Myostatin induces tumor necrosis factor-α expression in rheumatoid arthritis synovial fibroblasts through the PI3K–Akt signaling pathway

In rheumatoid arthritis (RA), a chronic inflammatory disease, loss of muscle mass is an important contributor to the loss of muscle strength in RA patients. Myostatin, a myokine involved in the process of muscle hypertrophy and myogenesis, enhances osteoclast differentiation and inflammation. Here, we investigated the mechanisms of myostatin in RA synovial inflammation. We found a positive correlation between myostatin and tumor necrosis factor-α (TNF-α), a well-known proinflammatory cytokine, in RA synovial tissue. Our in vitro results also showed that myostatin dose-dependently induced TNF-α expression through the phosphatidylinositol 3-kinase (PI3K)–Akt–AP-1 signaling pathway. Myostatin treatment of human MH7A cells stimulated AP-1-induced luciferase activity and activation of the c-Jun binding site on the TNF-α promoter. Our results indicated that myostatin increases TNF-α expression via the PI3K–Akt–AP-1 signaling pathway in human RA synovial fibroblasts. Myostatin appears to be a promising target in RA therapy.     

Interleukin-18 induces serum amyloid A (SAA) protein production from rheumatoid synovial fibroblasts

Interleukin-18 (IL-18) is a novel proinflammatory cytokine that was recently found in synovial fluids and synovial tissues from patients with rheumatoid arthritis (RA). To investigate the role of IL-18 in rheumatoid synovitis, the levels of IL-18 and serum amyloid A (SAA) were measured in synovial fluids from 24 patients with rheumatoid arthritis (RA) and 13 patients with osteoarthritis (OA). The levels of IL-18 and SAA in the synovial fluids were elevated in RA patients. In contrast, the levels of IL-18 in synovial fluids from OA patients were significantly lower compared to those of RA patients. SAA was not detected in synovial fluids from OA patients. The expression of SAA mRNA in rheumatoid synovial cells was also examined. SAA4 mRNA, which was constitutively expressed by rheumatoid synovial cells, was not affected by IL-18 stimulation. Although acute phase SAA (A-SAA, SAA1 + 2) mRNA was not detected in unstimulated synovial cells, its expression was induced by IL-18 stimulation. By immunoblot, we demonstrated that IL-18 induced the SAA protein synthesis from rheumatoid synovial cells in a dose-dependent manner. These results indicate a novel role for IL-18 in rheumatoid inflammation through the synovial SAA production.     

The role of LAIR-1 (CD305) in T cells and monocytes/macrophages in patients with rheumatoid arthritis

The LAIR-1 receptor is expressed on a majority of mononuclear leukocytes. It is used as a biomarker when testing synovial fluid for evidence of rheumatoid arthritis (RA). The primary objective of this study was to measure T cell- and monocyte/macrophage-specific LAIR-1 expression in RA patients and compare this to LAIR-1 expression in osteoarthritis (OA) patients and healthy individuals. LAIR-1 expression was significantly decreased in circulating CD4⁺ T cells in RA patients compared to both OA patients and healthy individuals. In contrast, LAIR-1 is high in CD14⁺ monocytes and local CD68⁺ macrophages in synovial tissues from RA patients. Upon stimulation with TNF-α, LAIR-1 expression decreased in T-helper (Th)1 and Th2 CD4⁺ T cells from healthy donors. These results indicate that LAIR-1 may exert different functions on T cells and monocytes/macrophages and suggest that LAIR-1 may be a novel therapeutic target for the treatment of RA.     

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

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

Blockade of Toll-like receptor 2 prevents spontaneous cytokine release from rheumatoid arthritis ex vivo synovial explant cultures

Introduction

The aim of this study was to examine the effect of blocking Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA) synovial cells.

Methods

RA synovial tissue biopsies, obtained under direct visualization at arthroscopy, were established as synovial explant cultures ex vivo or snap frozen for immunohistology. Mononuclear cell cultures were isolated from peripheral blood and synovial fluid of RA patients. Cultures were incubated with the TLR1/2 ligand, Pam3CSK4 (200 ng, 1 and 10 μg/ml), an anti-TLR2 antibody (OPN301, 1 μg/ml) or an immunoglobulin G (IgG) (1 μg/ml) matched control. The comparative effect of OPN301 and adalimumab (anti-tumour necrosis factor alpha) on spontaneous release of proinflammatory cytokines from RA synovial explants was determined using quantitative cytokine MSD multiplex assays or ELISA. OPN301 penetration into RA synovial tissue explants cultures was assessed by immunohistology.

Results

Pam3CSK4 significantly upregulated interleukin (IL)-6 and IL-8 in RA peripheral blood mononuclear cells (PBMCs), RA synovial fluid mononuclear cells (SFMCs) and RA synovial explant cultures (P < 0.05). OPN301 significantly decreased Pam3CSK4-induced cytokine production of tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, interferon (IFN)-γ and IL-8 compared to IgG control in RA PBMCs and SFMCs cultures (all P < 0.05). OPN301 penetration of RA synovial tissue cultures was detected in the lining layer and perivascular regions. OPN301 significantly decreased spontaneous cytokine production of TNF-α, IL-1β, IFN-γ and IL-8 from RA synovial tissue explant cultures (all P < 0.05). Importantly, the inhibitory effect of OPN on spontaneous cytokine secretion was comparable to inhibition by anti-TNFα monoclonal antibody adalimumab.

Conclusions

These findings further support targeting TLR2 as a potential therapeutic agent for the treatment of RA.     

Implication of cytosolic phospholipase A2 (cPLA2) in the regulation of human synoviocyte NADPH oxidase (Nox2) activity

NADPH oxidase Nox2 is involved in the production of superoxide by rheumatoid synovial cells, constitutively and after pro-inflammatory cytokine treatment. The aims of the study were to evaluate the capacity of these cells to produce the superoxide anion in response to arachidonic acid (AA), and to study the involvement of cytosolic phospholipase A(2) (cPLA(2)) in the cytokine regulation of Nox2. Superoxide production was quantified in synovial cells obtained from six patients with rheumatoid arthritis (RA) and six with osteoarthritis (OA), stimulated with (i) AA, and (ii) PLA(2) inhibitors prior to IL-1beta or TNF-alpha treatment. Total cellular AA concentrations and PLA(2) activity were measured; effects of cytokines and NADPH oxidase inhibitors on the AA-activatable proton channel opening were also studied. Our results demonstrated that AA enhanced superoxide production in RA and OA cells; this production was significantly inhibited by iodonium diphenyl and apocynin. cPLA(2) inhibitors inhibited both IL-1beta and TNF-alpha-induced superoxide production in RA and OA cells. Basal PLA(2) activity was significantly more important in RA cells than in OA cells; PLA(2) activity was increased in IL-1beta and TNF-alpha pre-treated RA cells, and cPLA(2) inhibitors inhibited this activity. Opening of the AA-activatable proton channel was amplified when RA cells were pre-treated with both IL-1beta and TNF-alpha, and iodonium diphenyl and apocynin inhibited these cytokine effects. We concluded that AA is an important cofactor for synovial NADPH oxidase activity. Despite their direct effects on p47-phox phosphorylation, cytokines can also regulate the Nox2 activity though the AA-activatable associated H(+) channel.     

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

Introduction

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

Methods

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

Results

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

Conclusions

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

Discrimination of osteoarthritic and rheumatoid human synovial cells in culture by nuclear image analysis.

Rheumatoid arthritic (RA) and osteoarthritic (OA) synovial cells in culture differ in their metabolic and proliferative behaviour. To assess links between these properties and nuclear changes, we used image analysis to study chromatin texture, together with nuclear morphometry and densitometry of OA and RA cells in primary culture. Chromatin pattern at the third day (D3) was heterogeneous and granular with chromatin clumps whereas at the final stage (D11) of culture a homogeneous and finely granular chromatin texture was observed. This evolution indicates global chromatin decondensation. These characteristics were more marked for RA than for OA nuclei. At each culture time, RA nuclei could be discriminated with high confidence from OA ones from parameters evaluating the organization of the chromatine texture. Nuclear image analysis is thus a useful tool for investigating synovial cell biology.     

DHA and its derived lipid mediators MaR1, RvD1 and RvD2 block TNF-α inhibition of intestinal sugar and glutamine uptake in Caco-2 cells

Tumor necrosis factor-alfa (TNF-α) is a pro-inflammatory cytokine highly-involved in intestinal inflammation. Omega-3 polyunsaturated fatty acids (n3-PUFAs) show anti-inflammatory actions. We previously demonstrated that the n3-PUFA EPA prevents TNF-α inhibition of sugar uptake in Caco-2 cells. Here, we investigated whether the n3-PUFA DHA and its derived specialized pro-resolving lipid mediators (SPMs) MaR1, RvD1 and RvD2, could block TNF-α inhibition of intestinal sugar and glutamine uptake. DHA blocked TNF-α-induced inhibition of α-methyl-D-glucose (αMG) uptake and SGLT1 expression in the apical membrane of Caco-2 cells, through a pathway independent of GPR120. SPMs showed the same preventive effect but acting at concentrations 1000 times lower. In diet-induced obese (DIO) mice, oral gavage of MaR1 reversed the up-regulation of pro-inflammatory cytokines found in intestinal mucosa of these mice. However, MaR1 treatment was not able to counteract the reduced intestinal transport of αMG and SGLT1 expression in the DIO mice. In Caco-2 cells, TNF-α also inhibited glutamine uptake being this inhibition prevented by EPA, DHA and the DHA-derived SPMs. Interestingly, TNF-α increased the expression in the apical membrane of the glutamine transporter B⁰AT1. This increase was partially blocked by the n-3 PUFAs. These data reveal DHA and its SPMs as promising biomolecules to restore intestinal nutrients transport during intestinal inflammation.     

Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-κB ligand (RANKL) expression in rheumatoid arthritis

Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-κB ligand (RANKL) in RA synoviocytes and CD4(+) T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4(+) T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4(+) T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-α in MH7A cells and CD4(+) T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4(+) T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.     

Peripheral blood but not synovial fluid natural killer T cells are biased towards a Th1-like phenotype in rheumatoid arthritis

Natural killer T (NKT) cells have been implicated in the regulatory immune mechanisms that control autoimmunity. However, their precise role in the pathogenesis of rheumatoid arthritis (RA) remains unclear. The frequency, cytokine profile and heterogeneity of NKT cells were studied in peripheral blood mononuclear cells (PBMCs) from 23 RA patients and 22 healthy control individuals, including paired PBMC–synovial fluid samples from seven and paired PBMC–synovial tissue samples from four RA patients. Flow cytometry revealed a decreased frequency of NKT cells in PBMCs from RA patients. NKT cells were present in paired synovial fluid and synovial tissue samples. Based on the reactivity of PBMC-derived NKT cells toward α-galactosylceramide, RA patients could be divided into responders (53.8%) and nonresponders (46.2%). However, NKT cells isolated from synovial fluid from both responders and nonresponders expanded upon stimulation with α-galactosylceramide. Analysis of the cytokine profile of CD4⁺ and CD4^- PBMC derived NKT cell lines from RA patients revealed a significantly reduced number of IL-4 producing cells. In contrast, synovial fluid derived NKT cell lines exhibited a Th0-like phenotype, which was comparable to that in healthy control individuals. This suggests that synovial fluid NKT cells are functional, even in patients with nonresponding NKT cells in their blood. We conclude that, because the number of Vα24⁺Vβ11⁺CD3⁺ NKT cells is decreased and the cytokine profile of blood-derived NKT cells is biased toward a Th1-like phenotype in RA patients, NKT cells might be functionally related to resistance or progression of RA. Providing a local boost to the regulatory potential of NKT cells might represent a useful candidate therapy for RA.     

15-deoxy-Δ(12,14) -prostaglandin-J2 and ciglitazone inhibit TNF-α-induced matrix metalloproteinase 13 production via the antagonism of NF-κB activation in human synovial fibroblasts

Collagenase-3 (matrix metalloproteinase, MMP-13) plays an important role in the degradation of cartilage in pathologic conditions. MMP-13 is elevated in joint tissues in both rheumatoid arthritis (RA) and osteoarthritis (OA). In addition, inflammation-stimulated synovial fibroblasts are able to release MMP-13 and other cytokines in these diseases. The peroxisome proliferator-activated receptor-γ (PPARγ) ligands are recently considered as new anti-inflammatory compounds and these ligands were reported to ameliorate inflammatory arthritis. The aim of this study is to evaluate the mechanisms how PPARγ ligands inhibit the inflammatory response in synovial fibroblasts. Two PPARγ ligands, cyclopentenone prostaglandin 15-deoxy-Δ(12,14) -prostaglandin-J2 (15d-PGJ2) and synthetic thiazolidinedione compound ciglitazone were examined in this study. Here we found that 15d-PGJ2 and ciglitazone markedly inhibited TNF-α-induced MMP-13 production in human synovial fibroblasts. In addition, activation of nuclear factor κB (NF-κB) is strongly associated with MMP-13 induction by TNF-α and the activation of NF-κB was determined by Western blot, reporter assay, and immunofluorescence. It was found that 15d-PGJ2 markedly attenuated the translocation of NF-κB by direct inhibition of the activation of IKK via a PPARγ-independent manner. Ciglitazone also inhibits TNF-α-induced MMP-13 expression by suppressing NF-κB activation mainly via the modulation of p38-MAPK. Collectively, our data demonstrate that 15d-PGJ2 and ciglitazone attenuated TNF-α-induced MMP-13 expression in synovial fibroblasts primarily through the modulation of NF-κB signaling pathways. These compounds may have therapeutic application in inflammatory arthritis.     

Resting CD4+ effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function

Background

Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.

Methods

Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4⁺CD45RO⁺, CCR7^-, CD49d^high population, and that these cells are derived from the effector memory CD4⁺ T cells in resting blood.

Results

After stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-α production from monocytes stimulated with CD4⁺CD45RO⁺ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-α production in RA synovial mononuclear cell cultures.

Conclusion

Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.     

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

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

Mechanism of inhibitory effect of atorvastatin on resistin expression induced by tumor necrosis factor-α in macrophages

Atorvastatin has been shown to reduce resistin expression in macrophages after pro-inflammatory stimulation. However, the mechanism of reducing resistin expression by atorvastatin is not known. Therefore, we sought to investigate the molecular mechanisms of atorvastatin for reducing resistin expression after proinflammatory cytokine, tumor necrosis factor-α (TNF-α) stimulation in cultured macrophages. Cultured macrophages were obtained from human peripheral blood mononuclear cells. TNF-α stimulation increased resistin protein and mRNA expression and atorvastatin inhibited the induction of resistin by TNF-α. Addition of mevalonate induced resistin protein expression similar to TNF-α stimulation. However, atorvastatin did not have effect on resistin protein expression induced by mevalonate. SP600125 and JNK small interfering RNA (siRNA) completely attenuated the resistin protein expression induced by TNF-α and mevalonate. TNF-α induced phosphorylation of Rac, while atorvastatin and Rac-1 inhibitor inhibited the phosphorylation of Rac induced by TNF-α. The gel shift and promoter activity assay showed that TNF-α increased AP-1-binding activity and resistin promoter activity, while SP600125 and atorvastatin inhibited the AP-1-binding activity and resistin promoter activity induced by TNF-α. Recombinant resistin and TNF-α significantly reduced glucose uptake in cultured macrophages, while atorvastatin reversed the reduced glucose uptake by TNF-α. In conclusion, JNK and Rac pathway mediates the inhibitory effect of atorvastatin on resistin expression induced by TNF-α.