Inhibition of adjuvant‐induced arthritis by systemic tissue inhibitor of metalloproteinases 4 gene delivery

Objective

An imbalance in the matrix metalloproteinase:tissue inhibitor of metalloproteinases (MMP:TIMP) ratio in favor of MMP appears to be an important determinant of tissue damage in arthritis. We undertook this study to explore whether reversal of this imbalance in favor of TIMP would alter this process and to examine the mechanism of this alteration.

Methods

We administered human TIMP‐4 by electroporation‐mediated intramuscular injection of naked DNA using the rat adjuvant‐induced arthritis (AIA) model.

Results

Intramuscular naked TIMP‐4 gene administration resulted in high circulating TIMP‐4 levels and completely abolished arthritis development in the rat AIA model. This inhibition was associated with significantly decreased MMP activity in the joint tissue as well as with significantly decreased serum and tissue tumor necrosis factor α levels and serum interleukin‐1α levels compared with animals with arthritis. The mutation of cysteine at position 1 of TIMP‐4 failed to block the development of AIA.

Conclusion

Our data indicate that TIMP‐4 is a potent antiinflammatory agent, and that its antiarthritis function may be mediated by MMPs. Arthritis‐inhibiting effects of TIMP‐4 may suggest a unique application of this gene therapy method for arthritis.

     

Paradoxical effects of tissue inhibitor of metalloproteinases 1 gene transfer in collagen‐induced arthritis

Objective

The imbalance between matrix metalloproteinases (MMPs) 1, 3, and 9 and their specific inhibitor, tissue inhibitor of metalloproteinases 1 (TIMP‐1), is a critical step in cartilage injury and angiogenesis in arthritis. To explore the therapeutic potential of TIMP‐1 gene transfer in erosive arthritis, the effects of an adenoviral vector (Ad‐TIMP‐1) were assessed in DBA/1 mice with collagen‐induced arthritis (CIA).

Methods

DBA/1 mice with CIA received an intravenous injection of replication‐deficient adenovirus containing the human TIMP‐1 gene or a control LacZ gene on day 28 postimmunization. The efficiency of gene transfer was determined by serum TIMP‐1 detection, measurements of paw swelling, as well as radiologic and histologic examination of the paws.

Results

A single administration of Ad‐TIMP‐1 resulted in detectable serum levels of the exogenous protein for at least 13 days. The incidence and onset of arthritis were not statistically modified after human TIMP‐1 gene transfer in DBA/1 mice compared with control mice. However, the severity of inflammation was statistically significantly increased in Ad‐TIMP‐1–treated mice and a similar trend was observed in the histologic and radiologic scores. With regard to the mechanisms of the worsened effect in the Ad‐TIMP‐1–treated mice, we observed 1) higher serum levels of anti–type II collagen IgG2a, 2) a significant increase in endogenous soluble tumor necrosis factor receptor I (TNFRI) in sera, and 3) increased labeling of mouse tumor necrosis factor α and TNFRI within arthritic joints.

Conclusion

These findings show that overexpression of TIMP‐1 does not prevent osteochondral injury in a mouse model of arthritis. Since MMPs have overlapping properties in terms of their roles in extracellular matrix degradation, angiogenesis, and shedding of cell surface adhesion molecules, cytokines, and cytokine receptors, the paradoxical results obtained suggest that TIMP‐1 is probably not the main inhibitor to target.

     

Involvement of matrix metalloproteinases and their inhibitors in peripheral synovitis and down‐regulation by tumor necrosis factor α blockade in spondylarthropathy

Objective

To investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in spondylarthropathy (SpA) synovitis.

Methods

Paired samples of synovial biopsy tissue as well as serum and synovial fluid (SF) from 41 patients with SpA and 20 patients with rheumatoid arthritis (RA) and serum samples from 20 healthy controls were analyzed by immunohistochemistry and enzyme‐linked immunosorbent assay for the presence of MMPs 1, 2, 3, and 9 and TIMPs 1 and 2. In addition, sera from 16 patients with ankylosing spondylitis (AS) and peripheral synovitis and 17 patients with AS and exclusively axial involvement were analyzed. An additional cohort of SpA patients was analyzed at baseline and after 12 weeks of infliximab treatment.

Results

Staining for MMPs and TIMPs showed a cellular and interstitial pattern in the synovial lining and sublining layers that was similar between the RA and SpA patients. Involvement of MMPs and TIMPs in SpA synovitis was suggested by the correlation with cellular infiltration, vascularization, and cartilage degradation. Higher serum levels of MMPs 3 and 9 were revealed in SpA and RA patients as compared with healthy controls. Production of MMP‐3, but not MMP‐9, in the serum reflected the presence of peripheral synovitis, as indicated by 1) the correlation between serum levels, SF levels (which were 1,000‐fold higher than the serum levels), and synovial expression of MMP‐3, 2) the increased levels of MMP‐3 in AS patients with peripheral disease and not exclusively axial involvement, and 3) the correlation of serum and SF MMP‐3 with parameters of synovial, but not systemic, inflammation. The modulation of the MMP/TIMP system by tumor necrosis factor α (TNFα) blockade was confirmed by the down‐regulation of all MMPs and TIMPs in the synovium and a pronounced and rapid decrease of serum MMP‐3.

Conclusion

MMPs and TIMPs are highly expressed in SpA synovitis and mirror both the inflammatory and tissue‐remodeling aspects of the local disease process. Serum MMP‐3, originating from the inflamed joint, represents a valuable biomarker for peripheral synovitis. Modulation of the MMP/TIMP system by infliximab could contribute to the antiinflammatory and tissue‐remodeling effects of TNFα blockade in SpA.

     

Heterogeneous requirement of IκB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: Implications for therapy

Objective

To investigate the potential role of IκB kinase 1 (IKK‐1) and IKK‐2 in the regulation of nuclear factor κB (NF‐κB) activation and the expression of tumor necrosis factor α (TNFα), as well as interleukin‐1β (IL‐1β), IL‐6, IL‐8, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), in rheumatoid arthritis (RA).

Methods

Recombinant adenoviruses expressing β‐galactosidase, dominant‐negative IKK‐1 and IKK‐2, or IκBα were used to infect ex vivo RA synovial membrane cultures and synovial fibroblasts obtained from patients with RA undergoing joint replacement surgery, or human dermal fibroblasts, human umbilical vein endothelial cells (HUVECs), and monocyte‐derived macrophages from healthy volunteers. Then, their effect on the spontaneous or stimulus‐induced release of inflammatory cytokines, VEGF, and MMPs from RA synovial membrane cells was examined.

Results

IKK‐2 was not required for lipopolysaccharide (LPS)–induced NF‐κB activation or TNFα, IL‐6, or IL‐8 production in macrophages, but was essential for this process in response to CD40 ligand, TNFα, and IL‐1. In synovial fibroblasts, dermal fibroblasts, and HUVECs, IKK‐2 was also required for LPS‐induced NF‐κB activation and IL‐6 or IL‐8 production. In RA synovial membrane cells, IKK‐2 inhibition had no effect on spontaneous TNFα production but significantly reduced IL‐1β, IL‐6, IL‐8, VEGF, and MMPs 1, 2, 3, and 13.

Conclusion

Our study demonstrates that IKK‐2 is not essential for TNFα production in RA. However, because IKK‐2 regulates the expression of other inflammatory cytokines (IL‐1β, IL‐6, and IL‐8), VEGF, and MMPs 1, 2, 3, and 13, which are involved in the inflammatory, angiogenic, and destructive processes in the RA joint, it may still be a good therapeutic target.

     

Demyelination occurring during anti–tumor necrosis factor α therapy for inflammatory arthritides

Objective

To review the occurrence of neurologic events suggestive of demyelination during anti–tumor necrosis factor α (anti‐TNFα) therapy for inflammatory arthritides.

Methods

The Adverse Events Reporting System of the Food and Drug Administration (FDA) was queried following a report of a patient with refractory rheumatoid arthritis who developed confusion and difficulty with walking after receiving etanercept for 4 months.

Results

Nineteen patients with similar neurologic events were identified from the FDA database, 17 following etanercept administration and 2 following infliximab administration for inflammatory arthritis. All neurologic events were temporally related to anti‐TNFα therapy, with partial or complete resolution on discontinuation. One patient exhibited a positive rechallenge phenomenon.

Conclusion

Further surveillance and studies are required to better define risk factors for and frequency of adverse events and their relationship to anti‐TNFα therapies. Until more long‐term safety data are available, consideration should be given to avoiding anti‐TNFα therapy in patients with preexisting multiple sclerosis and to discontinuing anti‐TNFα therapy immediately when new neurologic signs and symptoms occur, pending an appropriate evaluation.

     

Oncostatin M in combination with tumor necrosis factor α induces cartilage damage and matrix metalloproteinase expression in vitro and in vivo

Objective

To determine the effects of the proinflammatory cytokine combination of oncostatin M (OSM) and tumor necrosis factor α (TNFα) on cartilage destruction in both in vitro and in vivo model systems.

Methods

The release of collagen and proteoglycan was assessed in bovine cartilage explant cultures, while messenger RNA (mRNA) from bovine chondrocytes was analyzed by Northern blotting. Immunohistochemistry was performed on sections prepared from murine joints following injection of adenovirus vectors encoding murine OSM and/or murine TNFα.

Results

The combination of OSM + TNFα induced significant collagen release from bovine cartilage, accompanied by high levels of active collagenolytic activity. Northern blot analysis indicated that this cytokine combination synergistically induced matrix metalloproteinase 1 (MMP‐1), MMP‐3, and MMP‐13 mRNA. The in vivo data clearly indicated that OSM + TNFα overexpression increased MMP levels and decreased levels of tissue inhibitor of metalloproteinases 1 (TIMP‐1). Specifically, OSM + TNFα induced marked synovial hyperplasia, inflammation, and cartilage and bone destruction with a concomitant increase in MMP expression in both cartilage and synovium and decreased TIMP‐1 expression in the articular cartilage. These effects were markedly greater than those seen with either cytokine alone.

Conclusion

This study demonstrates that OSM + TNFα represents a potent proinflammatory cytokine combination that markedly induces MMP production in both cartilage and synovium, thus promoting joint destruction.

     

Effects of intraarticular glucocorticoids on macrophage infiltration and mediators of joint damage in osteoarthritis synovial membranes: Findings in a double‐blind,placebo‐controlled study

Objective

To investigate the effects of intraarticular glucocorticoid treatment on macrophage infiltration, the expression of the chemokines monocyte chemoattractant protein 1 (MCP‐1) and macrophage inflammatory protein 1α (MIP‐1α), and the expression of matrix metalloproteinases 1 and 3 (MMPs 1 and 3) and their inhibitors, the tissue inhibitors of metalloproteinases 1 and 2 (TIMPs 1 and 2), in osteoarthritis (OA) synovial membranes.

Methods

Forty patients underwent arthroscopic biopsy before and 1 month after intraarticular injection of glucocorticoids. Twenty‐one patients received 120 mg of methylprednisolone acetate (Depo‐Medrol; Upjohn, Kalamazoo, MI), and 20 patients received placebo (1 patient received placebo in 1 knee and methylprednisolone acetate in the other). Immunoperoxidase staining for the expression of CD68, MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 was performed, and the immunostaining was quantified by color video image analysis.

Results

CD68, MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 immunostaining was observed in all synovial membranes. Intraarticular glucocorticoid treatment was associated with a small (30%) but statistically significant (P = 0.048) reduction in CD68+ macrophage staining in the synovial lining layer, but there was no change in the CD68 expression in the synovial sublining layer. No significant differences were observed for MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 immunostaining in the synovial lining or sublining layers.

Conclusion

Intraarticular glucocorticoids may reduce CD68+ macrophage infiltration into the synovial lining layer, but not the expression of MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 in the synovial membrane, in patients with OA.

     

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.

     

Enhanced integrative repair of the porcine meniscus in vitro by inhibition of interleukin‐1 or tumor necrosis factor α

Objective

To examine the hypotheses that increasing concentrations of interleukin‐1 (IL‐1) or tumor necrosis factor α (TNFα) inhibit the integrative repair of the knee meniscus in an in vitro model system, and that inhibitors of these cytokines will enhance repair.

Methods

Explants (8 mm in diameter) were harvested from porcine medial menisci. To simulate a full‐thickness defect, a 4‐mm–diameter core was removed and reinserted. Explants were cultured for 14, 28, or 42 days in the presence of 0–1,000 pg/ml of IL‐1 or TNFα. Explants were also cultured in the presence of IL‐1 or TNFα with IL‐1 receptor antagonist (IL‐1Ra) or TNF monoclonal antibody (mAb). At the end of the culture period, biomechanical testing, cell viability, and histologic analyses were performed to quantify the extent of repair.

Results

Mechanical testing revealed increased repair strength, cell accumulation, and tissue formation at the interface over time under control conditions. Pathophysiologic concentrations of both IL‐1 and TNFα significantly decreased repair strength, cell migration, and tissue formation at the interface. The addition of IL‐1Ra or TNF mAb to explants prevented the effects of IL‐1 or TNFα, respectively.

Conclusion

Our findings document that physiologically relevant concentrations of IL‐1 and TNFα inhibit meniscal repair in vitro and therefore may also inhibit meniscal repair during arthritis or following joint injury. The finding that IL‐1Ra and TNF mAb promoted integrative meniscal repair in an inflammatory microenvironment suggests that intraarticular delivery of IL‐1Ra and/or TNF mAb may be useful clinically to promote meniscal healing following injury.

     

Tumor necrosis factor α–mediated cleavage and inactivation of SirT1 in human osteoarthritic chondrocytes

Objective

The protein deacetylase SirT1 positively regulates cartilage‐specific gene expression, while the proinflammatory cytokine tumor necrosis factor α (TNFα) negatively regulates these same genes. This study was undertaken to test the hypothesis that SirT1 is adversely affected by TNFα, resulting in altered gene expression.

Methods

Cartilage‐specific gene expression, SirT1 activity, and results of chromatin immunoprecipitation analysis at the α2(I) collagen enhancer site were determined in RNA, protein extracts, and nuclei of human osteoarthritic chondrocytes left untreated or treated with TNFα. Protein extracts from human chondrocytes transfected with epitope‐tagged SirT1 that had been left untreated or had been treated with TNFα were analyzed by immunoblotting with SirT1 and epitope‐specific antibodies. The 75‐kd SirT1‐reactive protein present in TNFα‐treated extracts was identified by mass spectroscopy, and its amino‐terminal cleavage site was identified via Edman sequencing. SirT1 activity was assayed following an in vitro cathepsin B cleavage reaction. Cathepsin B small interfering RNA (siRNA) was transfected into chondrocytes left untreated or treated with TNFα.

Results

TNFα‐treated chondrocytes had impaired SirT1 enzymatic activity and displayed 2 forms of the enzyme: a full‐length 110‐kd protein and a smaller 75‐kd fragment. The 75‐kd SirT1 fragment was found to lack the carboxy‐terminus. Cathepsin B was identified as the TNFα‐responsive protease that cleaves SirT1 at residue 533. Reducing cathepsin B levels via siRNA following TNFα exposure blocked the generation of the 75‐kd SirT1 fragment.

Conclusion

These data indicate that TNFα, a cytokine that mediates joint inflammation in arthritis, induces cathepsin B–mediated cleavage of SirT1, resulting in reduced SirT1 activity. This reduced SirT1 activity correlates with the reduced cartilage‐specific gene expression evident in these TNFα‐treated cells.

     

Therapeutic effects of a novel tylophorine analog,NK‐007, on collagen‐induced arthritis through suppressing tumor necrosis factor α production and Th17 cell differentiation

Objective

To analyze the effects of a novel compound, NK‐007, on the prevention and treatment of collagen‐induced arthritis (CIA) and the underlying mechanisms.

Methods

We determined the effect of NK‐007 on lipopolysaccharide (LPS)–triggered tumor necrosis factor α (TNFα) production by murine splenocytes and a macrophage cell line (RAW 264.7) by enzyme‐linked immunosorbent assay, intracellular cytokine staining, and Western blotting. The LPS‐boosted CIA model was adopted, and NK‐007 or vehicle was administered at different time points after immunization. Mice were monitored for clinical severity of arthritis, and joint tissues were used for histologic examination, cytokine detection, and immunohistochemical staining. Finally, stability of TNFα production and Th17 cell differentiation were studied using quantitative polymerase chain reaction and flow cytometry.

Results

NK‐007 significantly suppressed LPS‐induced TNFα production in vitro. Administration of NK‐007 completely blocked CIA development and delayed its progression. Furthermore, treatment with NK‐007 at the onset of arthritis significantly inhibited the progress of joint inflammation. Administration of NK‐007 also suppressed production of TNFα, interleukin‐6 (IL‐6), and IL‐17A in the joint and reduced percentages of IL‐17+ cells among CD4+ and γ/δ T cells in draining lymph nodes. We further demonstrated that NK‐007 acted on the stability of TNFα messenger RNA and reduced Th17 cell differentiation. In addition, it significantly inhibited levels of IL‐6 and IL‐17A in human coculture assay.

Conclusion

For its effects on the development and progression of CIA and for its therapeutic effect on CIA, NK‐007 has great potential to be a therapeutic agent for human rheumatoid arthritis.

     

The transcription factors hypoxia‐inducible factor 1α and Ets‐1 colocalize in the hypoxic synovium of inflamed joints in adjuvant‐induced arthritis

Objective

To determine the relationship between hypoxia and the expression of Ets‐1 and hypoxia‐inducible factor 1α (HIF‐1α) in both normal and inflamed joints. Adjuvant‐induced arthritis (AIA) was used as the model system, since it mirrors many aspects of the pathology of rheumatoid arthritis.

Methods

Adjuvant arthritis was induced in a group of 10 female Lewis rats. A second group of 10 uninjected female Lewis rats served as naive controls. When a maximum clinical joint score was achieved in the AIA group, all 20 rats were injected with the specific hypoxic cell marker Hypoxyprobe‐1 and subsequently killed. Hypoxyprobe‐1 adducts, Ets‐1, and HIF‐1α were localized in the joints of the hind feet from these groups using immunohistochemistry.

Results

Compared with the joints from control rats, inflamed joints contained markedly more cells with Hypoxyprobe‐1 adduct immunoreactivity, Ets‐1–immunoreactive nuclei, and nuclear immunoreactivity for both Ets‐1 and HIF‐1α.

Conclusion

Our results demonstrate the presence of hypoxia in inflamed joints in this experimental model of arthritis. The colocalization of Ets‐1 and HIF‐1α in these hypoxic areas suggests that hypoxia may induce Ets‐1 and HIF‐1α expression during joint inflammation.

     

Tumor necrosis factor α and RANKL blockade cannot halt bony spur formation in experimental inflammatory arthritis

Objective

To investigate the kinetics of bony spur formation and the relationship of bony spur formation to synovial inflammation and bone erosion in 2 rat arthritis models, and to address whether bony spur formation depends on the expression of tumor necrosis factor α (TNFα) or RANKL.

Methods

Analysis of the kinetics of synovial inflammation, bone erosion, osteoclast formation, and growth of bony spurs was performed in rat collagen‐induced arthritis (CIA) and adjuvant‐induced arthritis (AIA). In addition, inhibition experiments were performed to assess whether inhibition of TNFα and RANKL by pegylated soluble TNF receptor type I (pegTNFRI) and osteoprotegerin (OPG), respectively, affected bony spur formation.

Results

Bony spurs emerged from the periosteal surface close to joints, and initial proliferation of mesenchymal cells was noted as early as 3 days and 5 days after onset of CIA and AIA, respectively. Initiation of bony spur formation occurred shortly after the onset of inflammation and bone erosion. Neither pegTNFRI nor OPG could significantly halt the osteophytic responses in CIA and AIA.

Conclusion

These results suggest that bony spur formation is triggered by inflammation and initial structural damage in these rat models of inflammatory arthritis. Moreover, emergence of bony spurs depends on periosteal proliferation and is not affected by inhibition of either TNFα or RANKL. Bony spur formation can thus be considered a process that occurs independent of TNFα and RANKL and is triggered by destructive arthritis.

     

Tie2 receptor tyrosine kinase,a major mediator of tumor necrosis factor α–induced angiogenesis in rheumatoid arthritis

Objective

Rheumatoid arthritis (RA) is an inflammatory disease and an angiogenic disease. However, the molecular mechanisms promoting angiogenesis in RA are not clearly identified. Our objective was to study the role of an endothelium‐specific receptor tyrosine kinase, Tie2, in angiogenesis of inflammatory arthritis.

Methods

Expression of Tie2 and its ligand, angiopoietin 1 (Ang1), in human synovium was examined by immunohistochemistry and Western blot. A novel synovium vascular window model was established to study the role of Tie2 in angiogenesis in vivo. Primary cultured endothelial cells and synoviocytes were used to study tumor necrosis factor α (TNFα)–induced Tie2 and Ang1 expression.

Results

Tie2 was implicated in pathologic angiogenesis. We observed that Tie2 and Ang1 were elevated in human RA synovium. Using a novel collagen‐induced arthritis synovial window model, we demonstrated that Tie2 signaling regulated arthritis angiogenesis in vivo. We also showed that Tie2 mediated TNFα‐induced angiogenesis in a mouse cornea assay. In addition, we observed that TNFα can regulate Tie2 activation in multiple ways that may involve interactions between endothelial cells and synoviocytes. TNFα up‐regulates Tie2 in endothelial cells through nuclear factor κB, and it up‐regulates Ang1 in synoviocytes. These findings suggest paracrine regulation of angiogenesis between endothelial cells and synoviocytes.

Conclusion

This study demonstrates that Tie2 regulates angiogenesis in inflammatory synovium. Tie2 signaling is an important angiogenic mediator that links the proinflammatory cytokine TNFα to pathologic angiogenesis.

     

Endogenous interleukin‐6, but not tumor necrosis factor α, contributes to the development of toll‐like receptor 4/myeloid differentiation factor 88–mediated acute arthritis in mice

Objective

To generate a mouse model of reactive arthritis (ReA), an aseptic synovitis that develops in joints distant from the primary bacterial infection site, to examine roles for Toll‐like receptors (TLRs) that recognize bacterial components involved in the development of this arthritis, and to identify the cytokine(s) relevant to this arthritis.

Methods

Mice were treated with cell wall extract from Escherichia coli (ECW) gram‐negative bacterium by injection into the footpads. Seven days later, the mice were challenged with lipopolysaccharide (LPS), a TLR‐4 ligand, which was injected into the knee joint cavity. To investigate the cytokine(s) involved in this arthritis, mice deficient in various arthritogenic cytokines, such as interleukin‐6 (IL‐6), IL‐12, IL‐18, interferon‐γ, and tumor necrosis factor α (TNFα), were sequentially treated with ECW and LPS.

Results

ECW‐primed mice manifested acute severe arthritis after intraarticular challenge with ECW or LPS, while unprimed mice exhibited modest changes after these challenges. Mutant mice lacking functional TLR‐4 or myeloid differentiation factor 88 (MyD88), an adaptor molecule of TLR‐4 signaling, were resistant to this arthritis. Although both TNFα and IL‐6 were equally expressed in the joint after LPS challenge, Il6^−/− mice, but not Tnf^−/− mice, were resistant to ECW/LPS‐induced arthritis.

Conclusion

Our present results clearly indicate the importance of priming with ECW and the requirement of TLR‐4/MyD88–mediated IL‐6, but not TNFα, for the development of ECW/LPS‐induced arthritis. LPS‐induced IL‐6, in the absence of TNFα, mediates LPS‐induced arthritis. These results suggest that IL‐6 is a rational target for therapeutic regimens for inflammatory arthritis, including ReA and rheumatoid arthritis.