Rheumatoid Arthritis Fibroblasts Research Articles

STAT3-induced WNT5A signaling loop in embryonic stem cells, adult normal tissues, chronic persistent inflammation, rheumatoid arthritis and cancer (Review)

Leukemia inhibitory factor (LIF), oncostatin M, leptin, ciliary neurotrophic factor, cardiotrophin 1, cardiotrophin-like cytokine factor 1, interleukin 6 (IL6), interleukin 11 and interleukin 27 activate the gp130-JAK-STAT3 signaling cascade. Here, WNT5A was characterized as the evolutionarily conserved target of the STAT3 signaling cascade based on 11-bp-spaced tandem STAT3-binding sites within intron 4 of human, chimpanzee, cow, mouse and rat WNT5A orthologs. Canonical WNT5A signaling through Frizzled and LRP5/LRP6 receptors activates FGF20, WISP1, MYC and CCND1 transcription for the maintenance of stem/progenitor cells, while non-canonical WNT5A signaling through Frizzled and ROR2/PTK7/RYK receptors activates the RHOA, JNK, NLK and NFAT signaling cascades for the control of tissue polarity, cell adhesion or movement. LIF-induced Wnt5a activates canonical Wnt signaling in mouse embryonic stem cells for self-renewal. STAT3-induced Wnt5a activates non-canonical Wnt signaling in rat cardiac myocytes for N-cadherin-dependent aggregation. IL6, secreted from epithelial cells or macrophages, induces WNT5A upregulation in mesenchymal cells. WNT5A then activates canonical WNT signaling in epithelial cells. IL6-induced WNT5A activates canonical WNT signaling for autocrine proliferation of human synovial fibroblasts in rheumatoid arthritis. IL-6 signaling is activated during human chronic atrophic gastritis with Helicobacter pylori infection, and aberrant Stat3 signaling activation gives rise to mouse gastric tumors. WNT5A is frequently upregulated in human primary gastric cancer due to tumor-stromal interaction. WNT5A might be downregulated in advanced cancer with poorer prognosis due to genetic alterations compensating WNT5A signaling. Oncogenic WNT5A activates canonical WNT signaling in cancer stem cells for self-renewal, and non-canonical WNT signaling at the tumor-stromal interface for invasion and metastasis. SNP of genes encoding components of the cytokine-induced WNT5A signaling loop is a predicted risk factor for RA and cancer, especially diffuse-type gastric and pancreatic cancer. Humanized anti-IL6 receptor antibody and WNT5A mimetic small-molecule antagonist could be applied to personalized medicine for RA and cancer driven by the IL6-induced WNT5A signaling loop.

Expression and function of junctional adhesion molecule-C in human and experimental arthritis

Junctional adhesion molecule-C (JAM-C) is an adhesion molecule involved in transendothelial migration of leukocytes. In this study, we examined JAM-C expression in the synovium and investigated the role of this molecule in two experimental mouse models of arthritis. JAM-C expression was investigated by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of a monoclonal anti-JAM-C antibody were assessed in antigen-induced arthritis (AIA) and K/BxN serum transfer-induced arthritis. JAM-C was expressed by synovial fibroblasts in the lining layer and associated with vessels in the sublining layer in human and mouse arthritic synovial tissue. In human tissue, JAM-C expression was increased in rheumatoid arthritis (RA) as compared to osteoarthritis synovial samples (12.7 ± 1.3 arbitrary units in RA versus 3.3 ± 1.1 in OA; p < 0.05). Treatment of mice with a monoclonal anti-JAM-C antibody decreased the severity of AIA. Neutrophil infiltration into inflamed joints was selectively reduced as compared to T-lymphocyte and macrophage infiltration (0.8 ± 0.3 arbitrary units in anti-JAM-C-treated versus 2.3 ± 0.6 in isotype-matched control antibody-treated mice; p < 0.05). Circulating levels of the acute-phase protein serum amyloid A as well as antigen-specific and concanavalin A-induced spleen T-cell responses were significantly decreased in anti-JAM-C antibody-treated mice. In the serum transfer-induced arthritis model, treatment with the anti-JAM-C antibody delayed the onset of arthritis. JAM-C is highly expressed by synovial fibroblasts in RA. Treatment of mice with an anti-JAM-C antibody significantly reduced the severity of AIA and delayed the onset of serum transfer-induced arthritis, suggesting a role for JAM-C in the pathogenesis of arthritis.

Macrophage Migration Inhibitory Factor (MIF) Induced Stromal Cell-derived Factor 1 (SDF-1) Production Via Nuclear Factor KappaB (NF-κB) Signaling in Rheumatoid Arthritis Fibroblast Like Synoviocytes (RA-FLS)

Stromal cell-derived factor (SDF)-1 is a potent chemoattractant for activated T cells into the inflamed Rheumatoid arthritis (RA) synovium. To determine the effect of macrophage migration inhibitory factor (MIF) on the production of SDF-1 in the inflamed RA synovium. Methods: The expression of SDF-1 and MIF in RA and Osteoarthritis (OA) synovium was examined by immunohistochemical staining. The SDF-1 was quantified by RT-PCR and ELISA after RA fibroblast like synoviocyte (FLS) were treated with MIF in the presence and absence of inhibitors of intracellular signal molecules. The synovial fluid (SF) and serum levels of MIF and SDF-1 in RA, OA and healthy control were measured by ELISA. Results: Expression of SDF-1 and MIF in synovium was higher in RA patients than in OA patients. The production of SDF-1 was enhanced in RA FLS by MIF stimulation. Such effect of MIF was blocked by the inhibitors of NF-κB . Concentrations of SDF-1 in the serum and SF were higher in RA patients than in OA patients and healthy control. SDF-1 and MIF was overexpressed in RA FLS, and MIF could up-regulate the production of SDF-1 in RA FLS via NF-κB -mediated pathways. Conclusion: These results suggest that an inhibition of interaction between MIF from T cells and SDF-1 of FLS may provide a new therapeutic approach in the treatment of RA.

Toll-like receptor 2 ligand mediates the upregulation of angiogenic factor, vascular endothelial growth factor and interleukin-8/CXCL8 in human rheumatoid synovial fibroblasts

Rheumatoid arthritis (RA) is characterized by infiltrations of inflammatory cells accompanied by neovascularization in the joint. We hypothesized that cell activation via the toll-like receptor (TLR) may be involved in the induction of angiogenic molecules, which are relevant to the pathogenesis of RA. RA fibroblast like synoviocytes (FLS) were stimulated with TLR-2 ligand bacterial peptidoglycan (PGN), TLR-4 ligand lipopolysaccharide (LPS) and various cytokines. Vascular endothelial growth factor (VEGF) and IL-8 were measured by ELISA in culture supernatants; mRNA levels were assessed by RT-PCR and real time PCR. The levels of TLR-2, VEGF and IL-8 were analyzed by dual immunohistochemistry in RA synovium and compared with osteoarthritis (OA). Regulation of MyD88, IRAK4, IRAK1, IRAK-M and TRAF-6 mRNA expression levels by PGN were analyzed by RT-PCR. Phosphorylation of IκBα was evaluated by western blotting. Levels of VEGF and IL-8 were upregulated in culture supernatants of RA FLS stimulated with PGN, similar to the levels of IL-1β and IL-17 stimulation. Neutralization of TLR-2 with a blocking monoclonal antibody significantly reduced both VEGF and IL-8 levels ( P < 0.05), which reflected the functional relevance of TLR-2 activation to the induction of VEGF and IL-8 production. Downstream intracellular signaling following TLR-2 stimulation involved MyD88-IRAK-4-TRAF-6 pathways, resulting in NF-κB activation. Thus, TLR-2 activation in RA FLS by microbial constituents could be involved in the induction of VEGF and IL-8 and thereby promote inflammation either directly or via angiogenesis. This possibly contributes to the perpetuation of synovitis in patients with RA.

JAB1 Determines the Response of Rheumatoid Arthritis Synovial Fibroblasts to Tumor Necrosis Factor-α

Fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis (RA FLSs) exhibit prosurvival, rather than apoptotic, response to tumor necrosis factor (TNF)-alpha stimulation. Here, we show that JAB1 is a critical regulator of the TNF-alpha-mediated anti-apo-ptosis pathways in RA FLSs. We found that knockdown of JAB1 using small interfering (si)RNA led to restoration of the TNF-alpha-induced apoptosis response, reduction of nuclear factor-kappaB activity, delayed degradation of IkappaB-alpha, and inhibited phosphorylation of JNK. Analysis of the interactions of JAB1 by reciprocal co-immunoprecipitations and confocal microscopy revealed that JAB1 interacts with TNF receptor-associated-factor 2 (TRAF2). The generation of the anti-apoptotic signal on binding of TNF-alpha to the TNF receptor (TNFR)1 has been shown to be associated with the recruitment of TRAF2 to the TNFR1 in a process that requires ubiquitination of TRAF2 with lysine-63-linked polyubiquitin chains. We found that TNF-alpha stimulation of JAB1 siRNA-transfected RA FLSs failed to stimulate ubiquitination of TRAF2. Thus, we conclude that JAB1-regulated ubiquitination of TRAF2 is a novel mechanism whereby TNF-alpha can induce anti-apoptosis signaling and production of matrix metalloproteinases through activation of nuclear factor-kappaB and JNK in RA FLSs.

Regulation of neurokinin-1 receptor messenger RNA expression in synovial fibroblasts of patients with rheumatoid arthritis

We examined whether soluble mediators regulate the expression of tachykinin receptor mRNAs in synovial fibroblasts of patients with rheumatoid arthritis (RA). mRNAs encoding long and short isomers of neurokinin 1 receptor (NK1R), and neurokinin 2 receptor (NK2R) were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Level of long, but not the short, of NK1R mRNA was increased by treatment with 10-100 ng/ml basic fibroblast growth factor (bFGF) or 20 ng/ml tumor necrosis factor-alpha (TNF-alpha), but not with 1ng/ml interleukin 1beta (IL-1beta). TNF-alpha upregulated NK2R mRNA as well as long NK1R mRNA whereas bFGF had no effect on NK2R mRNA. Expression of neurokinin 3 receptor (NK3R) mRNA was not observed in RA fibroblasts, and its expression was not induced by bFGF and TNF-alpha. The basal and increased levels of long NK1R mRNA were inhibited by treatment with 20 microM SU5402, an inhibitor of the tyrosine kinase activity of FGF receptor 1 (FGFR1), or 10 ng/ml transforming growth factor-beta1 (TGF-beta1). SU5402 and TGF-beta1 had no effect on the basal level of short NK1R mRNA. Immunocytochemistry revealed the enhancement by bFGF of immunoreactive NK1Rs in the cells at 24 h after treatment. These results suggest that bFGF, TGF-beta1, and TNF-alpha in synovial tissue and fluid play a role in the regulation of long NK1R expression in synovial fibroblasts of RA patients. It appears that the pathway of downregulation by TGF-beta1 is more dominant in the long NK1R mRNA expression than that of upregulation by bFGF or TNF-alpha. Furthermore, the regulation of short NK1R mRNA expression seems to be performed via a different pathway from that of long isomer mRNA.

Das Immunsystem ist nicht alles

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that primarily affects the joints and results in their progressive destruction. Research during past years has shown that in addition to inflammatory cells and their mediators, resident fibroblasts of the synovial membrane play an important role in the pathogenesis of the disease. These cells exhibit features of stable cellular activation that is maintained in the absence of continuous inflammatory stimuli. In contrast to normal synovial fibroblasts or fibroblasts from patients with osteoarthritis, RA synovial fibroblasts show an upregulation of proto-oncogenes and transcription factors, which in a self-perpetuating manner mediate the expression of adhesion molecules and matrix degrading enzymes, and result in alterations in apoptosis. As a consequence, these activated fibroblasts attach to cartilage and bone and progressively destroy articular structures. A better understanding of the molecular mechanisms that lead to the stable activation of synovial fibroblasts in RA is, therefore, of utmost importance for elucidating the pathogenesis of RA as well as for the development of novel therapeutic strategies aimed at inhibiting joint destruction.

Expression and regulation of WISP2 in rheumatoid arthritic synovium

Numbers of growth factors expressed in the synovium deeply impact on the pathology of rheumatoid arthritis (RA). The WISP family was identified as growth factors, which are upregulated by WNT signaling. In the present study, we investigated expression pattern and regulatory mechanisms of WISPs in the synovium in patients with RA and osteoarthritis (OA). Among three members of WISP family, WISP2 mRNA was only preferentially detected in RA synovium by RT-PCR. WISP2 expression was immunohistochemically identified in RA fibroblasts in an extensive fibrotic area. WNT signaling-activated (s/aβ-catenin-expressing) synovial fibroblasts upregulated WISP2 at 2.9-fold, but -inactivated (Δβ-catenin-expressing) cells downregulated the expression. Quantitative RT-PCR demonstrated that WISP2 expression was increased upon 17-β-estradiol stimulation and synergistically enhanced by WNT signaling. These data demonstrate that the expression of WISP2 is synergistically upregulated in RA synovial fibroblasts by estrogen and WNT pathways, and suggest an involvement in the pathology of the disease.

The induction of matrix metalloproteinase and cytokine expression in synovial fibroblasts stimulated with immune cell microparticles

Rheumatoid arthritis is a chronic inflammatory disease characterized by destruction of cartilage and bone that is mediated by synovial fibroblasts. To determine the mechanisms by which these cells are activated to produce matrix metalloproteinases (MMPs), the effects of microparticles were investigated. Microparticles are small membrane-bound vesicles whose release from immune cells is increased during activation and apoptosis. Because microparticles occur abundantly in the synovial fluid in rheumatoid arthritis, they could represent novel stimulatory agents. Microparticles derived from T cells and monocytes strongly induced the synthesis of MMP-1, MMP-3, MMP-9, and MMP-13 in fibroblasts. The induction was time-dependent, with effects primarily observed after 36 h; under these conditions, MMP-2, MMP-14, and tissue inhibitor of MMP-1 (TIMP-1), TIMP-2, and TIMP-3 were not induced. Microparticles also increased the synthesis of inflammatory mediators including IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), and MCP-2. In Ikappa-B-transfected synovial fibroblasts, MMPs were less inducible by microparticles compared with wild-type fibroblasts. Blocking of TNFalpha and IL-1beta with antibodies against TNFalpha and with IL-1 receptor antagonist did not abrogate stimulation by microparticles. These data provide evidence for a novel mechanism by which vesicles derived from activated or apoptotic immune cells can promote the destructive activity of synovial fibroblasts in rheumatoid arthritis.

A Novel Model of Fibroblast‐Mediated Cartilage Destruction

In rheumatoid arthritis (RA), fibroblasts have been shown to be crucial for disease progression as well as joint destruction. In the model of human/murine SCID arthritis, synovial explants as well as fibroblasts from human rheumatoid synovial membrane induce destructive arthritis in immunodeficient mice. Hereby, the underlying cartilage destruction is accomplished by murine fibroblasts. Therefore, murine destructive fibroblasts represent a promising tool to investigate destruction of articular cartilage and bone. In this context, a novel destructive murine fibroblast line (LS48) was examined for morphological, ultrastructural, immunological and functional cellular parameters. These cells were injected into knees of SCID mice. Subsequently, the animals were monitored for joint swelling and serological parameters of arthritis by radiological methods. Finally, cartilage destruction was assessed morphologically. Cultured LS48 cells exhibit characteristic features that resemble those of activated synovial fibroblasts in human RA. Expression levels of inducible nitric oxide synthase, interleukin-6, tumour necrosis factor-alpha and matrix metalloproteinases were comparable to those detected in invasive human fibroblasts. The instillation of 5 x 10(5) LS48 cells into the knee joints of SCID mice initiated a rapid progressive process, that caused cartilage destruction within 10 days, and morphological examinations revealed that articular cartilage was infiltrated by the fibroblasts injected previously. In summary, the intra-articular application of LS48 cells represents a rapid and highly reproducible model to investigate the initiation and progression of cartilage destruction in connection with RA therapy and represents an easy-to-handle animal model.

Cooperation of Ras- and c-Myc-dependent pathways in regulating the growth and invasiveness of synovial fibroblasts in rheumatoid arthritis.

To study the specific contribution of MAP kinase activator c-Raf-1 and one of its downstream transcription factors, c-Myc, to the growth and invasive behavior of rheumatoid arthritis synovial fibroblasts (RASFs). RASFs were transduced with retroviral constructs expressing dominant-negative mutants of c-Raf-1 or c-Myc (DN c-Raf-1 or DN c-Myc, respectively) or with the mock vector. The expression of wild-type and mutant proteins was confirmed by Western blotting. Growth curves of RASFs were recorded, and apoptosis was measured by flow cytometry. Invasiveness of RASFs was assessed in the SCID mouse model of RA. Immunohistochemistry was used to study the effects of DN c-Raf-1 on phosphorylated c-Jun and matrix metalloproteinase 1 (MMP-1) in RASFs implanted into SCID mice. The phosphorylation of ERK and JNK in DN c-Raf-1- and mock-transduced RASFs was determined in vitro by Western blotting. The levels of MMPs in these cells were measured by quantitative polymerase chain reaction (PCR). Neither DN c-Raf-1 alone nor DN c-Myc alone significantly altered proliferation or apoptosis of RASFs, but both mutants together rapidly induced apoptosis. Inhibition of c-Raf-1 or c-Myc significantly reduced the invasiveness of RASFs in the SCID mouse model. DN c-Raf-1 decreased the phosphorylation of ERK and JNK in vitro and reduced the in vivo expression of phosphorylated c-Jun as well as the expression of disease-relevant MMPs. As determined by quantitative PCR, the inhibition was most pronounced for MMP-1 and MMP-3. The data demonstrate that Ras- and c-Myc-dependent signaling events cooperate to regulate the growth and invasiveness of RASFs. Targeting of both c-Raf-1 and c-Myc may constitute an interesting therapeutic approach in RA.

Synovial Fibroblasts from Rheumatoid Arthritis Patients Differ in their Regulation of IL-16 Gene Activity in Comparison to Osteoarthritis Fibroblasts

Background: In rheumatoid arthritis (RA), synovial fibroblast-like cells (SF) contribute significantly to articular inflammation. They express distinct patterns of genes associated with cell proliferation and differentiation and elevated levels of cytokines and chemoattractant factors, including IL-16. Here we investigated pathways regulating IL-16 expression in fibroblasts from RA patients in comparison to fibroblasts from osteoarthritis (OA) patients. Methods: Fibroblasts were isolated from dermal and articular biopsies, expanded and pathways of IL-16 induction were investigated by real time quantitative RT/PCR, immuno blot and ELISA. Results: Stimulation of cAMP dependent signal transduction by forskolin induced prominent IL-16 RT/PCR signals in OA-DF and OA-SF. In contrast, in RA-DF and RA-SF staurosporine significantly augmented IL-16 RT/PCR signals, but forskolin induced less IL-16 transcript amounts. Activation of protein kinase C by PMA induced a significant IL-16 response only in RA-SF. Addition of IL-1β or TNF-α did not upregulate IL-16 mRNA but secretion of the mature IL-16 cytokine was activated in serum starved cells in presence of IL-1β. Conclusion: Our results suggest that RA fibroblasts differ from OA fibroblasts with respect to their sensitivities to kinase/phospatase signal transduction pathways. The enhanced expression of IL-16 in the synovial membrane early in RA vs OA may be associated in part with these distinct signaling responses.

The role of COX-2 in angiogenesis and rheumatoid arthritis☆

Recent evidence suggests that cyclooxygenase (COX)-2 is a mediator of angiogenesis, and COX-2 activity is known to be upregulated in the rheumatoid arthritis (RA) synovium. We examined whether mediation of angiogenesis by COX-2 was occuring in cells of the RA synovium and in microvascular endothelial cells (ECs) that are similar to those found in the RA synovium. We demonstrate that rofecoxib, a selective COX-2 inhibitor, acts directly on human dermal microvascular ECs (HMVECs) to inhibit their chemotactic and tube forming ability. Likewise, pretreatment of HMVECs with rofecoxib significantly inhibited their ability to form tubes induced by conditioned media (CM) of activated RA synovial fibroblasts. When RA synovial fibroblasts were pretreated with rofecoxib for 16 h and then stimulated with interleukin (IL)-1β, their CM induced significantly less HMVEC tube formation when compared with CM from vehicle-treated RA synovial fibroblasts. ELISAs performed on activated RA fibroblast CM for known proangiogenic factors demonstrated a significant reduction in bFGF, in addition to the expected decrease in PGE 2. Our studies suggest that COX-2-induced angiogenic activity is an active mechanism within diseased synovium and may provide an additional rationale for the use of COX-2 inhibitors in RA.

Role of Macrophage Inflammatory Protein-3α and Its Ligand CCR6 in Rheumatoid Arthritis

We examined the expression and participation of CCR6 and its ligand MIP-3α in rheumatoid arthritis (RA) by ELISA, RT-PCR, real-time PCR (TaqMan) analysis, monocyte chemotaxis, and two- and four-color flow cytometry. We found that RA synovial fluid (SF) contained significantly more MIP-3α than osteoarthritis (OA), indicating a potential role for MIP-3α in RA. IL-1β, IL-18, and TNF-α stimulated RA fibroblast MIP-3α production at 48 hours of incubation in vitro. By TaqMan analysis, there were more CCR6 mRNA transcripts in RA synovial tissue (ST) than in OA or normal (NL) ST, and elevated MIP-3α mRNA expression in RA compared with NL ST. By FACS analysis, there were significantly elevated percentages of CD3+/CD4+/CD45RO+/CCR6+ memory lymphocytes found in RA peripheral blood (PB) compared with NL PB or RA SF. We also found that MIP-3α induced monocyte chemotactic activity at 1.25 pm, consistent with concentrations of MIP-3α found in RA SF. Furthermore, MIP-3α accounted for 40% of RA SF chemotactic activity for monocytes in modified Boyden chamber assays. We confirmed that MIP-3α may be binding a G-coupled protein receptor because in vitro monocyte chemotaxis was inhibited by preincubation of monocytes with pertussis toxin. RA patient clinical data revealed that CD3+ lymphocyte/CCR6 expression inversely correlated with the age of the patient, indicating that CCR6 expression may be important in the development of RA at a younger age. Overall, these studies indicate that MIP-3α and CCR6 may function to recruit monocytes and memory lymphocytes from the RA PB to the RA joint. These results further indicate that expression of CCR6, the receptor for MIP-3α, can be correlated with RA development.

Comparison of the inhibitory effects of two types (90 kDa and 190 kDa) of hyaluronic acid on the expression of fibrinolytic factors in human synovial fibroblasts

Hyaluronic acid (HA) has been shown to be clinically effective, and is currently used for the treatment of arthropathy. We previously reported that HA of molecular weight 90 kDa (90-HA) inhibits the fibrinolytic factors in human synovial fibroblasts. In the present study, we investigated the effect of high molecular weight (190 kDa) HA (190-HA) compared with 90-HA on the pericellular fibrinolytic system of human synovial fibroblasts in osteoarthritis (OA) and rheumatoid arthritis (RA). Human synovial fibroblasts were obtained from synovial tissues of OA and RA, and were cultured in the presence and absence of 90-HA and 190-HA. Antigens of urokinase-type plasminogen activator (u-PA) and PA inhibitor-1 (PAI-1) were measured by ELISA, and the u-PA activity of the cell surface fraction was evaluated by electrophoretic enzymography. The binding assay of u-PA and the immunocytochemical analysis of u-PA were performed to detect u-PA receptor (u-PAR). HA inhibited the secretion of both u-PA and PAI-1 antigens from the synovial fibroblasts of OA to their conditioned medium, and the degree of inhibition was more effective in OA than in RA. The u-RA binding assay to these cells showed that both 90-HA and 190-HA slightly decreased the maximal number of binding sites (Bmax) in OA. However, in RA, stimulation with 90-HA and 190-HA decreased Bmax by a half and a quarter, respectively. Immunohistochemical analysis showed that u-PAR was constitutively expressed in both synovial fibroblasts, but if these cells were treated with HA, the decrease in the staining of u-PAR was more pronounced in RA than in OA. Furthermore, the degree was more effective with 190-HA than with 90-HA. HA inhibited the pericellular fibrinolytic activity mediated by the u-PA/u-PAR system in synovial fibroblasts of OA and RA, and 190-HA inhibited it more effectively than 90-HA.

Comparison of the inhibitory effects of two types (90 kDa and 190 kDa) of hyaluronic acid on the expression of fibrinolytic factors in human synovial fibroblasts

Hyaluronic acid (HA) has been shown to be clinically effective, and is currently used for the treatment of arthropathy. We previously reported that HA of molecular weight 90 kDa (90-HA) inhibits the fibrinolytic factors in human synovial fibroblasts. In the present study, we investigated the effect of high molecular weight (190 kDa) HA (190-HA) compared with 90-HA on the pericellular fibrinolytic system of human synovial fibroblasts in osteoarthritis (OA) and rheumatoid arthritis (RA). Human synovial fibroblasts were obtained from synovial tissues of OA and RA, and were cultured in the presence and absence of 90-HA and 190-HA. Antigens of urokinase-type plasminogen activator (u-PA) and PA inhibitor-1 (PAI-1) were measured by ELISA, and the u-PA activity of the cell surface fraction was evaluated by electrophoretic enzymography. The binding assay of u-PA and the immunocytochemical analysis of u-PA were performed to detect u-PA receptor (u-PAR). HA inhibited the secretion of both u-PA and PAI-1 antigens from the synovial fibroblasts of OA to their conditioned medium, and the degree of inhibition was more effective in OA than in RA. The u-RA binding assay to these cells showed that both 90-HA and 190-HA slightly decreased the maximal number of binding sites (Bmax) in OA. However, in RA, stimulation with 90-HA and 190-HA decreased Bmax by a half and a quarter, respectively. Immunohistochemical analysis showed that u-PAR was constitutively expressed in both synovial fibroblasts, but if these cells were treated with HA, the decrease in the staining of u-PAR was more pronounced in RA than in OA. Furthermore, the degree was more effective with 190-HA than with 90-HA. HA inhibited the pericellular fibrinolytic activity mediated by the u-PA/u-PAR system in synovial fibroblasts of OA and RA, and 190-HA inhibited it more effectively than 90-HA.

Regulation by PGE2 of the production of interleukin-6, macrophage colony stimulating factor, and vascular endothelial growth factor in human synovial fibroblasts.

1. We examined the effects of endogenous prostaglandin E(2) (PGE(2)) on the production of interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF) by interleukin-1beta (IL-1beta)-stimulated human synovial fibroblasts. 2. NS-398 (1 microM), a cyclo-oxygenase-2 (COX-2) inhibitor, inhibited IL-6 and VEGF production (35+/-4% and 26+/-2%, respectively) but enhanced M-CSF production (38+/-4%) by IL-1beta (1 ng ml(-1)) in synovial fibroblasts isolated from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Exogenous PGE(2) completely abolished the effects of NS-398 on the production of each mediator by OA fibroblasts stimulated with IL-1beta. 3. 8-Bromo cyclic AMP and dibutyryl cyclic AMP, cyclic AMP analogues, mimicked the effects of PGE(2) on IL-6, M-CSF, and VEGF production by OA fibroblasts. 4. The EP(2) selective receptor agonist ONO-AE1-259 (2 nM) and the EP(4) selective receptor agonist ONO-AE1-329 (2 or 20 nM), but not the EP(1) selective receptor agonist ONO-DI-004 (1 microM) and the EP(3) selective receptor agonist ONO-AE-248 (1 microM), replaced the effects of PGE(2) on IL-6, M-CSF, and VEGF production by OA and RA fibroblasts stimulated with IL-1beta in the presence of NS-398. 5. Both OA and RA fibroblasts expressed mRNA encoding EP(2) and EP(4) but not EP(1) receptors. In addition, up-regulation of EP(2) and EP(4) receptor mRNAs was observed at 3 h after IL-1beta treatment. 6. These results suggest that endogenous PGE(2) regulates the production of IL-6, M-CSF, and VEGF by IL-1beta-stimulated human synovial fibroblasts through the activation of EP(2) and EP(4) receptors with increase in cyclic AMP.

The DING protein: an autocrine growth-stimulatory protein related to the human synovial stimulatory protein

A synovial stimulating protein (SSP) has previously been isolated from rheumatoid arthritis synovial fluid and from the culture fluid of rheumatoid arthritis synovial fibroblasts. We have previously isolated, from skin fibroblast cultures, a 40 kDa hirudin-binding protein, which had amino acid sequence homology with the SSP. We sought to clarify the relationship, if any, between the SSP and the hirudin-binding protein. We show that the hirudin-binding protein is immunologically cross-reactive with a protein identical with, or very similar to, the SSP. This hirudin-binding protein is produced by normal and rheumatoid arthritis fibroblasts in culture, and also by cervical carcinoma cells. Traces of an SSP-like protein, and of proteins intermediate in size between the SSP and the hirudin-binding protein, suggest that the hirudin-binding protein may be proteolytically derived from the SSP. An SSP-like protein of about 200 kDa is present in all synovial fluid samples, arthritic and normal, indicating that its presence is not a primary cause of rheumatoid arthritis. There is no evidence for the existence of smaller fragments of the SSP-like protein in synovial fluid. A cDNA sequence, coding for part of the 40 kDa protein, has been obtained. The derived amino acid sequence indicates that a domain, previously identified in the dishevelled gene from Drosophila melanogaster, is present in this protein. Peptides predicted from the cDNA sequence were used to raise antisera, which recognise both the 40 kDa protein and the SSP-like protein. One of the antibody preparations is a good inhibitor of fibroblast proliferation, which confirms the autocrine growth-stimulatory role originally proposed for these proteins.

Molecular profile of synovial fibroblasts in rheumatoid arthritis depends on the stage of proliferation

The aim of this study was to explore the molecular profile of proliferating rheumatoid arthritis synovial fibroblasts (RA-SF). Total RNA was extracted from two cultures of RA-SF (low-density [LD] proliferating cells and high-density [HD] nonproliferating cells) and suppression subtractive hybridization was performed to compare differential gene expression of these two cultures. Subtracted cDNA was subcloned, and nucleotide sequences were analyzed to identify each clone. Differential expression of distinct clones was confirmed by semiquantitative RT-PCR. The expression of certain genes in synovial tissues was examined by in situ hybridization. In both LD and HD cells, 44 clones were upregulated. Of the 88 total clones, 46 were identical to sequences that have previously been characterized. Twenty-nine clones were identical to cDNAs that have been identified, but with unknown functions so far, and 13 clones did not show any significant homology to sequences in GenBank (NCBI). Differential expression of distinct clones was confirmed by RT-PCR. In situ hybridization showed that certain genes, such as S100A4, NFAT5, unr and Fbx3, were also expressed predominantly in synovial tissues from patients with RA but not from normal individuals. The expression of distinct genes in proliferating RA-SF could also be found in RA synovium, suggesting that these molecules are involved in synovial activation in RA. Most importantly, the data indicate that the expression of certain genes in RA-SF depends on the stage of proliferation; therefore, the stage needs to be considered in any analysis of differential gene expression in SF.

Differential gene expression of proliferating synovial fibroblasts in rheumatoid arthritis

Differential gene expression of proliferating synovial fibroblasts in rheumatoid arthritis