Differences in synovial fluid cytokine levels but not in synovial tissue cell infiltrate between anti-citrullinated peptide/protein antibody-positive and –negative rheumatoid arthritis patients

Introduction

Comparative data on synovial cell infiltrate and cytokine levels in anti citrullinated peptide/protein antibody (ACPA)-positive and ACPA negative rheumatoid arthritis (RA) patients are scarce. Our aim was to analyze synovial cell infiltrate and synovial fluid (SF) levels of cytokines in patients with RA according to the presence or absence of ACPA in serum.

Methods

A cross-sectional study in a single center including consecutive RA patients was performed. Patients were defined as ''ACPA negative'' if serum was negative to two different ACPAs [second generation commercial anti-cyclic citrullinated peptide antibodies (CCP2) and chimeric fibrin/filaggrin citrullinated antibodies]. Parallel synovial tissue (ST) biopsies and SF were obtained by knee arthroscopy. Synovial cell infiltrate and endothelial cells were analyzed by immunohistochemistry and SF levels of Th1, Th2, Th17 and pro-inflammatory cytokines by Quantibody(R) Human Array.

Results

A total of 83 patients underwent arthroscopy, with a mean age of 55.9 ± 12 years, and mean disease duration of 45 months (interquartile range, IQR 10.8 to 122). 62% were female and 77% were ACPA positive. No significant differences were found in clinical variables, acute phase reactants, synovial cell infiltrate or lymphoid neogenesis (LN) between ACPA positive and negative patients. However ACPA positive patients had significantly higher levels of IL-1β, IL-10, IL-17 F and CC chemokine ligand 20 (CCL-20) than ACPA negative patients.

Conclusions

In our cohort of patients with RA no significant differences were found in synovial cell infiltrate or synovial LN according to ACPA status. However, ACPA positive patients had higher levels of T-cell derived and pro-inflammatory cytokines than ACPA negative patients. As systemic and local inflammation was similar in the two groups, these findings support a distinct synovial physiopathology.     

Therapeutic potential of human umbilical cord mesenchymal stem cells in the treatment of rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is a T-cell-mediated systemic autoimmune disease, characterized by synovium inflammation and articular destruction. Bone marrow mesenchymal stem cells (MSCs) could be effective in the treatment of several autoimmune diseases. However, there has been thus far no report on umbilical cord (UC)-MSCs in the treatment of RA. Here, potential immunosuppressive effects of human UC-MSCs in RA were evaluated.

Methods

The effects of UC-MSCs on the responses of fibroblast-like synoviocytes (FLSs) and T cells in RA patients were explored. The possible molecular mechanism mediating this immunosuppressive effect of UC-MSCs was explored by addition of inhibitors to indoleamine 2,3-dioxygenase (IDO), Nitric oxide (NO), prostaglandin E2 (PGE2), transforming growth factor β1 (TGF-β1) and interleukin 10 (IL-10). The therapeutic effects of systemic infusion of human UC-MSCs on collagen-induced arthritis (CIA) in a mouse model were explored.

Results

In vitro, UC-MSCs were capable of inhibiting proliferation of FLSs from RA patients, via IL-10, IDO and TGF-β1. Furthermore, the invasive behavior and IL-6 secretion of FLSs were also significantly suppressed. On the other hand, UC-MSCs induced hyporesponsiveness of T cells mediated by PGE2, TGF-β1 and NO and UC-MSCs could promote the expansion of CD4⁺ Foxp3⁺ regulatory T cells from RA patients. More importantly, systemic infusion of human UC-MSCs reduced the severity of CIA in a mouse model. Consistently, there were reduced levels of proinflammatory cytokines and chemokines (TNF-α, IL-6 and monocyte chemoattractant protein-1) and increased levels of the anti-inflammatory/regulatory cytokine (IL-10) in sera of UC-MSCs treated mice. Moreover, such treatment shifted Th1/Th2 type responses and induced Tregs in CIA.

Conclusions

In conclusion, human UC-MSCs suppressed the various inflammatory effects of FLSs and T cells of RA in vitro, and attenuated the development of CIA in vivo, strongly suggesting that UC-MSCs might be a therapeutic strategy in RA. In addition, the immunosuppressive activitiy of UC-MSCs could be prolonged by the participation of Tregs.     

Citrullinated fibronectin inhibits apoptosis and promotes the secretion of pro-inflammatory cytokines in fibroblast-like synoviocytes in rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is characterized by synovial lining hyperplasia, in which there may be an imbalance between the growth and death of fibroblast-like synoviocytes (FLSs). Antibodies against citrullinated proteins are proposed to induce RA. This study aimed to investigate the pathogenic role of citrullinated fibronectin (cFn) in RA.

Methods

The distribution of fibronectin (Fn) and cFn in synovial tissues from RA and osteoarthritis (OA) patients was examined by immunohistochemical and double immunofluorescence analysis. FLSs were isolated from RA and OA patients and treated with Fn or cFn. Apoptosis was detected by flow cytometry and TUNEL assay. The expression of survivin, caspase-3, cyclin-B1, Bcl-2 and Bax was detected by real-time PCR. The secretion of proinflammatory cytokines was measured by ELISA.

Results

Fn formed extracellular aggregates that were specifically citrullinated in synovial tissues of RA patients, but no Fn deposits were observed in those of OA patients. Fn induced the apoptosis of RA and OA FLSs while cFn inhibited the apoptosis of RA and OA FLSs. Fn significantly increased the expression of caspase-3 and decreased the expression of survivin and cyclin-B1 in FLSs from RA and OA patients. cFn significantly increased the expression of survivin in RA FLSs. Furthermore, cFn increased the secretion of TNF-α and IL-1 by FLSs.

Conclusions

cFn plays a potential pathophysiologic role in RA by inhibiting apoptosis and increasing proinflammatory cytokine secretion of FLSs.     

Chemerin activates fibroblast-like synoviocytes in patients with rheumatoid arthritis

Introduction

Chemerin is a chemotactic agonist identified as a ligand for ChemR23 that is expressed on macrophages and dendritic cells (DCs). In this study, we analyzed the expression of chemerin and ChemR23 in the synovium of rheumatoid arthritis (RA) patients and the stimulatory effects of chemerin on fibroblast-like synoviocytes (FLSs) from RA patients.

Methods

Chemerin and ChemR23 expression in the RA synovium was ascertained by immunohistochemistry and Western blot analysis. Chemerin expression on cultured FLSs was analyzed by ELISA. ChemR23 expression on FLSs was determined by immunocytochemistry and Western blot analysis. Cytokine production from FLSs was measured by ELISA. FLS cell motility was evaluated by utilizing a scrape motility assay. We also examined the stimulating effect of chemerin on the phosphorylation of mitogen-activated protein kinase (MAPK), p44/42 mitogen-activated protein kinase (ERK1/2), p38MAPK, c-Jun N-terminal kinase (JNK)1/2 and Akt, as well as on the degradation of regulator of NF-κB (IκBα) in FLSs, by Western blot analysis.

Results

Chemerin was expressed on endothelial cells and synovial lining and sublining cells. ChemR23 was expressed on macrophages, immature DCs and FLSs and a few mature DCs in the RA synovium. Chemerin and ChemR23 were highly expressed in the RA synovium compared with osteoarthritis. Chemerin and ChemR23 were expressed on unstimulated FLSs. TNF-α and IFN-γ upregulated chemerin production. Chemerin enhanced the production of IL-6, chemokine (C-C motif) ligand 2 and matrix metalloproteinase 3 by FLSs, as well as increasing FLS motility. The stimulatory effects of chemerin on FLSs were mediated by activation of ERK1/2, p38MAPK and Akt, but not by JNK1/2. Degradation of IκB in FLSs was not promoted by chemerin stimulation. Inhibition of the ERK1/2, p38MAPK and Akt signaling pathways significantly suppressed chemerin-induced IL-6 production. Moreover, blockade of the p38MAPK and Akt pathways, but not the ERK1/2 pathway, inhibited chemerin-enhanced cell motility.

Conclusions

The interaction of chemerin and ChemR23 may play an important role in the pathogenesis of RA through the activation of FLSs.     

Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins

Introduction  

Rheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients.     

Long noncoding RNA MEG3 regulates rheumatoid arthritis by targeting NLRC5

Rheumatoid arthritis (RA) is one of the chronic systemic autoimmune diseases that cardinally affect the joints. Many people all over the world suffer from the disease. Fibroblast-like synoviocytes (FLSs) play a significant role in the occurrence and development of RA. The long noncoding RNA maternally expressed gene 3 (MEG3) is an imprinted gene, which participates in various cancers as a tumor suppressor. Previous studies have shown that nucleotide oligomerization domain (NOD)-like receptors 5 (NLRC5) plays a key role in inflammatory and autoimmune diseases. Nonetheless, we know very little about the biofunctionality of MEG3 during the development of RA. In this paper, we used complete Freund's adjuvant (CFA)-induced rats as RA animal models. The level of MEG3 significantly reduced in CFA-induced synovial tissues and FLSs, whereas the NLRC5 levels were increased. Enforced expression of MEG3 may be responsible for the decreased level of NLRC5 and inflammatory cytokine level. The results of methylation-specific PCR suggested that the MEG3 gene promoter was significantly methylated in CFA-induced synovial tissues and FLSs. More important, hypermethylation of MEG3 promoter could be inhibited by 5-aza-2-deoxycytidine (5-azadC; methylation inhibitor). Besides, the expression of NLRC5 significantly decreased followed by 5-azadc. Furthermore, DNA methyltransferases 1 (DNMT1) increased in CFA-induced synovial tissues and cells. These results indicated that MEG3 regulates RA by targeting NLRC5 potentially.     

MLN51 and GM-CSF involvement in the proliferation of fibroblast-like synoviocytes in the pathogenesis of rheumatoid arthritis

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease of unclear etiology. This study was conducted to identify critical factors involved in the synovial hyperplasia in RA pathology. We applied cDNA microarray analysis to profile the gene expressions of RA fibroblast-like synoviocytes (FLSs) from patients with RA. We found that the MLN51 (metastatic lymph node 51) gene, identified in breast cancer, is remarkably upregulated in the hyperactive RA FLSs. However, growth-retarded RA FLSs passaged in vitro expressed small quantities of MLN51. MLN51 expression was significantly enhanced in the FLSs when the growth-retarded FLSs were treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or synovial fluid (SF). Anti-GM-CSF neutralizing antibody blocked the MLN51 expression even though the FLSs were cultured in the presence of SF. In contrast, GM-CSF in SFs existed at a significant level in the patients with RA (n = 6), in comparison with the other inflammatory cytokines, IL-1beta and TNF-alpha. Most RA FLSs at passage 10 or more recovered from their growth retardation when cultured in the presence of SF. The SF-mediated growth recovery was markedly impaired by anti-GM-CSF antibody. Growth-retarded RA FLSs recovered their proliferative capacity after treatment with GM-CSF in a dose-dependent manner. However, MLN51 knock-down by siRNA completely blocked the GM-CSF/SF-mediated proliferation of RA FLSs. Taken together, our results imply that MLN51, induced by GM-CSF, is important in the proliferation of RA FLSs in the pathogenesis of RA.     

Detection of autoantibodies to citrullinated BiP in rheumatoid arthritis patients and pro-inflammatory role of citrullinated BiP in collagen-induced arthritis

Introduction

Anti-citrullinated protein/peptide antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA) patients and are thought to have a close relationship with the pathogenesis of arthritis. Several proteins, including fibrinogen, vimentin, and alpha-enolase, were reported as ACPA-target antigens, and their importance in RA pathogenesis was widely proposed. We identified citrullinated immunoglobulin binding protein (citBiP) as another ACPA target in RA patients and examined its pro-inflammatory role in arthritis.

Methods

We measured the levels of anti-citBiP, anti-BiP, and anti-cyclic citrullinated peptide (CCP) antibodies in the serum of RA patients (n = 100), systemic lupus erythematosus (SLE) patients (n = 60), and healthy controls (n = 30) using ELISA and immunoblotting. Epitope mapping was performed using 27 citBiP-derived peptides. In the mouse study, after DBA/1J mice were immunized with BiP or citBiP, serum titers of ACPAs were measured by ELISA and immunohistochemistry. The development of collagen-induced arthritis (CIA) was observed in BiP- or citBiP-pre-immunized mice.

Results

The serum levels of anti-BiP and anti-citBiP antibodies were significantly increased in RA patients, although only anti-BiP antibodies were slightly increased in SLE patients. Interestingly, anti-citBiP antibody levels were higher than anti-BiP antibody levels in 72% of RA patients, whereas no significant increase in anti-citBiP antibody levels was detected in SLE patients and healthy controls. The serum levels of anti-CCP antibodies were correlated with those of anti-citBiP antibodies in RA patients (R² = 0.41). Several citrulline residues of citBiP were determined to be major epitopes of anti-citBiP antibodies, one of which showed cross-reactivity with CCP. Immunization of DBA/1J mice with citBiP induced several kinds of ACPAs, including anti-CCP and anti-citrullinated fibrinogen antibodies. Pre-immunization with citBiP exacerbated CIA, and anti-CCP antibody levels were increased in citBiP-pre-immunized CIA mice.

Conclusions

CitBiP is a newly described ACPA target that may play a pro-inflammatory role in arthritis.     

Elevated levels of fibrinogen-derived endogenous citrullinated peptides in synovial fluid of rheumatoid arthritis patients

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients.

Methods

Endogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry.

Results

Our data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints.

Conclusions

The increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.     

FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis in rheumatoid arthritis

Introduction

The FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis plays a fundamental role in proliferation and differentiation of dendritic cells (DCs). As DCs play an important role in rheumatoid arthritis (RA) immunopathology we studied in detail the Flt3L/CD135 axis in RA patients.

Methods

The levels of Flt3L in (paired) serum and synovial fluid (SF) were quantified by enzyme-link immunosorbent assay (ELISA). Expression of Flt3L and CD135 in paired peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) was quantified by fluorescence-activated cell sorting (FACS). The expression of Flt3L, CD135 and TNF-Converting Enzyme (TACE) in synovial tissues (STs) and in vitro polarized macrophages and monocyte-derived DCs (Mo-DCs) was assessed by quantitative PCR (qPCR). CD135 ST expression was evaluated by immunohistochemistry and TACE ST expression was assessed by immunofluorescence. Flt3L serum levels were assessed in RA patients treated with oral prednisolone or adalimumab.

Results

Flt3L levels in RA serum, SF and ST were significantly elevated compared to gout patients and healthy individuals (HI). RA SF monocytes, natural killer cells and DCs expressed high levels of Flt3L and CD135 compared to HI. RA ST CD68⁺ and CD163⁺ macrophages, CD55⁺ fibroblast-like synoviocytes (FLS), CD31⁺ endothelial cells or infiltrating monocytes and CD19⁺ B cells co-expressed TACE. IFN-γ-differentiated macrophages expressed higher levels of Flt3L compared to other polarized macrophages. Importantly, Flt3L serum levels were reduced by effective therapy.

Conclusions

The Flt3L/CD135 axis is active in RA patients and is responsive to both prednisolone and adalimumab treatment. Conceivably, this ligand receptor pair represents a novel therapeutic target.     

Medication persistence over 2 years of follow-up in a cohort of early rheumatoid arthritis patients: associated factors and relationship with disease activity and with disability

Introduction

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumour necrosis factor (TNF) family member capable of inducing apoptosis in many cell types.

Methods

Using immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and real-time PCR we investigated the expression of TRAIL, TRAIL receptors and several key molecules of the intracellular apoptotic pathway in human synovial tissues from various types of arthritis and normal controls. Synovial tissues from patients with active rheumatoid arthritis (RA), inactive RA, osteoarthritis (OA) or spondyloarthritis (SpA) and normal individuals were studied.

Results

Significantly higher levels of TRAIL, TRAIL R1, TRAIL R2 and TRAIL R4 were observed in synovial tissues from patients with active RA compared with normal controls (p < 0.05). TRAIL, TRAIL R1 and TRAIL R4 were expressed by many of the cells expressing CD68 (macrophages). Lower levels of TUNEL but higher levels of cleaved caspase-3 staining were detected in tissue from active RA compared with inactive RA patients (p < 0.05). Higher levels of survivin and x-linked inhibitor of apoptosis protein (xIAP) were expressed in active RA synovial tissues compared with inactive RA observed at both the protein and mRNA levels.

Conclusions

This study indicates that the induction of apoptosis in active RA synovial tissues is inhibited despite stimulation of the intracellular pathway(s) that lead to apoptosis. This inhibition of apoptosis was observed downstream of caspase-3 and may involve the caspase-3 inhibitors, survivin and xIAP.     

Activation of fibroblast-like synoviocytes derived from rheumatoid arthritis via lysophosphatidic acid–lysophosphatidic acid receptor 1 cascade

Introduction

Lysophosphatidic acid (LPA) is a bioactive lipid that binds to G protein–coupled receptors (LPA_1–6). Recently, we reported that abrogation of LPA receptor 1 (LPA₁) ameliorated murine collagen-induced arthritis, probably via inhibition of inflammatory cell migration, Th17 differentiation and osteoclastogenesis. In this study, we examined the importance of the LPA–LPA₁ axis in cell proliferation, cytokine/chemokine production and lymphocyte transmigration in fibroblast-like synoviocytes (FLSs) obtained from the synovial tissues of rheumatoid arthritis (RA) patients.

Methods

FLSs were prepared from synovial tissues of RA patients. Expression of LPA_1–6 was examined by quantitative real-time RT-PCR. Cell surface LPA₁ expression was analyzed by flow cytometry. Cell proliferation was analyzed using a cell-counting kit. Production of interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), chemokine (C-C motif) ligand 2 (CCL2), metalloproteinase 3 (MMP-3) and chemokine (C-X-C motif) ligand 12 (CXCL12) was measured by enzyme-linked immunosorbent assay. Pseudoemperipolesis was evaluated using a coculture of RA FLSs and T or B cells. Cell motility was examined by scrape motility assay. Expression of adhesion molecules was determined by flow cytometry.

Results

The expression of LPA₁ mRNA and cell surface LPA₁ was higher in RA FLSs than in FLSs from osteoarthritis tissue. Stimulation with LPA enhanced the proliferation of RA FLSs and the production of IL-6, VEGF, CCL2 and MMP-3 by FLSs, which were suppressed by an LPA₁ inhibitor (LA-01). Ki16425, another LPA₁ antagonist, also suppressed IL-6 production by LPA-stimulated RA FLSs. However, the production of CXCL12 was not altered by stimulation with LPA. LPA induced the pseudoemperipolesis of T and B cells cocultured with RA FLSs, which was suppressed by LPA₁ inhibition. In addition, LPA enhanced the migration of RA FLSs and expression of vascular cell adhesion molecule and intercellular adhesion molecule on RA FLSs, which were also inhibited by an LPA₁ antagonist.

Conclusions

Collectively, these results indicate that LPA–LPA₁ signaling contributes to the activation of RA FLSs.     

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.     

Characterization and Localization of Citrullinated Proteoglycan Aggrecan in Human Articular Cartilage

Background

Rheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA.

Methods

CitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry.

Findings

Sera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage.

Conclusions

CitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.     

Transition of healthy to diseased synovial tissue in rheumatoid arthritis is associated with gain of mesenchymal/fibrotic characteristics

The healthy synovial lining layer consists of a single cell layer that regulates the transport between the joint cavity and the surrounding tissue. It has been suggested that abnormalities such as somatic mutations in the p53 tumor-suppressor gene contribute to synovial hyperplasia and invasion in rheumatoid arthritis (RA). In this study, expression of epithelial markers on healthy and diseased synovial lining tissue was examined. In addition, we investigated whether a regulated process, resembling epithelial to mesenchymal transition (EMT)/fibrosis, could be responsible for the altered phenotype of the synovial lining layer in RA. Synovial tissue from healthy subjects and RA patients was obtained during arthroscopy. To detect signs of EMT, expression of E-cadherin (epithelial marker), collagen type IV (indicator of the presence of a basement membrane) and alpha-smooth muscle actin (alpha-sma; a myofibroblast marker) was investigated on frozen tissue sections using immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from healthy subjects were isolated and subjected to stimulation with synovial fluid (SF) from two RA patients and to transforming growth factor (TGF)-beta. To detect whether EMT/fibrotic markers were increased, expression of collagen type I, alpha-sma and telopeptide lysylhydroxylase (TLH) was measured by real time PCR. Expression of E-cadherin and collagen type IV was found in healthy and arthritic synovial tissue. Expression of alpha-sma was only found in the synovial lining layer of RA patients. Stimulation of healthy FLSs with SF resulted in an upregulation of alpha-sma and TLH mRNA. Collagen type I and TLH mRNA were upregulated after stimulation with TGF-beta. Addition of bone morphogenetic protein (BMP)-7 to healthy FLS stimulated with SF inhibited the expression of alpha-sma mRNA. The finding that E-cadherin and collagen type IV are expressed in the lining layer of healthy and arthritic synovium indicates that these lining cells display an epithelial-like phenotype. In addition, the presence of alpha-sma in the synovial lining layer of RA patients and induction of fibrotic markers in healthy FLSs by SF from RA patients indicate that a regulated process comparable to EMT might cause the alteration in phenotype of RA FLSs. Therefore, BMP-7 may represent a promising agent to counteract the transition imposed on synoviocytes in the RA joint.     

The involvement of follistatin-like protein 1 in osteoarthritis by elevating NF-κB-mediated inflammatory cytokines and enhancing fibroblast like synoviocyte proliferation

IntroductionOur previous work has revealed that expression of follistatin-like protein 1 (FSTL1) is elevated in the synovial tissues from osteoarthritis (OA) patients. The aim of this study was to elucidate the underlying molecular mechanisms by which FSTL1 plays a role in the pathogenesis of OA.MethodsCultured fibroblast-like synoviocytes (FLSs) from synovial tissues of OA patients were stimulated with human recombinant FSTL1, and then the expression of inflammatory cytokines in FLS and their concentrations in the cell supernatants were measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Nuclear factor kappa B (NF-κB) activation was examined by western blot and chromatin immunoprecipitation (ChIP) assay at the p65 binding site. Finally, the proliferation of FLSs and the expression level of the proliferation-related tumor suppressors (p53 and p21) were determined by MTS assay kit and western blot in the presence or absence of FSTL1, respectively.ResultsFSTL1 remarkably promoted expression levels of several inflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6)) in vitro. Western blot analysis showed that FSTL1 activated the inflammatory-related NF-κB signaling pathway, as validated by ChIP assay detecting p65-binding level on the cytokine promoter region. Moreover, FSTL1 promoted the proliferation of OA FLS by downregulating the expression of p53 and p21. Interestingly, the concentration of synovial fluid IL-6 was remarkably elevated in OA patients, and was correlated with synovial fluid and serum FSTL1 levels.ConclusionsThese findings show that FSTL1 functions as an important proinflammatory factor in the pathogenesis of OA by activating the canonical NF-κB pathway and enhancing synoviocytes proliferation, suggesting that FSTL1 may be a promising target for the treatment of OA.     

c-Fms-mediated differentiation and priming of monocyte lineage cells play a central role in autoimmune arthritis

Introduction

Tyrosine kinases are key mediators of multiple signaling pathways implicated in rheumatoid arthritis (RA). We previously demonstrated that imatinib mesylate--a Food and Drug Administration (FDA)-approved, antineoplastic drug that potently inhibits the tyrosine kinases Abl, c-Kit, platelet-derived growth factor receptor (PDGFR), and c-Fms--ameliorates murine autoimmune arthritis. However, which of the imatinib-targeted kinases is the principal culprit in disease pathogenesis remains unknown. Here we examine the role of c-Fms in autoimmune arthritis.

Methods

We tested the therapeutic efficacy of orally administered imatinib or GW2580, a small molecule that specifically inhibits c-Fms, in three mouse models of RA: collagen-induced arthritis (CIA), anti-collagen antibody-induced arthritis (CAIA), and K/BxN serum transfer-induced arthritis (K/BxN). Efficacy was evaluated by visual scoring of arthritis severity, paw thickness measurements, and histological analysis. We assessed the in vivo effects of imatinib and GW2580 on macrophage infiltration of synovial joints in CIA, and their in vitro effects on macrophage and osteoclast differentiation, and on osteoclast-mediated bone resorption. Further, we determined the effects of imatinib and GW2580 on the ability of macrophage colony-stimulating factor (M-CSF; the ligand for c-Fms) to prime bone marrow-derived macrophages to produce tumor necrosis factor (TNF) upon subsequent Fc receptor ligation. Finally, we measured M-CSF levels in synovial fluid from patients with RA, osteoarthritis (OA), or psoriatic arthritis (PsA), and levels of total and phosphorylated c-Fms in synovial tissue from patients with RA.

Results

GW2580 was as efficacious as imatinib in reducing arthritis severity in CIA, CAIA, and K/BxN models of RA. Specific inhibition of c-Fms abrogated (i) infiltration of macrophages into synovial joints of arthritic mice; (ii) differentiation of monocytes into macrophages and osteoclasts; (iii) osteoclast-mediated bone resorption; and (iv) priming of macrophages to produce TNF upon Fc receptor stimulation, an important trigger of synovitis in RA. Expression and activation of c-Fms in RA synovium were high, and levels of M-CSF were higher in RA synovial fluid than in OA or PsA synovial fluid.

Conclusions

These results suggest that c-Fms plays a central role in the pathogenesis of RA by mediating the differentiation and priming of monocyte lineage cells. Therapeutic targeting of c-Fms could provide benefit in RA.