Interleukin‐20 antibody is a potential therapeutic agent for experimental arthritis

Objective

Interleukin‐20 (IL‐20) is a proinflammatory cytokine involved in the pathogenesis of rheumatoid arthritis (RA). We investigated whether anti–IL‐20 antibody treatment would modulate the severity of the disease in a collagen‐induced arthritis (CIA) rat model.

Methods

We generated a CIA model by immunizing rats with bovine type II collagen. Rats with CIA were treated subcutaneously with anti–IL‐20 antibody 7E, with the tumor necrosis factor (TNF) blocker etanercept, or with 7E in combination with etanercept. Arthritis severity was determined according to the hind paw thickness, arthritis severity score, degree of cartilage damage, bone mineral density, and cytokine production, which were evaluated using radiologic scans, microfocal computed tomography, and enzyme‐linked immunosorbent assay. To analyze gene regulation by IL‐20, rat synovial fibroblasts (SFs) were isolated and analyzed for the expression of RANKL, IL‐17, and TNFα. We also used real‐time quantitative polymerase chain reaction analysis and flow cytometry to determine IL‐20–regulated RANKL in mouse osteoblastic MC3T3‐E1 cells and Th17 cells.

Results

In vivo, treatment with 7E alone or in combination with etanercept significantly reduced the severity of arthritis by decreasing the hind paw thickness and swelling, preventing cartilage damage and bone loss, and reducing the expression of IL‐20, IL‐1β, IL‐6, RANKL, and matrix metalloproteinases (MMPs) in synovial tissue. In vitro, IL‐20 induced TNFα expression in SFs from rats with CIA. IL‐20 markedly induced RANKL production in SFs, osteoblasts, and Th17 cells.

Conclusion

Selectively blocking IL‐20 inhibited inflammation and bone loss in rats with CIA. Treatment with 7E combined with etanercept protected rats from CIA better than treatment with etanercept alone. Our findings provide evidence that IL‐20 is a novel target and that 7E may be a potential therapeutic agent for RA.

     

Synovial fibroblasts self‐direct multicellular lining architecture and synthetic function in three‐dimensional organ culture

Objective

To define the intrinsic capacity of fibroblast‐like synoviocytes (FLS) to establish a 3‐dimensional (3‐D) complex synovial lining architecture characterized by the multicellular organization of the compacted synovial lining and the elaboration of synovial fluid constituents.

Methods

FLS were cultured in spherical extracellular matrix (ECM) micromasses for 3 weeks. The FLS micromass architecture was assessed histologically and compared with that of dermal fibroblast controls. Lubricin synthesis was measured via immunodetection. Basement membrane matrix and reticular fiber stains were performed to examine ECM organization. Primary human and mouse monocytes were prepared and cocultured with FLS in micromass to investigate cocompaction in the lining architecture. Cytokine stimuli were applied to determine the capacity for inflammatory architecture rearrangement.

Results

FLS, but not dermal fibroblasts, spontaneously formed a compacted lining architecture over 3 weeks in the 3‐D ECM micromass organ cultures. These lining cells produced lubricin. FLS rearranged their surrounding ECM into a complex architecture resembling the synovial lining and supported the survival and cocompaction of monocyte/macrophages in the neo–lining structure. Furthermore, when stimulated by cytokines, FLS lining structures displayed features of the hyperplastic rheumatoid arthritis synovial lining.

Conclusion

This 3‐D micromass organ culture method demonstrates that many of the phenotypic characteristics of the normal and the hyperplastic synovial lining in vivo are intrinsic functions of FLS. Moreover, FLS promote survival and cocompaction of primary monocytes in a manner remarkably similar to that of synovial lining macrophages. These findings provide new insight into inherent functions of the FLS lineage and establish a powerful in vitro method for further investigation of this lineage.

     

Autoimmunity and inflammation are independent of class II transactivator type PIV–dependent class II major histocompatibility complex expression in peripheral tissues during collagen‐induced arthritis

Objective

To determine the regulation of class II major histocompatibility complex (MHC) expression in fibroblast‐like synoviocytes (FLS) in order to investigate their role as nonprofessional antigen‐presenting cells in collagen‐induced arthritis (CIA).

Methods

Expression of class II MHC, class II MHC transactivator (CIITA), and Ciita isoforms PI, PIII, and PIV was examined by real‐time quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry in human synovial tissues, arthritic mouse joints, and human and murine FLS. CIA was induced in mice in which isoform PIV of Ciita was knocked out (PIV^−/−), in PIV^−/− mice transgenic for CIITA in the thymus (K14 CIITA), and in their control littermates.

Results

HLA–DRA, total CIITA, and CIITA PIII messenger RNA levels were significantly increased in synovial tissue samples from patients with rheumatoid arthritis compared with the levels in tissue from patients with osteoarthritis. Human FLS expressed surface class II MHC via CIITA PIII and PIV, while class II MHC expression in murine FLS was entirely mediated by PIV. Mice with a targeted deletion of CIITA PIV lack CD4+ T cells and were protected against CIA. The expression of CIITA was restored in the thymus of PIV^−/− K14 CIITA–transgenic mice, which had a normal CD4+ T cell repertoire and normal surface levels of class II MHC on professional antigen‐presenting cells, but did not induce class II MHC on FLS. Synovial inflammation and immune responses against type II collagen were similar in PIV^−/− K14 CIITA–transgenic mice and control mice with CIA, but bone erosion was significantly reduced in the absence of PIV.

Conclusion

Overexpression of class II MHC is tightly correlated with CIITA expression in arthritic synovium and in FLS. Selective targeting of Ciita PIV in peripheral tissues abrogates class II MHC expression by murine FLS but does not protect against inflammation and autoimmune responses in CIA.

     

Presentation of arthritogenic peptide to antigen‐specific T cells by fibroblast‐like synoviocytes

Objective

To assess the ability of rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS) to function as antigen‐presenting cells (APCs) for arthritogenic autoantigens found within inflamed joint tissues.

Methods

Human class II major histocompatibility complex (MHC)–typed FLS were used as APCs for murine class II MHC–restricted CD4 T cell hybridomas. Interferon‐γ (IFNγ)–treated, antigen‐loaded FLS were cocultured with T cell hybridomas specific for immunodominant portions of human cartilage gp‐39 (HC gp‐39) or human type II collagen (CII). T cell hybridoma activation was measured by enzyme‐linked immunosorbent assay of culture supernatants for interleukin‐2. Both synthetic peptide and synovial fluid (SF) were used as sources of antigen. APC function in cocultures was inhibited by using blocking antibodies to human class II MHC, CD54, or CD58, or to murine CD4, CD11a, or CD2.

Results

Human FLS could present peptides from the autoantigens HC gp‐39 and human CII to antigen‐specific MHC‐restricted T cell hybridomas. This response required pretreatment of FLS with IFNγ, showed MHC restriction, and was dependent on human class II MHC and murine CD4 for effective antigen presentation. Furthermore, FLS were able to extract and present antigens found within human SF to both the HC gp‐39 and human CII T cell hybridomas in an IFNγ‐dependent and MHC‐restricted manner.

Conclusion

RA FLS can function as APCs and are able to present peptides derived from autoantigens found within joint tissues to activated T cells in vitro. In the context of inflamed synovial tissues, FLS may be an important and hitherto overlooked subset of APCs that could contribute to autoreactive immune responses.

     

Fibroblast‐like synoviocytes derived from patients with rheumatoid arthritis show the imprint of synovial tissue heterogeneity: Evidence of a link between an increased myofibroblast‐like phenotype and high‐inflammation synovitis

Objective

Given the heterogeneity of gene expression patterns and cellular distribution between rheumatoid arthritis (RA) synovial tissues, we sought to determine whether this variability was also reflected at the level of the fibroblast‐like synoviocyte (FLS) cultured from RA synovial tissues.

Methods

Gene expression profiles in FLS cultured from synovial tissues obtained from 19 RA patients were analyzed using complementary DNA microarrays and hierarchical cluster analysis. To validate the subclassification, we performed prediction analysis and principal components analysis. Genes that differed significantly in their expression between FLS cultures were selected using Statistical Analysis of Microarrays software. Real‐time quantitative polymerase chain reaction was performed to validate the microarray data. Immunocytochemistry was applied to study the expression of the genes of interest in FLS and synovial tissues.

Results

Hierarchical clustering identified 2 main groups of FLS characterized by distinctive gene expression profiles. FLS from high‐inflammation synovial tissues revealed increased expression of a transforming growth factor β/activin A–inducible gene profile that is characteristic of myofibroblasts, a cell type considered to be involved in wound healing, whereas increased production of growth factor (insulin‐like growth factor 2/insulin‐like growth factor binding protein 5) appeared to constitute a characteristic feature of FLS derived from low‐inflammation synovial tissues. The molecular feature that defines the myofibroblast‐like phenotype was reflected as an increased proportion of myofibroblast‐like cells in the heterogeneous FLS population. Myofibroblast‐like cells were also found upon immunohistochemical analysis of synovial tissue.

Conclusion

Our findings support the notion that heterogeneity between synovial tissues is reflected in FLS as a stable trait, and provide evidence of a possible link between the behavior of FLS and the inflammation status of RA synovium.

     

Amelioration of collagen‐induced arthritis in rats by nanogold

Objective

Angiogenesis plays a part in the pathogenesis of rheumatoid arthritis (RA), and nanogold inhibits the activity of an angiogenic factor, vascular endothelial growth factor (VEGF). We therefore investigated whether intraarticular delivery of nanogold ameliorates collagen‐induced arthritis (CIA) in rats.

Methods

Binding of 13‐nm nanogold to VEGF in human RA synovial fluid (SF) and its effects on RA SF–induced endothelial cell proliferation and migration were assessed. Nanogold was administered intraarticularly to rats with CIA before the onset of arthritis. Progression of CIA was monitored by measures of clinical, radiologic, and histologic changes. In addition, the microvessel density and extent of infiltrating macrophages as well as levels of tumor necrosis factor α (TNFα) and interleukin‐1β (IL‐1β) in the ankle joints were determined.

Results

Nanogold bound to VEGF in RA SF, resulting in inhibition of RA SF–induced endothelial cell proliferation and migration. Significant reductions in ankle circumference, articular index scores, and radiographic scores were observed in the nanogold‐treated rats with CIA compared with their control counterparts. In addition, the histologic score (of synovial hyperplasia, cartilage erosion, and leukocyte infiltration), microvessel density, macrophage infiltration, and levels of TNFα and IL‐1β were also significantly reduced in the ankle joints of nanogold‐treated rats.

Conclusion

Our results are the first to demonstrate that intraarticular administration of nanogold ameliorates the clinical course of CIA in rats. Nanogold exerted antiangiogenic activities and subsequently reduced macrophage infiltration and inflammation, which resulted in attenuation of arthritis. These results demonstrate proof of principle for the use of nanogold as a novel therapeutic agent for the treatment of RA.

     

Function of interleukin‐20 as a proinflammatory molecule in rheumatoid and experimental arthritis

Objective

The pathogenesis of rheumatoid arthritis (RA) reflects an ongoing imbalance between proinflammatory and antiinflammatory cytokines. Interleukin‐20 (IL‐20) has proinflammatory properties for keratinocytes. In this study, we sought to determine whether IL‐20 is involved in RA.

Methods

We analyzed IL‐20 levels in synovial fluid from RA patients. IL‐20 and its receptors were detected in RA synovial fibroblasts (RASFs), using immunohistochemical staining. The effect of IL‐20 on endothelial cells, neutrophils, and RASFs was investigated using MTT and migration assays. The expression of IL‐20 and its receptors in healthy rats and in rats with collagen‐induced arthritis (CIA) was also analyzed. Soluble IL‐20 receptor type I (sIL‐20RI) or sIL‐20RII was administered to rats with CIA by intramuscular electroporation, and the severity of arthritis was monitored.

Results

RA patients expressed significantly higher levels of synovial fluid IL‐20 than did the rheumatic disease controls. IL‐20 and its receptors were expressed in the synovial membranes and RASFs. IL‐20 induced RASFs to secrete monocyte chemoattractant protein 1, IL‐6, and IL‐8, and it promoted neutrophil chemotaxis, RASF migration, and endothelial cell proliferation. Both IL‐20 and IL‐20RI were up‐regulated in the rat CIA model. In vivo, electroporated sIL‐20RI plasmid DNA decreased the severity of arthritis in the rats with CIA.

Conclusion

IL‐20 was up‐regulated in the synovial fluid of RA patients and acted as a chemokine that attracted the migration of neutrophils and RASFs in vitro. The rat CIA model demonstrated that IL‐20 was involved in the pathogenesis of arthritis, because sIL‐20RI significantly reduced arthritis in rats with CIA. Thus, IL‐20 may modulate the incidence and severity of arthritis and play important roles at local sites of inflammation.

     

Fractalkine is a novel chemoattractant for rheumatoid arthritis fibroblast‐like synoviocyte signaling through MAP kinases and Akt

Objective

Fibroblast‐like synoviocytes (FLS) are a major constituent of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Fractalkine (FKN/CX₃CL1) expression is up‐regulated in RA synovium and RA synovial fluid. While RA FLS express the FKN receptor, CX₃CR1, the pathophysiologic relevance of FKN stimulation of RA FLS is not understood. This study was undertaken to better characterize the relationship between FKN and the RA FLS that both produce it and express its receptor.

Methods

RA FLS were subjected to chemotaxis and proliferation assays, Western blotting, enzyme‐linked immunosorbent assays, and filamentous actin staining to characterize the relationship between FKN and RA FLS.

Results

FKN secretion by RA FLS was regulated mainly by tumor necrosis factor α. Stimulation of RA FLS with FKN led to significant cytoskeletal rearrangement but no proliferation. Chemotaxis assays revealed that FKN was a novel chemoattractant for RA FLS. Stimulation of RA FLS with FKN resulted in activation of MAP kinases and Akt. JNK, ERK‐1/2, and Akt (at both Ser‐473 and Thr‐308) were each up‐regulated in a time‐dependent manner. Inhibition of ERK‐1/2–mediated signaling, but not JNK or Akt, significantly repressed FKN‐induced RA FLS migration.

Conclusion

These findings indicate a novel role of FKN in regulating RA FLS cytoskeletal structure and migration. FKN specifically induces RA FLS phosphorylation of the MAP kinases JNK and ERK‐1/2, as well as full activation of Akt.

     

Modulation of interleukin‐6 and matrix metalloproteinase 2 expression in human fibroblast‐like synoviocytes by functional ionotropic glutamate receptors

Objective

Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology.

Methods

Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast‐like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin‐6 (IL‐6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP‐2] and MMP‐9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP‐1] and TIMP‐2). IL‐6 concentrations were determined by enzyme‐linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL‐6 or MMP‐2.

Results

Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N‐methyl‐D ‐aspartate (NMDA) receptors in RA FLS increased proMMP‐2 release, whereas non‐NMDA ionotropic glutamate receptor antagonists reduced IL‐6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors.

Conclusion

Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL‐6 expression.

     

Suppression of leukocyte infiltration and cartilage degradation by selective inhibition of pre–B cell colony‐enhancing factor/visfatin/nicotinamide phosphoribosyltransferase: Apo866‐mediated therapy in human fibroblasts and murine collagen‐induced arthritis

Objective

To assess the ability of pre–B cell colony‐enhancing factor (PBEF) to regulate inflammation and degradative processes in inflammatory arthritis, using the small molecule inhibitor APO866 in human fibroblasts in vitro and in murine collagen‐induced arthritis (CIA).

Methods

Enzyme‐linked immunosorbent assays were used to examine regulation of expression of metalloproteinases and chemokines in human fibroblasts. The role of PBEF was further examined using APO866 in mice with CIA, with effects on disease activity assessed using radiography, histology, in vivo imaging, and quantitative polymerase chain reaction (qPCR).

Results

In vitro activation of human fibroblasts with PBEF promoted expression of matrix metalloproteinase 3 (MMP‐3), CCL2, and CXCL8, an effect inhibited by APO866. In mice with CIA, early intervention with APO866 inhibited synovial inflammation, including chemokine‐directed leukocyte infiltration, and reduced a systemic marker of inflammation, serum hyaluronic acid. APO866 blockade led to reduced expression of MMP‐3 and MMP‐13 in joint extracts and to a reduction in a systemic marker of cartilage erosion, serum cartilage oligomeric matrix protein. Radiologic images revealed that APO866 protected against bone erosion, while qPCR demonstrated inhibition of RANKL expression. In mice with established disease, APO866 reduced synovial inflammation and cartilage destruction, and halted bone erosion. In addition, APO866 reduced the activity of MMP‐3, CCL2, and RANKL in vivo, and inhibited production of CCL2 and RANKL in synovial explants from arthritic mice, a result that was reversed with nicotinamide mononucleotide.

Conclusion

These findings confirm PBEF to be an important regulator of inflammation, cartilage catabolism, and bone erosion, and highlight APO866 as a promising therapeutic agent for targeting PBEF activity in inflammatory arthritis.

     

Early elevation in circulating levels of C‐telopeptides of type II collagen predicts structural damage in articular cartilage in the rodent model of collagen‐induced arthritis

Objective

To investigate changes in the circulating levels of the C‐telopeptide of type II collagen (CTX‐II) with relation to disease onset and structural damage of cartilage in a rodent model of collagen‐induced arthritis (CIA), and to investigate immunolocalization of the CTX‐II epitope in the articular cartilage of affected joints.

Methods

Seven‐week‐old female Lewis rats were immunized with type II collagen and monitored using blood sampling at weekly intervals. At study termination (day 23), the animals were killed, synovial fluid was collected, and the affected joints were scored macroscopically for disease severity and underwent immunohistochemical evaluation.

Results

At the time of disease onset (day 15), which was characterized by redness and swelling of the affected joints (mean ± SD macroscopic severity score 9.1 ± 1.6), there was a 355% increase in serum CTX‐II levels. The early change in serum CTX‐II from day 0 to day 15 showed a significant association with the severity of cartilage damage (r = 0.61, P < 0.01). Immunostaining revealed extensive presence of the CTX‐II epitope in the damaged, uncalcified cartilage tissue.

Conclusion

The elevation in serum CTX‐II concomitant with the onset of disease and proportional to cartilage damage demonstrates that CTX‐II is a sensitive diagnostic tool for monitoring joint disease in the rodent model of CIA. Furthermore, the immunohistochemical findings are consistent with the concept that the major source of serum CTX‐II is the damaged articular cartilage.

     

A comparative genetic analysis between collagen‐induced arthritis and pristane‐induced arthritis

Objective

To compare the genetic regulation of collagen‐induced arthritis (CIA) with that of pristane‐induced arthritis (PIA) in rats.

Methods

A genome‐wide linkage analysis of an (E3 × DA)DA backcross of rats with CIA (n = 364 male rats; the same strain combinations as previously used to determine the genetic control of PIA) was performed. The strongest loci in both CIA and PIA (i.e., Cia12/Pia4 and Cia13/Pia7) were isolated in congenic strains. Susceptibility in both congenic strains was tested in rats with CIA and in rats with PIA.

Results

We found a striking, although not complete, similarity of the arthritis‐controlling loci in CIA and in PIA, as well as the previously defined loci associated with cartilage destruction, antibody production, and the acute‐phase response. All major PIA quantitative trait loci (QTLs) identified in early severe arthritis were also strong regulators of CIA. The 2 strongest QTLs, Cia12/Pia4 on chromosome 12 and Cia13/Pia7 on chromosome 4, were also analyzed in congenic strains with DA or E3 as the background genome. Consistent with the results of linkage analysis, the congenic strain experiments showed that the chromosome 4 locus was more penetrant in CIA than in PIA, while the chromosome 12 locus almost completely dominated the control of PIA severity.

Conclusion

The underlying genetic control of CIA was found to have many, but not all, pathogenic mechanisms in common with PIA, despite the use of a cartilage‐specific antigen (type II collagen) to induce CIA but not PIA.

     

Protein Tyrosine Phosphatase Expression Profile of Rheumatoid Arthritis Fibroblast‐like Synoviocytes: A Novel Role of SH2 Domain–Containing Phosphatase 2 as a Modulator of Invasion and Survival

Objective

The fibroblast‐like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage‐degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS.

Methods

A comparative screening of the expression of the PTPome in FLS from patients with RA and patients with osteoarthritis (OA) was conducted. The functional effect on RA FLS of SH2 domain–containing phosphatase 2 (SHP‐2), a PTP that was up‐regulated in RA, was then analyzed by knockdown using cell‐permeable antisense oligonucleotides.

Results

PTPN11 was overexpressed in RA FLS compared to OA FLS. Knockdown of PTPN11, which encodes SHP‐2, reduced the invasion, migration, adhesion, spreading, and survival of RA FLS. Additionally, signaling in response to growth factors and inflammatory cytokines was impaired by SHP‐2 knockdown. RA FLS that were deficient in SHP‐2 exhibited decreased activation of focal adhesion kinase and mitogen‐activated protein kinases.

Conclusion

These findings indicate that SHP‐2 has a novel role in mediating human FLS function and suggest that it promotes the invasiveness and survival of RA FLS. Further investigation may reveal SHP‐2 to be a candidate therapeutic target for RA.

     

Successful treatment of collagen‐induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal protein 1 activity

Objective

Extracellular high mobility group box chromosomal protein 1 (HMGB‐1) is a recently identified, endogenous, potent tumor necrosis factor– and interleukin‐1 (IL‐1)–inducing protein detectable in inflamed synovia in both human and experimental disease. In the present study, we examined clinical effects in collagen‐induced arthritis (CIA) using therapeutic administration of neutralizing HMGB‐1 antibodies or truncated HMGB‐1–derived A‐box protein, a specific, competitive antagonist of HMGB‐1.

Methods

CIA was induced in DBA/1j mice or dark agouti rats, and animals were examined daily for signs of arthritis. Treatment with polyclonal anti–HMGB‐1 antibodies or the A‐box protein was initiated at the onset of disease and was administered intraperitoneally twice daily for 7 days. Animals were killed 8 days after initiation of therapy, and immunohistochemical analysis of synovial tissue specimens was performed.

Results

Systemic administration of anti–HMGB‐1 antibodies or A‐box protein significantly reduced the mean arthritis score, the disease‐induced weight loss, and the histologic severity of arthritis. Beneficial effects were observed in both mice and rats. Immunohistochemical analysis revealed pronounced synovial IL‐1β expression and articular cartilage destruction in vehicle‐treated mice. Both these features were significantly less manifested in animals treated with anti–HMGB‐1 antibodies or A‐box protein.

Conclusion

Counteracting extracellular HMGB‐1 with either neutralizing antibodies or a specific HMGB‐1 antagonist may offer a new method for the successful treatment of arthritis. Inflammation and tissue destruction were suppressed in CIA after HMGB‐1 blockade.

     

Down‐regulation of FLIP sensitizes rheumatoid synovial fibroblasts to Fas‐mediated apoptosis

Objective

Hyperplasia of fibroblast‐like synoviocytes (FLS) contributes to chronic inflammation and joint destruction in rheumatoid arthritis (RA). FLICE‐inhibitory protein (FLIP) is an antiapoptotic protein that might prevent apoptotic elimination of FLS in response to death ligands such as tumor necrosis factor α (TNFα) or Fas ligand, which are present in RA synovium. Previous studies on FLIP expression by osteoarthritis (OA) and RA FLS have shown variable results, and the specific role of FLIP as an apoptosis inhibitor in these cells remains unclear. We undertook this study to investigate the expression and antiapoptotic function of FLIP in FLS.

Methods

We studied the expression of FLIP by immunohistochemistry and immunoblotting in synovial tissues or cultured FLS from RA and OA patients. FLS apoptosis was induced by an agonistic anti‐Fas monoclonal antibody and FLS were then quantified. We studied the effects of cycloheximide (CHX), TNFα, and FLIP antisense oligonucleotide on FLIP expression and FLS apoptotic susceptibility.

Results

FLIP_L was the isoform mainly expressed in lining synoviocytes and cultured FLS. Synovial tissues and cultured FLS from OA and RA tissues displayed similar patterns and levels of expression of FLIP. Fas‐induced apoptosis was variable in different FLS lines, but differences between OA and RA groups were not detected. TNFα induced increases in FLIP_L and FLIP_S expression and protected RA FLS from apoptosis, while CHX induced the opposite effects. Down‐regulation of FLIP by antisense oligonucleotide strongly sensitized RA FLS to Fas‐mediated apoptosis.

Conclusion

Apoptosis susceptibility and FLIP expression are similar in OA and RA FLS. Down‐regulation of FLIP sensitizes RA FLS to Fas‐mediated apoptosis and may be a valuable tool for targeting RA FLS hyperplasia.

     

Mechanisms and clinical relevance of TRAIL‐triggered responses in the synovial fibroblasts of patients with rheumatoid arthritis

Objective

Results of studies in mice suggest a protective role for TRAIL in arthritis. The aim of this study was to investigate the role of TRAIL in patients with rheumatoid arthritis (RA).

Methods

In the present study, we compared RA fibroblast‐like synoviocytes (FLS) that were resistant or sensitive to TRAIL‐induced apoptosis and the expression of TRAIL receptors in these cells, and also investigated the clinical features of the patients from whom the FLS were derived. Furthermore, we evaluated the levels of TRAIL and its soluble decoy receptor osteoprotegerin (OPG) in patients with RA, patients with osteoarthritis (OA), and patients with spondylarthritis (SpA).

Results

Sensitivity to TRAIL‐induced apoptosis varied in FLS from different patients, and the severity of disease in patients with RA was inversely correlated with the susceptibility of their FLS to TRAIL‐induced apoptosis. TRAIL‐sensitive cells expressed significantly lower levels of TRAILR‐1, and silencing of TRAILR‐1 increased TRAIL‐induced apoptosis in RA FLS. TRAIL levels were elevated in the arthritic joints of patients with established RA, and TRAIL levels in the synovial fluid of these patients were elevated compared with levels in the synovial fluid of patients with OA or SpA. At baseline, a low OPG‐to‐TRAIL ratio in the sera of patients with early RA was associated with a better evolution of disease activity, but high serum levels of TRAIL at followup were associated with joint damage.

Conclusion

These findings suggest that TRAIL has a dual role in RA, and that the resistance of RA FLS to TRAIL‐induced apoptosis is associated with a disease‐promoting activity of TRAIL in RA.