TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.     

Rheumatoid arthritis synovial stromal cells inhibit apoptosis and up-regulate Bcl-xL expression by B cells in a CD49/CD29-CD106-dependent mechanism

Inflammatory sites, such as rheumatoid arthritis (RA) synovial tissue, contain large numbers of activated B cells and plasma cells. However, the mechanisms maintaining B cell viability and promoting their differentiation are not known, but interactions with stromal cells may play a role. To examine this, purified human peripheral B cells were cultured with a stromal cell line (SCL) derived from RA synovial tissue, and the effects on apoptosis and expression of Bcl-2-related proteins were analyzed. As a control, B cells were also cultured with SCL from osteoarthritis synovium or skin fibroblasts. B cells cultured with medium alone underwent spontaneous apoptosis. However, B cells cultured with RA SCL cells exhibited less apoptosis and greater viability. Although SCL from osteoarthritis synovium and skin fibroblasts also rescued B cells from apoptosis, they were less effective than RA SCL. B cell expression of Bcl-xL was markedly increased by RA SCL in a contact-dependent manner, whereas B cell expression of Bcl-2 was unaffected. Protection of B cells from apoptosis and up-regulation of Bcl-xL by RA SCL were both blocked by mAbs to CD106 (VCAM-1), but not CD54 (ICAM-1). Furthermore, cross-linking of CD49d/CD29 (very late Ag-4) on the surface of B cells rescued them from apoptosis and up-regulated Bcl-xL expression. These results indicate that SCL derived from RA synovial tissue play a role in promoting B cell survival by inducing Bcl-xL expression and blocking B cell apoptosis in a CD49d/CD29-CD106-dependent manner.     

Inhibition of epidermal growth factor receptor tyrosine kinase ameliorates collagen-induced arthritis

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the formation of pannus and the destruction of cartilage and bone in the synovial joints. Although immune cells, which infiltrate the pannus and promote inflammation, play a prominent role in the pathogenesis of RA, other cell types also contribute. Proliferation of synovial fibroblasts, for example, underlies the formation of the pannus, while proliferation of endothelial cells results in neovascularization, which supports the growth of the pannus by supplying it with nutrients and oxygen. The synovial fibroblasts also promote inflammation in the synovium by producing cytokines and chemokines. Finally, osteoclasts cause the destruction of bone. In this study, we show that erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), reduces the severity of established collagen-induced arthritis, a mouse model of RA, and that it does so by targeting synovial fibroblasts, endothelial cells, and osteoclasts. Erlotinib-induced attenuation of autoimmune arthritis was associated with a reduction in number of osteoclasts and blood vessels, and erlotinib inhibited the formation of murine osteoclasts and the proliferation of human endothelial cells in vitro. Erlotinib also inhibited the proliferation and cytokine production of human synovial fibroblasts in vitro. Moreover, EGFR was highly expressed and activated in the synovium of mice with collagen-induced arthritis and patients with RA. Taken together, these findings suggest that EGFR plays a central role in the pathogenesis of RA and that EGFR inhibition may provide benefits in the treatment of RA.     

RANK ligand, RANK, and OPG expression in type II collagen-induced arthritis mouse

Rheumatoid arthritis (RA) is a systemic disorder characterized by synovial inflammation and subsequent destruction and deformity of synovial joints. The articular lesions start with synovitis, focal erosion of unmineralized cartilage, and then culminate in the destruction of subarticular bone by pannus tissue. Periarticular osteopenia and systemic osteoporosis follow as late complications of RA. Osteoclasts, specialized cells that resorb bone, play a central role in developing these osteolytic lesions. To elucidate the mechanism of osteoclastogenesis and bone destruction in autoimmune arthritis, we investigated the expression of RANK ligand (RANKL), RANK, and osteoprotegerin (OPG) mRNA in a mouse type II collagen-induced arthritis (CIA) model by in situ hybridization. The results indicated that most of the TRAP-positive mono- and multinucleated cells in the inflamed and proliferating synovium and in the pannus were RANK-positive authentic osteoclasts and their precursors. In the inflamed synovium and pannus of the mouse CIA model, synovial fibroblastic cells around these RANK-positive cells were strongly positive for RANKL. Moreover, RANKL-positive osteoblasts on the endosteal bone surface, at a distance from the affected synovial joints, increased significantly in the mouse CIA model prior to periarticular osteopenia and systemic osteoporosis. These data indicated that the RANKL-RANK system plays an important role for osteoclastogenesis in both local and systemic osteolytic lesions in autoimmune arthritis, and can therefore be a good target for therapeutic intervention.     

Role of C-reactive protein in osteoclastogenesis in rheumatoid arthritis

IntroductionC-reactive protein (CRP) is one of the biomarkers for the diagnosis and assessment of disease activity in rheumatoid arthritis (RA). CRP is not only the by-product of inflammatory response, but also plays proinflammatory and prothrombotic roles. The aim of this study was to determine the role of CRP on bone destruction in RA.MethodsCRP levels in RA synovial fluid (SF) and serum were measured using the immunoturbidimetric method. The expression of CRP in RA synovium was assessed using immunohistochemical staining. CD14+ monocytes from peripheral blood were cultured with CRP, and receptor activator of nuclear factor-κB ligand (RANKL) expression and osteoclast differentiation were evaluated using real-time PCR, counting tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and assessing bone resorbing function. CRP-induced osteoclast differentiation was also examined after inhibition of Fcγ receptors.ResultsThere was a significant correlation between CRP levels in serum and SF in RA patients. The SF CRP level was correlated with interleukin (IL)-6 levels, but not with RANKL levels. Immunohistochemical staining revealed that compared with the osteoarthritis synovium, CRP was more abundantly expressed in the lining and sublining areas of the RA synovium. CRP stimulated RANKL production in monocytes and it induced osteoclast differentiation from monocytes and bone resorption in the absence of RANKL.ConclusionsCRP could play an important role in the bony destructive process in RA through the induction of RANKL expression and direct differentiation of osteoclast precursors into mature osteoclasts. In the treatment of RA, lowering CRP levels is a significant parameter not only for improving disease activity but also for preventing bone destruction.     

Detection of high levels of heparin binding growth factor-1 (acidic fibroblast growth factor) in inflammatory arthritic joints

The synovium from patients with rheumatoid arthritis (RA) and LEW/N rats with streptococcal cell wall (SCW) arthritis, an experimental model resembling RA, is characterized by massive proliferation of synovial connective tissues and invasive destruction of periarticular bone and cartilage. Since heparin binding growth factor (HBGF)-1, the precursor of acidic fibroblast growth factor (FGF), is a potent angiogenic polypeptide and mitogen for mesenchymal cells, we sought evidence that it was involved in the synovial pathology of RA and SCW arthritis. HBGF-1 mRNA was detected in RA synovium using the polymerase chain reaction technique, and its product was immunolocalized intracellularly in both RA and osteoarthritis (OA) synovium. HBGF-1 staining was more extensive and intense in synovium of RA patients than OA and correlated with the extent and intensity of synovial mononuclear cell infiltration. HBGF-1 staining also correlated with c-Fos protein staining. In SCW arthritis, HBGF-1 immunostaining was noted in bone marrow, bone, cartilage, synovium, ligamentous and tendinous structures, as well as various dermal structures and developed early in both T-cell competent and incompetent rats. Persistent high level immunostaining of HBGF-1 was only noted in T-cell competent rats like the disease process in general. These observations implicate HBGF-1 in a multitude of biological functions in inflammatory joint diseases.     

Elimination of rheumatoid synovium in situ using a Fas ligand 'gene scalpel'

Surgical synovectomy to remove the inflammatory synovium can temporarily ameliorate rheumatoid inflammation and delay the progress of joint destruction. An efficient medically induced programmed cell death (apoptosis) in the rheumatoid synovium might play a role similar to synovectomy but without surgical tissue damage. Gene transfer of Fas ligand (FasL) has increased the frequency of apoptotic cells in mouse and rabbit arthritic synovium. In this study, we investigated whether repeated FasL gene transfer could remove human inflammatory synovial tissue in situ and function as a molecular synovectomy. Briefly, specimens of human synovium from joint replacement surgeries and synovectomies of rheumatoid arthritis (RA) patients were grafted subcutaneously into male C.B-17 severe combined immunodeficiency (SCID) mice. Injections of a recombinant FasL adenovirus (Ad-FasL) into the grafted synovial tissue at the dosage of 10(11) particles per mouse were performed every two weeks. Three days after the fifth virus injection, the mice were euthanized by CO2 inhalation and the human synovial tissues were collected, weighed and further examined. Compared to the control adenovirus-LacZ (Ad-LacZ) and phosphate buffered saline (PBS) injected RA synovium, the Ad-FasL injected RA synovium was dramatically reduced in size and weight (P < 0.005). The number of both synoviocytes & mononuclear cells was significantly reduced. Interestingly, an approximate 15-fold increased frequency of apoptotic cells was observed in RA synovium three days after Ad-FasL injection, compared with control tissues. In summary, our in vivo investigation of gene transfer to human synovium in SCID mice suggests that repeated intra-articular gene transfer of an apoptosis inducer, such as FasL, may function as a 'gene scalpel' for molecular synovectomy to arrest inflammatory synovium at an early stage of RA.     

Inhibition of protein geranylgeranylation induces apoptosis in synovial fibroblasts

Statins, competitive inhibitors of hydroxymethylglutaryl-CoA reductase, have recently been shown to have a therapeutic effect in rheumatoid arthritis (RA). In RA, synovial fibroblasts in the synovial lining, are believed to be particularly important in the pathogenesis of disease because they recruit leukocytes into the synovium and secrete angiogenesis-promoting molecules and proteases that degrade extracellular matrix. In this study, we show a marked reduction in RA synovial fibroblast survival through the induction of apoptosis when the cells were cultured with statins. Simvastatin was more effective in RA synovial fibroblasts than atorvastatin, and both statins were more potent on tumor necrosis factor-α-induced cells. In contrast, in osteoarthritis synovial fibroblasts, neither the statin nor the activation state of the cell contributed to the efficacy of apoptosis induction. Viability of statin-treated cells could be rescued by geranylgeraniol but not by farnesol, suggesting a requirement for a geranylgeranylated protein for synovial fibroblast survival. Phase partitioning experiments confirmed that in the presence of statin, geranylgeranylated proteins are redistributed to the cytoplasm. siRNA experiments demonstrated a role for Rac1 in synovial fibroblast survival. Western blotting showed that the activated phosphorylated form of Akt, a protein previously implicated in RA synovial fibroblast survival, was decreased by about 75%. The results presented in this study lend further support to the importance of elevated pAkt levels to RA synovial fibroblast survival and suggest that statins might have a beneficial role in reducing the aberrant pAkt levels in patients with RA. The results may also partly explain the therapeutic effect of atorvastatin in patients with RA.     

Regulation of osteoprotegerin secretion from primary cultures of human bone marrow stromal cells

Osteoprotegerin (OPG) is a soluble receptor for receptor activator of NF kappa B-ligand, a factor required for osteoclastogenesis. OPG secreted from bone marrow stromal cells is believed to inhibit osteoclast differentiation and several agents known to influence bone resorption have been demonstrated to regulate mRNA levels of OPG. In this report we have investigated the secretion of OPG protein from primary cultures of human bone marrow stromal cells. An ELISA was developed for measuring the concentration of OPG in culture medium. OPG secretion was decreased by 50% when the human bone marrow stromal cells were treated with 1 microM of prostaglandin E(2), possibly through activation of the protein kinase A-pathway since stimulation of protein kinase A by forskolin also inhibited OPG secretion. Treatment with phorbol 12,13 di butyrate, an activator of the protein kinase C-pathway, potently stimulated the secretion of OPG from human bone marrow stromal cells. The cells were also stimulated with inflammatory mediators and glucocorticoids. Treatment with interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) stimulated OPG secretion to 500% and 400% of control whereas dexamethasone decreased OPG production by 40%. In conclusion, an ELISA measuring OPG in cell culture media was developed. Using this ELISA, the amount of OPG secreted from human bone marrow stromal cells was clearly detectable, and the secretion of OPG-protein was potently regulated by prostaglandin E(2), forskolin, phorbol 12,13 di butyrate, IL-1 alpha, TNF-alpha, and dexamethasone.     

A novel therapy of murine collagen-induced arthritis with soluble T1/ST2

Rheumatoid arthritis is characterized by chronic inflammatory infiltration of the synovium, leading to eventual cartilage and bone destruction. Previously, we have reported that soluble T1/ST2 (sST2), a member of the IL-1R gene family, inhibits LPS-induced macrophage proinflammatory cytokine production. In this study, we report the therapeutic effect of sST2-Fc in the murine model of collagen-induced arthritis. A short term administration of sST2-Fc fusion protein significantly attenuated disease severity compared with controls treated with normal IgG. Histological examination revealed that while control IgG-treated mice developed severe cellular infiltration in the joints, synovial hyperplasia, and joint erosion, this pathology was profoundly reduced in sST2-Fc-treated animals. Treatment of sST2-Fc also down-regulated serum levels of IL-6, IL-12, and TNF-alpha. Spleen cells from the sST2-Fc-treated mice produced significantly less IFN-gamma, TNF-alpha, IL-6, and IL-12 compared with cells from the control mice when cultured with collagen in vitro. Finally, pretreatment with ST2-Fc markedly inhibited the ability of human monocytic THP1 cells to release TNF-alpha when cocultured with peripheral blood T cells from rheumatoid patients. Together these results demonstrate that sST2-Fc may provide a novel approach in treating chronic autoimmune conditions by inhibiting the release of proinflammatory cytokines.     

Characterization and functional consequences of underexpression of clusterin in rheumatoid arthritis

We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IkappaBalpha. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-kappaB activation and survival of the synoviocytes.     

Susceptibility of rheumatoid arthritis synovial fibroblasts to FasL- and TRAIL-induced apoptosis is cell cycle-dependent

Introduction  

The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro.     

Geldanamycin induces apoptosis and inhibits inflammation in fibroblast-like synoviocytes isolated from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease of the joints characterized by synovial hyperplasia and chronic inflammation. Fibroblasts-like synoviocytes (FLS), major cells in the synovium, together with infiltrated leukocytes, contribute greatly to RA progression. In our study, we hypothesized that geldanamycin (GA), a cancer drug might be able to inhibit RA FLS growth. To test the idea, RA FLS were isolated and cultured for cancer drug test. The results showed that GA can specifically inhibit the growth of RA FLS compared with normal FLS. Essentially, GA was found to promote reactive oxygen species production in RA FLS and induce programmed cell death. The annexinV/propidium iodide and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining confirmed that GA can directly induce apoptosis and subsequently inhibit the growth of RA FLS, which was also confirmed by Western blot assay. In addition, our data demonstrated that inflammation was inhibited by suppressed nuclear factor κB signaling pathway. The therapeutic effect of GA was explored in collagen-induced arthritis mice. In short, GA was a promising drug for the treatment of RA by specifically inhibiting the proliferation and inflammation of RA FLS.     

Expression of CD147 on monocytes/macrophages in rheumatoid arthritis: its potential role in monocyte accumulation and matrix metalloproteinase production

Monocytes/macrophages play an important role in rheumatoid arthritis (RA) pathogenesis. They can activate fibroblasts through many molecules, including IL-1 and tumor necrosis factor-alpha, but there have been very few reports on the role of CD147 in RA. In our study, the results of flow cytometry reveal that the mean fluorescence intensity (MFI) of CD147 expression on CD14+ monocytes of peripheral blood from RA patients was higher than that in normal control and ankylosing spondylitis (AS) patients. The MFI of CD147 expression on the CD14+ monocytes in RA synovial fluid was higher than that in RA peripheral blood. Immunohistochemical staining shows that CD147 expression in RA synovium correlated with matrix metalloproteinase (MMP)-1 expression. A double immunofluorescent assay shows that CD147 was expressed on CD68+ cells in RA synovium. The potential role of CD147 in cyclophilin A (CyPA)-mediated cell migration was studied using a chemotaxis assay in vitro and it was found that the addition of anti-CD147 antibody or a CD147 antagonistic peptide significantly decreased the chemotactic index of the mononuclear cells. The role of CD147 in MMP production and cell invasion in vitro were studied through the co-culture of human CD14+ monocytes or monocytic line THP-1 cells and human fibroblasts, as well as by gel zymography and an invasion assay. Significantly elevated release and activation of MMP-9 and/or MMP-2 were seen in the co-culture of human monocytes/THP-1 cells and fibroblasts compared with cultures of the cells alone. An increased number of cells invading through the filters in the invasion assays was also observed in the co-cultured cells. The addition of CD147 antagonistic peptide had some inhibitory effect, not only on MMP production but also on cell invasion in the co-culture. Our study demonstrates that the increased expression of CD147 on monocytes/macrophages in RA may be responsible for elevated MMP secretion, cell invasion and CyPA-mediated cell migration into the joints, all of which may contribute to the cartilage and bone destruction of RA. These findings, together with a better understanding of CD147, CyPA and RA, will help in the development of innovative therapeutic interventions for RA.     

Involvement of thioredoxin reductase 1 in the regulation of redox balance and viability of rheumatoid synovial cells

Rheumatoid arthritis (RA), a chronic and systemic disease of unknown etiology, is characterized by hyperplasia of synovial cells, which ultimately lead to the destruction of cartilage and bone. To elucidate the molecular mechanisms that lead to RA, we analyzed synovial cells established from patients with RA by oligonucleotide microarrays. Gene expression profiles clearly suggested that oxidative stress is enhanced in RA synovial cells, which was confirmed by measuring cellular levels of reactive oxygen species. One of the highly up-regulated proteins in RA synovial cells was thioredoxin reductase 1 (TRXR1), a protein that plays an important role in antioxidant defense system. Subsequent analysis demonstrated that TRXR1 suppresses hydrogen peroxide and inhibits apoptosis of RA synovial cells. Thus, our results reveal a novel pathophysiologic function of RA synovial cells as a generator of oxidative stress, and a self-defense mechanism against self-generated oxidative stress.     

Regulation of apoptosis and cell cycle activity in rheumatoid arthritis

The regulation of proliferation and cell death is vital for homeostasis, but the mechanisms that coordinately balances these two events in rheumatoid arthritis (RA) remains largely unknown. In RA, the synovial lining increases through enhanced proliferation, migration, and/or decreased cell death. The aberrant decrease in apoptosis or increased cell cycle activity of fibroblast-like or macrophage-like synoviocytes is responsible for the synovial hyperplasia and contributes to the destruction of cartilage and bone. Recently, numerous molecules that modulate apoptosis and cell cycle have been implicated to play a role in RA. This review will describe the current understanding of the molecular mechanisms that govern apoptosis and cell cycle and their relationship to RA pathogenesis.     

The role of T-cell interleukin-17 in conducting destructive arthritis: lessons from animal models

Interleukin-17 (IL-17) is a T cell cytokine spontaneously produced by cultures of rheumatoid arthritis (RA) synovial membranes. High levels have been detected in the synovial fluid of patients with RA. The trigger for IL-17 is not fully identified; however, IL-23 promotes the production of IL-17 and a strong correlation between IL-15 and IL-17 levels in synovial fluid has been observed. IL-17 is a potent inducer of various cytokines such as tumor necrosis factor (TNF)-alpha, IL-1, and receptor activator of NF-kappaB ligand (RANKL). Additive or even synergistic effects with IL-1 and TNF-alpha in inducing cytokine expression and joint damage have been shown in vitro and in vivo. This review describes the role of IL-17 in the pathogenesis of destructive arthritis with a major focus on studies in vivo in arthritis models. From these studies in vivo it can be concluded that IL-17 becomes significant when T cells are a major element of the arthritis process. Moreover, IL-17 has the capacity to induce joint destruction in an IL-1-independent manner and can bypass TNF-dependent arthritis. Anti-IL-17 cytokine therapy is of interest as an additional new anti-rheumatic strategy for RA, in particular in situations in which elevated IL-17 might attenuate the response to anti-TNF/anti-IL-1 therapy.