RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via toll‐like receptor 3

Objective

To assess the expression of Toll‐like receptor 3 (TLR‐3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR‐3 ligands.

Methods

TLR‐3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence‐activated cell sorting and real‐time polymerase chain reaction techniques. TLR‐3 signaling was assessed by incubating RASFs with poly(I‐C), lipopolysaccharide, palmitoyl‐3‐cysteine‐serine‐lysine‐4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon‐β (IFNβ), CXCL10, CCL5, and interleukin‐6 (IL‐6) protein production in the culture supernatants was performed by enzyme‐linked immunosorbent assays.

Results

TLR‐3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR‐3 expression was localized predominantly in the synovial lining, with a majority of the TLR‐3–expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR‐3 ligand poly(I‐C) resulted in the production of high levels of IFNβ, CXCL10, CCL5, and IL‐6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up‐regulation of these cytokines and chemokines in a TLR‐3–dependent manner.

Conclusion

Our findings demonstrate the expression of TLR‐3 in RA synovial tissue and the activation of RASFs in vitro by the TLR‐3 ligand poly(I‐C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR‐3 ligand for the stimulation of proinflammatory gene expression in RASFs.

     

Rheumatoid arthritis synovial fluid fibroblasts express TRAIL‐R2 (DR5) that is functionally active

Objective

To examine fibroblasts grown from the synovial fluid of rheumatoid arthritis (RA) patients for TRAIL‐R2 expression, and for susceptibility to apoptosis induced by an agonistic anti–TRAIL‐R2 monoclonal antibody (mAb).

Methods

The expression of TRAIL‐R2 (DR5) was determined by flow cytometry on fibroblasts grown from the synovial fluid of patients with RA, osteoarthritis (OA), seronegative arthritis, and unclassified monarthritis or oligoarthritis, and on fibroblasts from the synovial membrane of RA and OA patients. Susceptibility to apoptosis mediated by an agonistic anti–TRAIL‐R2 mAb was determined by alamar blue bioassay, fluorescence microscopy (annexin V/propidium iodide staining), and caspase activation.

Results

Fibroblasts grew from 35 of 50 RA synovial fluid samples, of which 26 were DR5+ (mean [±SD] fluorescence intensity [MFI] 18.74 ± 2.5). Fibroblasts also grew from 15 of 30 seronegative arthritis synovial fluid samples, 28 of 40 OA synovial fluid samples, and 8 of 20 unclassified monarthritis or oligoarthritis synovial fluid samples; all of these were DR5− (MFI 0.32 ± 0.02). All 10 of the fibroblast lines from joint replacement surgery or synovectomy specimens of RA patients were DR5+ (MFI 20.3 ± 3.2). All fibroblast lines from the synovium of 10 OA patients were DR5−, as were fibroblasts from the skin of 5 healthy subjects. DR5+ fibroblast cultures underwent apoptosis when treated in vitro with an agonistic anti‐DR5 antibody.

Conclusion

Fibroblasts grown from the synovial fluid and synovial membrane of RA patients express TRAIL‐R2 that is functionally active. An agonistic anti–TRAIL‐R2 antibody that does not induce hepatocyte toxicity may be an alternative strategy for treatment of RA.

     

Analysis of vascular gene expression in arthritic synovium by laser‐mediated microdissection

Objective

In rheumatoid arthritis (RA), formation of new blood vessels is necessary to meet the nutritional and oxygen requirements of actively proliferating synovial tissue. The aim of this study was to analyze the specific synovial vascular expression profiles of several angiogenesis‐related genes as well as CD82 in RA compared with osteoarthritis (OA), using laser‐mediated microdissection (LMM).

Methods

LMM and subsequent real‐time polymerase chain reaction were used in combination with immunohistochemical analysis for area‐specific analysis of messenger RNA (mRNA) and protein expression of vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR‐1), VEGFR‐2, hypoxia‐inducible factor 1α (HIF‐1α), HIF‐2α, platelet‐derived growth factor receptor α (PDGFRα), PDGFRβ, inhibitor of DNA binding/differentiation 2 (Id2), and CD82 in RA and OA synovial microvasculature and synovial lining.

Results

Expression of Id2 mRNA was significantly lower in RA synovial vessels compared with OA synovial vessels (P = 0.0011), whereas expression of VEGFR‐1 was significantly higher in RA (P = 0.0433). No differences were observed for the other parameters. At the protein level, no statistically significant differences were observed for any parameter, although Id2 levels were 2.5‐fold lower in RA (P = 0.0952). However, the number of synovial blood vessels and the number of VEGFR‐2–expressing blood vessels were significantly higher in RA compared with OA.

Conclusion

Our results underscore the importance of area‐specific gene expression analysis in studying the pathogenesis of RA and support LMM as a robust tool for this purpose. Of note, our results indicate that previously described differences between RA and OA in the expression of angiogenic molecules are attributable to higher total numbers of synovial and vascular cells expressing these molecules in RA rather than higher expression levels in the individual cells.

     

Bacterial peptidoglycans but not CpG oligodeoxynucleotides activate synovial fibroblasts by toll‐like receptor signaling

Objective

To test the hypothesis that bacterial products acting as adjuvants, such as CpG oligodeoxynucleotides (ODNs) and peptidoglycans (PGs), are able to activate synoviocytes, and to determine the involvement of Toll‐like receptors (TLRs) in this activation process.

Methods

Cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) were stimulated with CpG ODNs or PGs. The expression of various integrins was determined by fluorescence‐activated cell sorting. TLR and matrix metalloproteinase (MMP) messenger RNA (mRNA) was measured by real‐time polymerase chain reaction. Additionally, levels of interleukin‐6 (IL‐6) and IL‐8 in the culture supernatants were assessed by enzyme‐linked immunosorbent assay. Blocking experiments were performed by adding anti–TLR‐2 and anti–TLR‐4 monoclonal antibodies to cultures stimulated with bacterial PGs.

Results

Incubation of synovial fibroblasts with CpG ODNs resulted in neither up‐regulation of the expression of integrins on the cell surface, up‐regulation of MMP mRNA expression, nor IL‐6 and IL‐8 production. However, incubation of RA synovial fibroblasts as well as OA synovial fibroblasts with staphylococcal PGs led to an up‐regulation of CD54 (ICAM‐1) surface expression and to increased expression of MMP‐1, MMP‐3, and MMP‐13 mRNA. Furthermore, production of the proinflammatory cytokines IL‐6 and IL‐8 was increased by treatment with PGs. We demonstrated that cultured synovial fibroblasts express low levels of TLR‐2 and TLR‐9 mRNA. TLR‐2 was up‐regulated after stimulation with PGs, whereas TLR‐9 mRNA remained at baseline levels after stimulation with CpG ODNs. Anti–TLR‐2 monoclonal antibodies significantly inhibited production of IL‐6 and IL‐8 induced by stimulation with PGs.

Conclusion

We demonstrate that bacterial PGs activate synovial fibroblasts, at least partially via TLR‐2, to express integrins, MMPs, and proinflammatory cytokines. Inhibition of TLR signaling pathways might therefore have a beneficial effect on both joint inflammation and joint destruction.

     

Stimulation of collagenase 3 expression in synovial fibroblasts of patients with rheumatoid arthritis by contact with a three‐dimensional collagen matrix or with normal cartilage when coimplanted in NOD/SCID mice

Objective

To study the expression of collagenase 3 (matrix metalloproteinase 13 [MMP‐13]) and collagenase 1 (MMP‐1) in synovial fibroblasts from patients with rheumatoid arthritis (RA) when cultured within 3‐dimensional collagen gels or coimplanted with normal cartilage in immunodeficient NOD/SCID mice.

Methods

Messenger RNA (mRNA) and protein expression of collagenase 3 and collagenase 1 were characterized in synovial and skin fibroblasts by Northern blot and Western blot analysis. The mRNA expression of both collagenases in cell–cartilage implants in NOD/SCID mice was investigated by in situ hybridization in combination with immunohistochemistry of human fibroblasts.

Results

Synovial fibroblasts coimplanted with normal cartilage in NOD/SCID mice deeply invaded adjacent cartilage tissue. In this in vivo system of cartilage destruction, collagenase 3 mRNA was induced in synovial fibroblasts at sites of cartilage erosion, while the expression of collagenase 1 mRNA could not be detected. Culture of synovial fibroblasts within 3‐dimensional collagen gels was a ssociated with a marked increase in collagenase 3 mRNA expression and proenzyme production. This stimulatory effect was 1 order of magnitude higher in comparison with a 2–4‐fold increase upon treatment with interleukin‐1 β or tumor necrosis factor α. In contrast, mRNA expression and proenzyme production of collagenase 1 were increased strongly, and to a similar extent, either by contact with 3‐dimensional collagen or by proinflammatory cytokines.

Conclusion

The expression of collagenase 3, in contrast to that of collagenase 1, is preferentially stimulated in synovial fibroblasts by 3‐dimensional collagen rather than by proinflammatory cytokines. The induction of collagenase 3 by cell–matrix interactions represents a potential mechanism contributing to the invasive phenotype of synovial fibroblasts at sites of synovial invasion into cartilage in RA.

     

Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed‐polymerase chain reaction

Objective

There is increasing evidence that T cell‐independent pathways, such as the up‐regulation of protooncogenes and the production of growth factors and matrix‐degrading enzymes, lead to progressive destruction of affected joints. Therefore, identification of differentially regulated genes restricted to rheumatoid arthritis (RA) synovial fibroblasts is essential. A combination of RNA arbitrarily primed–polymerase chain reaction (RAP‐PCR) and complementary DNA (cDNA) array with defined genes was used for a highly sensitive differential screening using small amounts of RNA.

Methods

RNA was extracted from cultured synovial fibroblasts obtained from 6 patients with RA and 6 patients with osteoarthritis (OA). RAP‐PCR was performed using different arbitrary primers for first‐ and second‐strand synthesis. PCRs were hybridized to cDNA array membranes. RA samples were compared with OA samples for differentially expressed genes.

Results

In contrast to standard cDNA array, the identification of 12 differentially expressed genes in RA compared with OA (∼6%) was possible. Differentially expressed genes of interest were confirmed using semiquantitative RT‐PCR and in situ hybridization.

Conclusion

Numerous variants of the differential display method and continuous improvements, including RAP‐PCR, have proven to be both efficient and reliable for examining differentially regulated genes. Our results show that RAP‐PCR combined with cDNA arrays is a suitable method for identifying differentially expressed genes in rheumatoid synovial fibroblasts, using very small amounts of RNA.

     

Down‐regulation of myeloid cell leukemia 1 by epigallocatechin‐3‐gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor α–induced apoptosis

Objective

Overexpression of the antiapoptotic protein myeloid cell leukemia 1 (Mcl‐1) in rheumatoid arthritis (RA) synovial fibroblasts is a major cause of their resistance to tumor necrosis factor α (TNFα)–induced apoptosis. This study was undertaken to evaluate the efficacy of epigallocatechin‐3‐gallate (EGCG) in down‐regulating Mcl‐1 expression and its mechanism of RA synovial fibroblast sensitization to TNFα‐induced apoptosis.

Methods

EGCG effects on cultured RA synovial fibroblast cell morphology, proliferation, and viability over 72 hours were determined by microscopy and a fluorescent cell enumeration assay. Caspase 3 activity was determined by a colorimetric assay. Western blotting was used to evaluate the apoptosis mediators poly(ADP‐ribose) polymerase (PARP), Mcl‐1, Bcl‐2, Akt, and nuclear translocation of NF‐κB.

Results

In RA synovial fibroblasts, EGCG (5–50 μM) inhibited constitutive and TNFα‐induced Mcl‐1 protein expression in a concentration‐ and time‐dependent manner (P < 0.05). Importantly, EGCG specifically abrogated Mcl‐1 expression in RA synovial fibroblasts and affected Mcl‐1 expression to a lesser extent in osteoarthritis and normal synovial fibroblasts or endothelial cells. Inhibition of Mcl‐1 by EGCG triggered caspase 3 activity in RA synovial fibroblasts, which was mediated via down‐regulation of the TNFα‐induced Akt and NF‐κB pathways. Caspase 3 activation by EGCG also suppressed RA synovial fibroblast growth, and this effect was mimicked by Akt and NF‐κB inhibitors. Interestingly, Mcl‐1 degradation by EGCG sensitized RA synovial fibroblasts to TNFα‐induced PARP cleavage and apoptotic cell death.

Conclusion

Our findings indicate that EGCG itself induces apoptosis and further sensitizes RA synovial fibroblasts to TNFα‐induced apoptosis by specifically blocking Mcl‐1 expression and, hence, may be of promising adjunct therapeutic value in regulating the invasive growth of synovial fibroblasts in RA.

     

Increased intraarticular interleukin‐7 in rheumatoid arthritis patients stimulates cell contact–dependent activation of CD4+ T cells and macrophages

Objective

To determine the level of intraarticular expression of interleukin‐7 (IL‐7) in patients with rheumatoid arthritis (RA) and to investigate the mechanisms by which IL‐7 facilitates activation of CD4+ T cells and monocyte/macrophages in RA.

Methods

IL‐7 levels were measured in synovial fluid obtained from patients with RA and patients with osteoarthritis (OA). Immunohistologic analysis was used to assess the expression of IL‐7 in synovial tissue from patients with RA. Proliferation and activation markers were determined in order to measure the effect of IL‐7 on mononuclear cells, isolated CD4+ T cells, and monocyte/macrophages from the peripheral blood and synovial fluid. Cocultures of CD4+ T cells and monocytic cells in the absence or presence of a semipermeable membrane were performed to assess the extent to which IL‐7 induces its effects, either contact dependently or via soluble mediators.

Results

IL‐7 levels were increased in synovial fluid from patients with RA compared with the levels in synovial fluid from patients with OA. Macrophages, fibroblasts, and endothelial cells in the joint lining tissue expressed abundant IL‐7. In vitro, synovial fluid CD4+ T cells and macrophages were hyperresponsive to IL‐7 when compared with peripheral blood cells. Furthermore, IL‐7 enhanced cell contact–dependent activation of CD4+ T cells and monocyte/macrophages.

Conclusion

The abundant intraarticular expression of IL‐7 and the stimulation by IL‐7 of contact‐dependent activation of CD4+ T cells and monocytic cells indicate that this cytokine plays an important proinflammatory role in RA synovitis. Further identification of IL‐7–induced pathways may improve understanding of the important interactive role of CD4+ T cells and monocytic cells in RA.

     

Blocking ERK‐1/2 reduces tumor necrosis factor α–induced interleukin‐18 bioactivity in rheumatoid arthritis synovial fibroblasts by induction of interleukin‐18 binding protein A

Objective

To examine the mechanism of regulation of interleukin‐18 (IL‐18) bioactivity by IL‐18 binding protein (IL‐18BP) induction.

Methods

Levels of IL‐18 and IL‐18BPa in synovial fluid samples from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) were determined by enzyme‐linked immunosorbent assays (ELISAs), followed by calculation of free IL‐18. IL‐18 and IL‐18BPa synthesis in RA synovial fibroblasts that had been treated with proinflammatory and antiinflammatory cytokines were assessed by quantitative real‐time polymerase chain reaction and ELISA, respectively, followed by IL‐18 bioactivity determination using KG‐1 cells. Chemical signaling inhibitors were used for determination of the signal transduction pathways involved in IL‐18BPa/IL‐18 regulation. Tumor necrosis factor α (TNFα)–induced caspase 1 activity was determined by a colorimetric assay.

Results

IL‐18BPa was lower in RA synovial fluid than in OA synovial fluid (P < 0.05; n = 8), and free IL‐18 was higher in RA synovial fluid than in OA synovial fluid. TNFα induced RA synovial fibroblast IL‐18BPa and IL‐18 in a time‐dependent manner (P < 0.05). Evaluation of signaling pathways suggested that TNFα induced IL‐18 production through the ERK‐1/2, protein kinase Cδ (PKCδ), and Src pathways, whereas IL‐18BPa synthesis was mediated through the NFκB, PKC, Src, and JNK pathways. Furthermore, addition of exogenous IL‐18BPa‐Fc reduced the RA synovial fibroblast phosphorylation of ERK‐1/2 induced by TNFα.

Conclusion

These results suggest that IL‐18BPa reduces IL‐18 bioactivity induced by TNFα, by regulating the ERK‐1/2 pathway in RA synovial fibroblasts. Targeting IL‐18 bioactivity by induction or addition of IL‐18BPa may provide another therapeutic option in the management of RA.

     

Rheumatoid arthritis synovial fluid macrophages express decreased tumor necrosis factor–related apoptosis‐inducing ligand R2 and increased decoy receptor tumor necrosis factor–related apoptosis‐inducing ligand R3

Objective

To characterize the expression pattern of tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) and its cognate receptors (TRAIL R1, R2, R3, and R4) on rheumatoid arthritis (RA) synovial fluid (SF) lymphocytes and monocyte/macrophages and on cultured RA synovial fibroblasts.

Methods

The expression of TRAIL and TRAIL receptors on RA SF lymphocytes and monocyte/macrophages, normal macrophages, and RA synovial fibroblasts was examined by flow cytometry with previously characterized monoclonal antibodies. The ability of adenoviral‐mediated delivery of TRAIL to induce macrophage or RA synovial fibroblast apoptosis was examined by flow cytometry.

Results

By flow cytometry, neither TRAIL nor its cognate receptors was detectable on RA SF lymphocytes or RA synovial fibroblasts. In contrast, RA SF macrophages expressed TRAIL R3, a decoy receptor (P < 0.01 versus isotype control), but not TRAIL, or TRAIL R1, R2, or R4. Normal peripheral blood–derived monocyte‐differentiated macrophages expressed TRAIL R2 (P < 0.01), but not TRAIL or the other TRAIL receptors. Adenoviral‐mediated delivery of TRAIL had no effect on the survival of normal macrophages or RA synovial fibroblasts but readily induced apoptosis in the prostate cancer cell line (PC‐3) that expressed TRAIL R1 and R2.

Conclusion

TRAIL R1 and R2, which are required for signal transmission by TRAIL, were not detected on RA SF lymphocytes, macrophages, or synovial fibroblasts. These observations do not support a potential therapeutic role for TRAIL in RA.

     

NF‐κB–regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor α–mediated apoptosis

Objective

Little apoptosis has been observed in rheumatoid arthritis (RA) synovial tissues. Tumor necrosis factor α (TNFα) is expressed in the joints of patients with RA, yet RA synovial fibroblasts are relatively resistant to apoptosis induced by TNFα. Recently, we demonstrated that FLIP is highly expressed in the RA joint. These studies were performed to determine if TNFα‐induced NF‐κB controls the expression of FLIP long (FLIP_L) and FLIP short (FLIP_S) in RA synovial fibroblasts and to determine the role of FLIP in the control of TNFα‐induced apoptosis.

Methods

RA synovial fibroblasts were isolated from RA synovial tissues and used between passages 3 and 9. RA synovial or control fibroblasts were sham infected or infected with a control adenovirus vector or one expressing the super‐repressor IκBα (srIκBα). The cells were stimulated with TNFα or a control vehicle, and expression of FLIP_L and FLIP_S was determined by isoform‐specific real‐time polymerase chain reaction and Western blot analysis. Cell viability was determined by XTT cleavage, and apoptosis was determined by annexin V staining, DNA fragmentation, and activation of caspases 8 and 3.

Results

TNFα induced the expression of both isoforms of FLIP messenger RNA (mRNA) in RA synovial fibroblasts; however, FLIP_L was the dominant isoform detected by Western blot analysis. In control fibroblasts, TNFα induced the expression of FLIP_L and FLIP_S mRNA and protein. The TNFα‐induced, but not the basal, expression of FLIP was regulated by NF‐κB. When NF‐κB activation was suppressed by the expression of srIκBα, TNFα‐mediated apoptosis was induced. TNFα‐induced apoptotic cell death was mediated by caspase 8 activation and was prevented by the ectopic expression of FLIP_L or the caspase 8 inhibitor CrmA.

Conclusion

The TNFα‐induced, but not the basal, expression of FLIP is regulated by NF‐κB in RA synovial fibroblasts. The resistance of RA synovial fibroblasts to TNFα‐induced apoptosis is mediated by the NF‐κB–regulated expression of FLIP. These observations support the role of NF‐κB and FLIP as attractive therapeutic targets in RA.