Fc fusion protein diminished the invasive capacity of fibroblast-like synoviocytes. Conclusion The results of these in vitro studies implicate a role for cadherin 11 in promoting cell invasion and contribute insight into the invasive nature of fibroblast-like synoviocytes in chronic synovitis and rheumatoid arthritis. [source] Tumor necrosis factor ,,induced interleukin-32 is positively regulated via the Syk/protein kinase C,/JNK pathway in rheumatoid synovial fibroblastsARTHRITIS & RHEUMATISM, Issue 3 2009Se Hwan Mun Objective Interleukin-32 (IL-32) is a recently discovered cytokine that appears to play a critical role in human rheumatoid arthritis (RA). It is highly expressed in synovium and fibroblast-like synoviocytes (FLS) from RA patients, but not in patients with osteoarthritis (OA). This study was undertaken to assess IL-32 levels in RA synovial fluid (SF) and to investigate the secretion and regulation of IL-32 in RA FLS. Methods FLS and SF were obtained from the joints of RA patients. The secretion and expression of IL-32 and activation of signaling molecules were examined by enzyme-linked immunosorbent assay, immunoblotting, immunoprecipitation, reverse transcriptase,polymerase chain reaction, and small interfering RNA (siRNA) transfection. Results IL-32 levels were high in RA SF compared with OA SF. Furthermore, RA FLS expressed and secreted IL-32 when stimulated with tumor necrosis factor , (TNF,). TNF,-induced expression of IL-32 was significantly suppressed, in a dose-dependent manner, by inhibitors of Syk, protein kinase C, (PKC,), and JNK and by knockdown of these kinases and c-Jun with siRNA. We also observed that PKC, mediated the activation of JNK and c-Jun, and experiments using specific inhibitors and siRNA demonstrated that Syk was the upstream kinase for the activation of PKC,. Conclusion The present findings suggest that IL-32 may be a newly identified prognostic biomarker in RA, thereby adding valuable knowledge to the understanding of this disease. The results also demonstrate that the production of IL-32 in RA FLS is regulated by Syk/PKC,-mediated signaling events. [source] Chondroitin sulfate increases hyaluronan production by human synoviocytes through differential regulation of hyaluronan synthases: Role of p38 and AktARTHRITIS & RHEUMATISM, Issue 3 2009Maha David-Raoudi Objective To uncover the mechanism by which chondroitin sulfate (CS) enhances hyaluronan (HA) production by human osteoarthritic (OA) fibroblast-like synoviocytes (FLS). Methods The production of HA was investigated by exposing human OA FLS to CS in the presence or absence of interleukin-1, (IL-1,). HA levels were determined by enzyme-linked immunosorbent assay, and levels of messenger RNA (mRNA) for HA synthase 1 (HAS-1), HAS-2, and HAS-3 were determined by real-time polymerase chain reaction analysis. The effect of CS and IL-1, on signaling pathways was assessed by Western blotting. Specific inhibitors were used to determine their effects on both HA production and HAS expression. The molecular size of HA was analyzed by high-pressure liquid chromatography. Results CS increased HA production by FLS through up-regulation of the expression of HAS1 and HAS2. This was associated with activation of ERK-1/2, p38, and Akt, although to a lesser extent. Both p38 and Akt were involved in CS-induced HA accumulation. IL-1, increased HA production and levels of mRNA for HAS1, HAS2, and HAS3. CS enhanced the IL-1,,induced level of HAS2 mRNA and reduced the level of HAS3 mRNA. IL-1,,induced activation of p38 and JNK was slightly decreased by CS, whereas that of ERK-1/2 and Akt was enhanced. More high molecular weight HA was found in CS plus IL-1,,treated FLS than in FLS treated with IL-1, alone. Conclusion CS stimulates the synthesis of high molecular weight HA in OA FLS through up-regulation of HAS1 and HAS2. It reduces the IL-1,,enhanced transcription of HAS3 and increases the production of HA of large molecular sizes. These effects may be beneficial for maintaining viscosity and antiinflammatory properties in the joint. [source] Role of placenta growth factor and its receptor flt-1 in rheumatoid inflammation: A link between angiogenesis and inflammationARTHRITIS & RHEUMATISM, Issue 2 2009Seung-Ah Yoo Objective To investigate the direct effects of placenta growth factor (PlGF) and its specific receptor, flt-1, which are known to mediate angiogenesis, on the inflammatory process of rheumatoid arthritis (RA). Methods Expression of PlGF and flt-1 in the synovial tissue of RA patients was examined using immunohistochemistry. Enzyme-linked immunosorbent assay was used to determine the concentrations of PlGF, tumor necrosis factor , (TNF,), and interleukin-6 (IL-6) in culture supernatants of either mononuclear cells or synoviocytes. The flt-1 expression level in mononuclear cells was analyzed by flow cytometry. Experimental arthritis was induced in mice either by immunization with type II collagen (CII) or by injection of anti-CII antibody. Results PlGF was highly expressed in the synovium of RA patients, and its primary source was fibroblast-like synoviocytes (FLS). When stimulated with IL-1,, FLS from RA patients produced higher amounts of PlGF than did FLS from patients with osteoarthritis. Exogenous PlGF specifically increased the production of TNF, and IL-6 in mononuclear cells from RA patients (but not those from healthy controls) via a calcineurin-dependent pathway. The response to PlGF was associated with increased expression of flt-1 on RA monocytes, which could be induced by IL-1, and TNF,. A novel anti,flt-1 hexapeptide, GNQWFI, abrogated the PlGF-induced increase in TNF, and IL-6 production, and also suppressed CII-induced arthritis and serum IL-6 concentrations in mice. Moreover, genetic ablation of PlGF prevented the development of anti-CII antibody,induced arthritis in mice. Conclusion Our data suggest that enhanced expression of PlGF and flt-1 may contribute to rheumatoid inflammation by triggering production of proinflammatory cytokines. The use of the novel anti,flt-1 peptide, GNQWFI, may be an effective strategy for the treatment of RA. [source] | ||||||||||||||||||||||||||||