Effectiveness of the soluble form of the interleukin‐1 receptor accessory protein as an inhibitor of interleukin‐1 in collagen‐induced arthritis

Objective

To investigate whether the soluble form of interleukin‐1 (IL‐1) receptor accessory protein (sIL‐1RAcP), whose physiologic function remains to be established, can serve as a specific inhibitor of IL‐1 signaling in vitro, and to evaluate its applicability in collagen‐induced arthritis (CIA).

Methods

Soluble IL‐1RAcP was cloned from murine liver complementary DNA and expressed by the use of either an adenoviral vector (AdRGD) for sIL‐1RAcP or a stable‐transfected NIH3T3 fibroblast cell line. The ability of affinity‐purified sIL‐1RAcP to inhibit IL‐1 signaling was tested on NF‐κB luciferase reporter fibroblasts and quantified by luminometer. To investigate therapeutic efficacy, sIL‐1RAcP was both locally (knee joint) and systemically overexpressed in collagen‐immunized male DBA/1 mice. Severity of arthritis was monitored visually, and the pathologic process in the joint was examined histologically. Serum was obtained from mice to quantify IL‐6 and anti–bovine type II collagen (BCII) antibody levels.

Results

Incubation of the NF‐κB reporter fibroblast with purified sIL‐1RAcP protein showed a marked reduction of IL‐1–induced, but not tumor necrosis factor–induced, NF‐κB activation. This showed a novel role for sIL‐1RAcP as a specific inhibitor of IL‐1 signaling. Local transplantation of sIL‐1RAcP–producing NIH3T3 fibroblasts into the knee before onset of CIA had little or no effect on general disease severity in these mice. Histologic evaluation of the knee joints receiving sIL‐1RAcP cell transplantation showed a marked reduction in both joint inflammation and bone and cartilage erosion. Local treatment with sIL‐1RAcP had no profound effect on serum levels of IL‐6 and anti‐BCII antibodies, which is indicative of the ongoing presence of arthritis in distal joints. In contrast to local treatment, systemic treatment with the AdRGD for sIL‐1RAcP markedly ameliorated CIA in all joints.

Conclusion

In this study we demonstrated that sIL‐1RAcP is a biologically active and innovative inhibitor of IL‐1, and treatment of mice with sIL‐1RAcP had a profound prophylactic effect on collagen‐induced arthritis.

     

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.

     

Treatment with a neutralizing anti‐murine interleukin‐17 antibody after the onset of collagen‐induced arthritis reduces joint inflammation,cartilage destruction,and bone erosion

Objective

Interleukin‐17 (IL‐17) is a proinflammatory cytokine that is expressed in the synovium of rheumatoid arthritis (RA) patients. This T cell cytokine is implicated in the initiation phase of arthritis. However, the role of IL‐17 during the effector phase of arthritis has still not been identified; this was the objective of the present study.

Methods

Mice with collagen‐induced arthritis (CIA) were treated with polyclonal rabbit anti‐murine IL‐17 (anti–IL‐17) antibody–positive serum or normal rabbit serum after the first signs of arthritis. In addition, during a later stage of CIA mice were selected and treated with anti–IL‐17 antibody or control serum. Arthritis was monitored visually, and joint pathology was examined radiologically and histologically. Systemic IL‐6 levels were measured by enzyme‐linked immunosorbent assay, and local synovial IL‐1 and receptor activator of NF‐κB ligand (RANKL) expression was analyzed using specific immunohistochemistry.

Results

Treatment with a neutralizing anti–IL‐17 antibody after the onset of CIA significantly reduced the severity of CIA. Radiographic analysis revealed marked suppression of joint damage in the knee and ankle joints. Histologic analysis confirmed the suppression of joint inflammation and showed prevention of cartilage and bone destruction after anti–IL‐17 antibody therapy. Systemic IL‐6 levels were significantly reduced after anti–IL‐17 antibody treatment. Moreover, fewer IL‐1β–positive and RANKL‐positive cells were detected in the synovium after treatment with neutralizing IL‐17. Interestingly, initiation of anti–IL‐17 antibody therapy during a later stage of CIA, using mice with higher clinical arthritis scores, still significantly slowed the progression of the disease.

Conclusion

IL‐17 plays a role in early stages of arthritis, but also later during disease progression. Systemic IL‐6 was reduced and fewer synovial IL‐1–positive and RANKL‐positive cells were detected after neutralizing endogenous IL‐17 treatment, suggesting both IL‐1–dependent and IL‐1–independent mechanisms of action. Our data strongly indicate that IL‐17 neutralization could provide an additional therapeutic strategy for RA, particularly in situations in which elevated IL‐17 may attenuate the response to anti–tumor necrosis factor/anti–IL‐1 therapy.

     

Induction of cartilage damage by overexpression of T cell interleukin‐17A in experimental arthritis in mice deficient in interleukin‐1

Objective

To examine the capacity of T cell interleukin‐17A (IL‐17A; referred to hereinafter as IL‐17) to induce cartilage damage during experimental arthritis in the absence of IL‐1.

Methods

Local IL‐17 gene transfer was performed in the knee joint of IL‐1–deficient mice and wild‐type controls during streptococcal cell wall (SCW)–induced arthritis. Knee joints were isolated at various time points for histologic analysis of cartilage proteoglycan (PG) depletion. Expression of messenger RNA for inducible nitric oxide synthase, matrix metalloproteinases (MMPs) 3, 9, and 13, and ADAMTS‐4 was determined by quantitative polymerase chain reaction analysis. VDIPEN staining was analyzed to study MMP‐mediated cartilage damage. In addition, systemic anti–IL‐1α/β antibody treatment was performed in mice immunized with type II collagen and injected locally with an adenoviral vector expressing IL‐17 or with control adenovirus. Knee joints were isolated and analyzed for cartilage PG depletion, chondrocyte death, and cartilage surface erosion.

Results

During SCW‐induced arthritis, local T cell IL‐17 gene transfer turned this acute, macrophage‐driven joint inflammation into a severe, chronic arthritis accompanied by aggravated cartilage damage. Of high interest, the IL‐1 dependency of cartilage PG depletion was fully abrogated when IL‐17 was locally overexpressed in the joint. Moreover, local IL‐17 gene transfer increased MMP expression without the need for IL‐1, although IL‐1 remained essential for part of the cartilage VDIPEN expression. Furthermore, when IL‐17 was overexpressed in the knee joints of mice with collagen‐induced arthritis, anti–IL‐1 treatment did not reduce the degree of chondrocyte death or cartilage surface erosion.

Conclusion

These data show the capacity of IL‐17 to replace the catabolic function of IL‐1 in cartilage damage during experimental arthritis.

     

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.

     

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.

     

T cell dependence of chronic destructive murine arthritis induced by repeated local activation of toll‐like receptor–driven pathways: Crucial role of both interleukin‐1β and interleukin‐17

Objective

The pathogenesis of rheumatoid arthritis is often linked to bacterial infections. The present study was undertaken to develop a mouse model of chronic destructive arthritis induced by repeated intraarticular (IA) exposure to bacterial cell wall fragments and to investigate the cytokine dependence of this model.

Methods

Mice that were deficient in various cytokines were injected IA with cell wall fragments of Streptococcus pyogenes on days 0, 7, 14, and 21. The development of chronic destructive arthritis was compared between groups of mice lacking different cytokines, to assess which cytokines were crucial for development of chronic destructive arthritis.

Results

Repeated exposure of a joint to S pyogenes cell wall fragments resulted in the development of chronic destructive arthritis. In mice deficient in recombination‐activating gene 2, streptococcal cell wall (SCW)–directed T cell reactivity was found and chronic arthritis did not develop, implicating T cells in the generation of chronic SCW‐induced arthritis. Interleukin‐17 (IL‐17) receptor–deficient mice showed a reduction of joint destruction in the chronic stage, implicating a detrimental role of the recently discovered IL‐17–producing T helper cells (Th17 cells). IL‐23 expression was apparent during the late stages of arthritis. Joint swelling was no longer dependent on tumor necrosis factor α (TNFα) after the last flare, and pronounced cartilage damage was found after 28 days in TNFα‐deficient mice. In contrast, IL‐1β–deficient mice were fully protected against joint swelling and cartilage and bone destruction during the late stages of disease.

Conclusion

These findings indicate that the TNFα dependence of arthritis is lost during the erosive stage, when Th17 cells become crucial. IL‐1β dependence remains strong, consistent with its pivotal role in the generation of Th17 cells.

     

Gamma/delta T cells are the predominant source of interleukin‐17 in affected joints in collagen‐induced arthritis,but not in rheumatoid arthritis

Objective

Although interleukin‐17 (IL‐17)–producing γ/δ T cells were reported to play pathogenic roles in collagen‐induced arthritis (CIA), their characteristics remain unknown. The aim of this study was to clarify whether γ/δ T cells or CD4+ T cells are the predominant IL‐17–producing cells, and to determine what stimulates γ/δ T cells to secret IL‐17 in mice with CIA. The involvement of IL‐17–producing γ/δ T cells in SKG mice with autoimmune arthritis and patients with rheumatoid arthritis (RA) was also investigated.

Methods

IL‐17–producing cells in the affected joints of mice with CIA were counted by intracellular cytokine staining during 6 distinct disease phases, and these cells were stimulated with various combinations of cytokines or specific antigens to determine the signaling requirements. Similar studies were performed using SKG mice with arthritis and patients with RA.

Results

Gamma/delta T cells were the predominant population in IL‐17–producing cells in the swollen joints of mice with CIA, and the absolute numbers of these cells increased in parallel with disease activity. IL‐17–producing γ/δ T cells expressed CC chemokine receptor 6, were maintained by IL‐23 but not by type II collagen in vitro, and were induced antigen independently in vivo. Furthermore, IL‐17 production by γ/δ T cells was induced by IL‐1β plus IL‐23 independently of T cell receptor. In contrast to what was observed in mice with CIA, IL‐17–producing γ/δ T cells were nearly absent in the affected joints of SKG mice and patients with RA, and Th1 cells were predominant in the joints of patients with RA.

Conclusion

Gamma/delta T cells were antigen independently stimulated by inflammation at affected joints and produced enhanced amounts of IL‐17 to exacerbate arthritis in mice with CIA but not in SKG mice with arthritis or patients with RA.

     

Inhibition of interleukin‐33 signaling attenuates the severity of experimental arthritis

Objective

Interleukin‐33 (IL‐33; or, IL‐1F11) was recently identified as the ligand of the IL‐1 family receptor T1/ST2. The aim of this study was to examine IL‐33 production in human and mouse joints and to investigate the role of IL‐33 and T1/ST2 in experimental arthritis.

Methods

IL‐33 expression was examined in human synovial tissue, rheumatoid arthritis (RA) synovial fibroblasts, and arthritic mouse joints. Mice with collagen‐induced arthritis (CIA) were treated with blocking anti‐ST2 antibody or control antibody beginning at the onset of disease. Arthritis severity was assessed by clinical and histologic scoring. Draining lymph node (LN) cell responses were examined ex vivo, and joint messenger RNA (mRNA) was used for expression profiling.

Results

IL‐33 was highly expressed in human RA synovium. In cultured synovial fibroblasts, IL‐33 expression was strongly induced by IL‐1β and/or tumor necrosis factor α. Furthermore, IL‐33 mRNA was detected in the joints of mice with CIA and increased during the early phase of the disease. Administration of a blocking anti‐ST2 antibody at the onset of disease attenuated the severity of CIA and reduced joint destruction. Anti‐ST2 antibody treatment was associated with a marked decrease in interferon‐γ production as well as with a more limited reduction in IL‐17 production by ex vivo–stimulated draining LN cells. Finally, RANKL mRNA levels in the joint were reduced by anti‐ST2 treatment.

Conclusion

IL‐33 is produced locally in inflamed joints, and neutralization of IL‐33 signaling has a therapeutic effect on the course of arthritis. These observations suggest that locally produced IL‐33 may contribute to the pathogenesis of joint inflammation and destruction.

     

Plasmid encoding interleukin‐4 in the amelioration of murine collagen‐induced arthritis

Objective

To evaluate the therapeutic effect of the administration of plasmid encoding interleukin‐4 (IL‐4) via gene‐gun delivery and via intradermal injection on collagen‐induced arthritis (CIA).

Methods

IL‐4 plasmid was administered by gene‐gun delivery and intradermal injection to DBA/1 mice immunized with type II collagen (CII). The therapeutic effect on the development of CIA was evaluated clinically with a visual scoring method for arthritis and serologically by enzyme‐linked immunosorbent assays and polymerase chain reaction.

Results

Treatment with IL‐4–expressing plasmid significantly reduced the incidence and severity of CIA, including a reduction in the anti‐CII antibody level. In particular, gene‐gun delivery had a higher immunosuppressive effect on CIA compared with intradermal injection. As shown by in vitro stimulation assay, the spleen cells from mice immunized with CII and treated with IL‐4 plasmid via gene gun exhibited higher Th2 cytokine responses compared with cells treated with control plasmid after in vitro stimulation with CII.

Conclusion

The results of this study suggest that treatment with IL‐4 plasmid may constitute a new clinical use of cytokine gene therapy for rheumatoid arthritis.

     

The parasitic helminth product ES‐62 suppresses pathogenesis in collagen‐induced arthritis by targeting the interleukin‐17–producing cellular network at multiple sites

Objective

Among many survival strategies, parasitic worms secrete molecules that modulate host immune responses. One such product, ES‐62, is protective against collagen‐induced arthritis (CIA), a model of rheumatoid arthritis (RA). Since interleukin‐17 (IL‐17) has been reported to play a pathogenic role in the development of RA, this study was undertaken to investigate whether targeting of IL‐17 may explain the protection against CIA afforded by ES‐62.

Methods

DBA/1 mice progressively display arthritis following immunization with type II collagen. The protective effects of ES‐62 were assessed by determination of cytokine levels, flow cytometric analysis of relevant cell populations, and in situ analysis of joint inflammation in mice with CIA.

Results

ES‐62 was found to down‐regulate IL‐17 responses in mice with CIA. First, it acted to inhibit priming and polarization of IL‐17 responses by targeting a complex IL‐17–producing network, involving signaling between dendritic cells and γ/δ or CD4+ T cells. In addition, ES‐62 directly targeted Th17 cells by down‐regulating myeloid differentiation factor 88 expression to suppress responses mediated by IL‐1 and Toll‐like receptor ligands. Moreover, ES‐62 modulated the migration of γ/δ T cells and this was reflected by direct suppression of CD44 up‐regulation and, as evidenced by in situ analysis, dramatically reduced levels of IL‐17–producing cells, including lymphocytes, infiltrating the joint. Finally, there was strong suppression of IL‐17 production by cells resident in the joint, such as osteoclasts within the bone areas.

Conclusion

Our findings indicate that ES‐62 treatment of mice with CIA leads to unique multisite manipulation of the initiation and effector phases of the IL‐17 inflammatory network. ES‐62 could be exploited in the development of novel therapeutics for RA.

     

Interferon‐γ regulates susceptibility to collagen‐induced arthritis through suppression of interleukin‐17

Objective

The enhanced expression of experimental arthritis in the absence of interferon‐γ (IFNγ) suggests that IFNγ suppresses arthritis. Interleukin‐17 (IL‐17) is a pivotal T cell cytokine in arthritis, and in vitro studies have indicated that IFNγ suppresses IL‐17 production. We undertook this study to test the hypothesis that resistance to collagen‐induced arthritis (CIA) in C57BL/6 (B6) mice is regulated by IFNγ‐mediated suppression of IL‐17.

Methods

Wild‐type (WT) B6 mice, IFNγ‐knockout (KO) B6 mice, and DBA/1 mice were immunized with type II collagen (CII) in Freund's complete adjuvant (CFA). Lymphocytes from immunized mice were analyzed for cytokine production ex vivo by intracellular staining or restimulation with CII and enzyme‐linked immunosorbent assays. In vivo blockade of IL‐17 was achieved with an anti–IL‐17 monoclonal antibody (mAb).

Results

CII restimulation of T cells from CII/CFA‐immunized mice resulted in an ∼5‐fold increase in IL‐17 production in IFNγ‐KO B6 mice compared with WT B6 mice. Neutralization of IFNγ increased IL‐17 production in WT B6 mice, and neutralization of IL‐4 had a synergistic effect. Interestingly, the prototypical CIA‐susceptible strain DBA/1 also demonstrated a high IL‐17 and a low IFNγ cytokine profile compared with WT B6 mice. Administration of the anti–IL‐17 mAb attenuated arthritis in DBA/1 mice and almost completely prevented expression of arthritis in IFNγ‐KO B6 mice.

Conclusion

These results indicate that sensitivity of IFNγ‐deficient B6 mice to CIA is associated with high IL‐17 production and that this cytokine is required for expression of arthritis in this strain.

     

The combination of tumor necrosis factor α blockade with interleukin‐1 and interleukin‐17 blockade is more effective for controlling synovial inflammation and bone resorption in an ex vivo model

Objective

Anti–tumor necrosis factor α (anti‐TNFα) therapy has shown efficacy in the treatment of rheumatoid arthritis (RA). Since interleukin‐1 (IL‐1), TNFα, and IL‐17 have many additive and/or synergistic effects in vitro, we tested whether their combined inhibition by soluble receptors would lead to an enhanced effect on ex vivo models of synovial inflammation and bone destruction.

Methods

RA synovium and bone explants were cultured for 7 days in the presence of 1 μg/ml soluble TNFα receptor (STNFR; as in current therapy), type II soluble IL‐1 receptor (sIL‐1RII), or sIL‐17R either alone or in combination. Their effects on the production of IL‐6 and the release of C‐telopeptide of type I collagen (CTX), a marker of type I collagen destruction, were measured by enzyme‐linked immunosorbent assay.

Results

In synovium, each soluble receptor alone decreased IL‐6 production and CTX release by ∼35% and ∼55%, respectively. The combination of all 3 receptors was more effective, inhibiting IL‐6 production and collagen degradation by up to 70%. Neither sIL‐17R, sIL‐1RII, or sTNFR alone had no effect (or an effect of <20% inhibition) on IL‐6 production in 18%, 33%, and 22%, respectively, of the samples. In bone, sIL‐17R, sIL‐1RII, and sTNFR decreased IL‐6 production by 23%, 50%, and 37%, respectively, while the combination decreased IL‐6 production by 75%. A 50% inhibition of CTX release was obtained with sIL‐1RII for 63% of the samples versus 38% of the samples with either sTNFR or sIL‐17R. However, the combination of all 3 receptors was not more potent than sIL‐1RII alone.

Conclusion

The inhibitory effect of sTNFR on IL‐6 production and collagen degradation in RA synovium and bone was increased in combination with sIL‐17R and sIL‐1RII. These results support the concept of combination therapy, which may increase the percentage of responding patients as well as the degree of individual patient response.

     

Prostaglandin E2 exacerbates collagen‐induced arthritis in mice through the inflammatory interleukin‐23/interleukin‐17 axis

Objective

Recently, Th17 cells, a new subset of CD4+ T cells, emerged as major players in inflammation/autoimmunity. Maintenance of the Th17 phenotype requires interleukin‐23 (IL‐23), whereas the Th1‐promoting cytokine IL‐12p70 exerts a negative effect on Th17 cell differentiation. The lipid mediator prostaglandin E₂ (PGE₂) acts primarily as a proinflammatory agent in autoimmune conditions, through mechanisms that remain to be elucidated. The aim of this study was to investigate whether PGE₂ released in inflammatory foci activates resident dendritic cells (DCs) to express IL‐23 (at the expense of IL‐12) and IL‐6, resulting in a shift toward Th17 cell responses.

Methods

The effect of PGE₂ on IL‐23 production by DCs and subsequent induction of T cell–derived IL‐17 was assessed in vitro and in vivo. The effect of the stable PGE analog misoprostol was evaluated in a murine model of rheumatoid arthritis, in conjunction with IL‐23 and IL‐17 expression in affected joints and draining lymph nodes.

Results

In vivo administration of PGE₂ induced IL‐23–dependent IL‐17 production. Administration of misoprostol exacerbated collagen‐induced arthritis (CIA). CIA exacerbation was associated with increased levels of IL‐23p19/p40 messenger RNA and reduced expression of IL‐12p35, and with increased levels of the proinflammatory cytokines IL‐17, IL‐1β, IL‐6, and tumor necrosis factor in the affected joint. Following ex vivo restimulation, draining lymph node cells from misoprostol‐treated mice secreted higher levels of IL‐17 and lower levels of interferon‐γ.

Conclusion

Our results indicate that PGE₂ enhances DC‐derived IL‐6 production and induces a shift in the IL‐23/IL‐12 balance in favor of IL‐23, resulting in increased IL‐17 production, presumably through the amplification of self‐reactive Th17 cells.

     

Interleukin‐17 receptor deficiency results in impaired synovial expression of interleukin‐1 and matrix metalloproteinases 3, 9, and 13 and prevents cartilage destruction during chronic reactivated streptococcal cell wall–induced arthritis

Objective

To examine the role of interleukin‐17 receptor (IL‐17R) signaling in cartilage destruction and its interrelationship with synovial IL‐1 expression during chronic reactivated streptococcal cell wall (SCW)–induced arthritis.

Methods

SCW arthritis was repeatedly induced in wild‐type (WT) and IL‐17R–deficient (IL‐17R^–/–) mice. At different time points, joint inflammation was assessed by using calipers to measure joint swelling. On day 42, mice were killed, and knee joints were removed for histologic analysis. Quantitative polymerase chain reaction (PCR) analyses for different proinflammatory mediators and matrix metalloproteinases (MMPs) were performed on inflamed synovium from WT and IL‐17R^–/– mice after 5 repeated injections of SCW fragments.

Results

IL‐17R signaling did not play a significant role in acute joint swelling induced by a single injection of SCW fragments directly into the joint. However, repeated local injections of SCW fragments into the knee joints of IL‐17R^–/– mice resulted in fewer infiltrating cells in the joint compared with WT mice. Moreover, histologic analysis on day 42 revealed a significant suppression of the degree of chondrocyte death and an absence of cartilage surface erosion in IL‐17R^–/– mice. Quantitative PCR analysis revealed impaired synovial expression of IL‐1, IL‐6, cyclooxygenase 2, stromelysin (MMP‐3), gelatinase B (MMP‐9), and collagenase 3 (MMP‐13) in IL‐17R^–/– mice.

Conclusion

These data show a critical role of IL‐17R signaling in driving the synovial expression of proinflammatory and catabolic mediators, such as IL‐1 and different MMPs, during progression from an acute, macrophage‐driven joint inflammation to a chronic, cartilage‐destructive, T cell–mediated synovitis. Prevention of IL‐17R signaling warrants consideration as a therapeutic target in chronic destructive arthritis.

     

Important role of interleukin‐3 in the early phase of collagen‐induced arthritis

Objective

Activation of basophils contributes to memory immune responses and results in exacerbation of collagen‐induced arthritis (CIA). We undertook the present study to analyze the production and biologic effects of interleukin‐3 (IL‐3), a strong activator of basophils, in CIA.

Methods

Arthritis was induced by immunization with type II collagen. Mice were treated with blocking monoclonal antibodies against IL‐3 or with recombinant IL‐3. Clinical scoring, histologic analysis, fluorescence‐activated cell sorter analysis, enzyme‐linked immunosorbent assay, and cell culturing were performed to assess disease activity and IL‐3 production.

Results

IL‐3 was produced in large quantities by collagen‐specific CD4+ T cells in the spleen and was present in the synovial tissue during onset of arthritis, but was down‐regulated in paws with severe inflammation. Blockade of IL‐3 during the time of arthritis onset resulted in profound improvement of the disease, with reductions in synovial leukocyte and cytokine levels, peripheral blood basophil levels, and anticollagen antibody titers. Blockade of IL‐3 during the late phase of arthritis had no beneficial effect. Administration of recombinant IL‐3 during onset of arthritis induced a marked exacerbation of the disease, with increased peripheral blood basophil and plasma IL‐6 levels and increased titers of anticollagen antibody. In studies of the regulation of IL‐3 expression in CD4+ T cells, IL‐6 and IL‐4 suppressed the release of IL‐3 by activated CD4+ T cells, whereas lipopolysaccharide and CpG DNA up‐regulated IL‐3 secretion in activated CD4+ T cells by acting on costimulatory cells.

Conclusion

Taken together, the present results demonstrate for the first time that IL‐3 has an important role in the early phase of CIA.

     

Protective effect of RC‐3095, an antagonist of the gastrin‐releasing peptide receptor,in experimental arthritis

Objective

To evaluate the antiinflammatory effects of RC‐3095 in 2 experimental models of arthritis, collagen‐induced arthritis (CIA) and antigen‐induced arthritis (AIA), and to determine the mechanisms of action involved.

Methods

RC‐3095 was administered daily to mice with CIA and mice with AIA, after induction of disease with methylated bovine serum albumin. Disease incidence and severity were assessed using a clinical index and evaluation of histologic features, respectively. In mice with CIA, gastrin‐releasing peptide receptor (GRPR) was detected by immunohistochemical analysis, while in mice with AIA, migration of neutrophils, presence of glycosaminoglycans, and lymphocyte proliferation, determined using the MTT assay, were assessed. Expression of cytokines interleukin‐17 (IL‐17), IL‐1β, and tumor necrosis factor α (TNFα) was evaluated in all mouse knees using enzyme‐linked immunosorbent assay. Treg cell production was assessed by flow cytometry in the joints of mice with AIA.

Results

In mice with AIA, administration of RC‐3095 reduced neutrophil migration, mechanical hypernociception, and proteoglycan loss. These findings were associated with inhibition of the levels of all 3 proinflammatory cytokines, decreased lymphocyte proliferation, and increased Treg cell numbers. In the CIA model, treatment with RC‐3095 led to a significant reduction in arthritis clinical scores and the severity of disease determined histologically. Synovial inflammation, synovial hyperplasia, pannus formation, and extensive erosive changes were all dramatically reduced in the arthritic mice treated with RC‐3095. Furthermore, arthritic mice treated with RC‐3095 showed a significant reduction in the concentrations of IL‐17, IL‐1β, and TNFα, and showed a diminished expression of GRPR.

Conclusion

These findings suggest that the GRP pathway has a significant role in chronic arthritis, and its inhibition can be explored as a possible therapeutic strategy in rheumatoid arthritis.

     

Enhanced Th1 and Th17 responses and arthritis severity in mice with a deficiency of myeloid cell–specific interleukin‐1 receptor antagonist

Objective

The balance between interleukin‐1 (IL‐1) and its specific inhibitor, the IL‐1 receptor antagonist (IL‐1Ra), plays a major role in the development of arthritis. The purpose of this study was to investigate the role of IL‐1Ra produced specifically by myeloid cells in the control of collagen‐induced arthritis (CIA) by using myeloid cell–specific IL‐1Ra–deficient mice (IL‐1Ra^ΔM).

Methods

IL‐1Ra^ΔM mice were generated by using the loxP/Cre recombinase system. CIA was induced in IL‐1Ra^ΔM mice and littermate control mice by a single immunization with bovine type II collagen (CII) in Freund's complete adjuvant. Arthritis severity was assessed by clinical and histologic scoring. Draining lymph node (DLN) cell responses were examined ex vivo, and ankle extracts were used in the quantification of cytokines and chemokines.

Results

Clinical and histopathologic evaluations revealed an early disease onset and a severe form of CIA in IL‐1Ra^ΔM mice. This was characterized by increased production of interferon‐γ (IFNγ) and IL‐17 by CII‐stimulated DLN cells. We also observed that the CII‐specific CD4+ T cell response shifted in vivo, from a dominant Th1 response early in the course of the arthritis to the presence of both Th1 and Th17 cytokines later in the disease course. Interestingly, IL‐1Ra levels were higher in the arthritic joints of IL‐1Ra^ΔM mice as compared with the controls, indicating that nonmyeloid cells strongly contribute to the local production of IL‐1Ra. However, this enhanced IL‐1Ra production was not sufficient to limit joint inflammation and tissue damage.

Conclusion

Our results suggest that myeloid cell–derived IL‐1Ra plays a critical role in the control of the development and the severity of CIA by modulating Th1 and Th17 responses in lymphoid organs.