IL-17B and IL-17C are associated with TNF-alpha production and contribute to the exacerbation of inflammatory arthritis

IL-17A is a T cell-derived proinflammatory cytokine that contributes to the pathogenesis of rheumatoid arthritis. Recently, six related molecules have been identified to form the IL-17 family, as follows: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. Whereas IL-17A and IL-17F up-regulate IL-6 in synovial fibroblasts, IL-17B and IL-17C are reported to stimulate the release of TNF-alpha and IL-1beta from the monocytic cell line, THP-1 cell. However, their detailed function remains to be elucidated. We report in this study the effects of IL-17 family on the collagen-induced arthritis (CIA) progression by T cell gene transfer and bone marrow chimeric mice. The mRNA expressions of IL-17 family (IL-17A, IL-17B, IL-17C, and IL-17F) and their receptor (IL-17R and IL-17Rh1) genes in the arthritic paws of CIA mice were elevated compared with controls. Although IL-17A and IL-17F were expressed in CD4(+) T cells, IL-17B and IL-17C were expressed in the cartilage and in various cell populations in the CIA arthritic paws, respectively. In vitro, IL-17A, IL-17B, IL-17C, and IL-17F induced TNF-alpha production in mouse peritoneal exudate cells. In vivo, adoptive transfer of IL-17B- and IL-17C-transduced CD4(+) T cells evidently exacerbated arthritis. Bone marrow chimeric mice of IL-17B and IL-17C exhibited elevated serum TNF-alpha concentration and the high arthritis score upon CIA induction. Moreover, neutralization of IL-17B significantly suppressed the progression of arthritis and bone destruction in CIA mice. Therefore, not only IL-17A, but also IL-17B and IL-17C play an important role in the pathogenesis of inflammatory arthritis.     

Schistosoma mansoni infection reduces severity of collagen-induced arthritis via down-regulation of pro-inflammatory mediators

Various experimental and epidemiological studies have demonstrated that helminth infections affect outcomes of allergic or autoimmune disorders. Here, we examined the effects of Schistosoma mansoni infection on mouse collagen-induced arthritis, one of the most widely used animal models for rheumatoid arthritis. Male DBA/1 mice were infected with S. mansoni 2 weeks prior to being immunized with type II collagen (IIC). Cytokine mRNA expression in mouse paws, cytokine production by ConA-stimulated spleen cells, and anti-IIC antibodies were evaluated in addition to the severity of arthritis. S. mansoni infection significantly reduced the severity of arthritis. Anti-IIC IgG and IgG2a levels were lower in infected than uninfected mice. With regard to cytokine producing potentials in the infected mice, the down-regulation of Th1 (IFNγ) and pro-inflammatory cytokines (TNFα and IL-17A), and up-regulation of Th2 (IL-4) and an anti-inflammatory cytokine (IL-10) were observed. In addition, real-time PCR revealed that the augmentation of pro-inflammatory mediators such as IL-1β, IL-6 and receptor activator of NFκB in inflamed paws was abrogated by S. mansoni infection. In conclusion, schistosome infection reduced the severity of autoimmune arthritis via systemic and local suppression of pro-inflammatory mediators, suggesting the potential of parasite-derived materials as therapeutic agents against rheumatoid arthritis.     

Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws

Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery to diseased joints offers the prospect of achieving high, local concentrations of a therapeutic gene product in a sustained manner, while minimizing exposure of nontarget organs. We report that a single administration of a modified adenovirus encoding the Epstein-Barr-derived homologue of IL-10 can suppress the development of disease for extended periods of time when injected locally within the periarticular tissue surrounding the ankle joints of mice with collagen type II-induced arthritis. Furthermore, we show that injection of an adenoviral vector carrying the IL-10 gene into a single paw can suppress development of arthritis in other, noninjected paws of the same individual. The systemic protection resulting from local gene therapy occurred in the absence of detectable levels of viral IL-10 in the serum. Circulating Ab levels to heterologous collagen were unaffected; however, treatment with viral IL-10 significantly suppressed the development of Abs to autologous mouse type II collagen. Thus, the treatment of a single joint by local delivery of the vIL-10 gene may protect multiple joints of the same individual while avoiding deleterious side effects often associated with systemic therapy.     

Systemic Delivery of Small Interfering RNA Targeting the Interleukin-2/15 Receptor β Chain Prevents Disease Progression in Experimental Arthritis

The role of interleukin (IL)-15 in the pathogenesis of rheumatoid arthritis (RA) is well established; however, systemic knockdown of IL-15 receptor (IL-15R) for reduction in inflammation at local sites has not been demonstrated. In this study, the therapeutic effect of intravenously administered siRNA targeting the β chain of IL-15R which is shared by the receptor for IL-2 was examined in rats with adjuvant-induced arthritis (AA). Polyethylenimine (PEI)-complexed siRNA nanoparticles could easily accumulate in arthritic paws of AA rats. In the paws, the nanoparticles were avidly taken up by macrophages and to a lesser extent by T cells. Weekly administered IL-2/15Rβ siRNA polyplexes were capable of decreasing disease progression in AA rats, with striking inhibition of clinical, radiologic, and histologic features of RA. The observed therapeutic effect was associated with reduced expression of proinflammatory mediators in the inflamed joints. Thus, this study provides evidence that IL-2/15Rβ could be targeted for the treatment of RA.     

Protection against collagen-induced arthritis by electrotransfer of an expression plasmid for the interleukin-4

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, leading to cartilage and bone destruction. We investigated whether the electrotransfer of IL-4 DNA could regulate the disease progress of murine collagen-induced arthritis (CIA). The maximum serum level of mIL-4 was measured by 340 pg/ml on day 1 following DNA transfer. The onset of severe CIA and the degree of synovitis and cartilage erosion were significantly reduced in mice treated with IL-4 DNA (P<0.05). The beneficial effect of IL-4 gene transfer lasted for at least 17 days subsequent to treatment. The expression of IL-1beta was considerably decreased in the paws by IL-4 DNA transfer (P<0.01). On the contrary, the ratio of TIMP2 to MMP2 significantly increased in the IL-4 DNA-treated group (P<0.01). These data demonstrated that electroporation-mediated gene transfer could provide a new approach as an IL-4 therapy for autoimmune arthritis.     

The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

Six novel members of the IL-1 family of cytokines were recently identified, primarily through the use of DNA database searches for IL-1 homologues, and were named IL-1F5 to IL-1F10. In the present study, we investigated the effect of IL-1F8 on primary human joint cells, and examined the expression of the new IL-1 family members in human and mouse joints. Human synovial fibroblasts (hSFs) and human articular chondrocytes (hACs) expressed the IL-1F8 receptor (IL-1Rrp2) and produced pro-inflammatory mediators in response to recombinant IL-1F8. IL-1F8 mRNA expression was increased in hSFs upon stimulation with proinflammatory cytokines, whereas in hACs IL-1F8 mRNA expression was constitutive. However, IL-1F8 protein was undetectable in hSF and hAC culture supernatants. Furthermore, although IL-1beta protein levels were increased in inflamed human and mouse joint tissue, IL-1F8 protein levels were not. IL-1F8 levels in synovial fluids were similar to or lower than those in matched serum samples, suggesting that the joint itself is not a major source of IL-1F8. Serum levels of IL-1F8 were similar in healthy donors, and patients with rheumatoid arthritis, osteoarthritis and septic shock, and did not correlate with inflammatory status. Interestingly however, we observed high IL-1F8 levels in several serum samples in all groups. In conclusion, IL-1F8 exerts proinflammatory effects in primary human joint cells. Joint and serum IL-1F8 protein levels did not correlate with inflammation, but they were high in some human serum samples tested, including samples from patients with rheumatoid arthritis. It remains to be determined whether circulating IL-1F8 can contribute to joint inflammation in rheumatoid arthritis.     

Adenoviral transfer of cyclin-dependent kinase inhibitor genes suppresses collagen-induced arthritis in mice

In rheumatoid synovial tissues, synovial fibroblasts are activated by proinflammatory cytokines and proliferate to develop hyperplastic pannus tissues, which irreversibly damage the affected joints. We recently reported that the cyclin-dependent kinase inhibitors p16(INK4a) and p21(Cip1) are not expressed in vivo in rheumatoid synovial fibroblasts, but are readily inducible in vitro. This observation was followed by the successful treatment of rat adjuvant arthritis by local p16(INK4a) gene transfer, showing that the inhibition of the cell cycle of the synovial cells ameliorates the arthritis. In this study, we show that another animal model of rheumatoid arthritis, murine collagen-induced arthritis, can be effectively treated by local gene transfer of p21(Cip1) as well as that of p16(INK4a). The anti-arthritic effects were observed even when the treatment was conducted after the arthritis had developed. Furthermore, the effects included suppression of the expression of proinflammatory cytokines such as IL-1ss, IL-6, and TNF-alpha. Our results demonstrate that the ectopic expression of cyclin-dependent kinase inhibitors not only prevents synovial overgrowth but also ameliorates the proinflammatory milieu in the affected joints. The induction of p21(Cip1) in rheumatoid synovial tissues by pharmacological agents may also be an effective strategy to treat rheumatoid arthritis.     

Gene therapy for established murine collagen-induced arthritis by local and systemic adenovirus-mediated delivery of interleukin-4

To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis.     

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.     

Role of IL-17, transforming growth factor-beta, and IL-6 in the development of arthritis and production of anti-outer surface protein A borreliacidal antibodies in Borrelia-vaccinated and -challenged mice.

We showed recently that the adaptive immune events leading to the development of arthritis in Borrelia burgdorferi isolate 297-vaccinated and Borrelia bissettii-challenged mice involve IL-17. Here, we show in Borrelia-vaccinated and -challenged mice that two cytokines known to induce the production of IL-17, IL-6 and transforming growth factor (TGF)-beta, are also involved in the development of arthritis. Vaccinated and challenged mice administered either anti-TGF-beta or anti-IL-6 antibodies developed histopathologic changes of the hind paws similar to or greater than untreated control mice. By contrast, simultaneous blockage of these cytokines reduced the severity of arthritis in Borrelia-vaccinated and -challenged mice. Moreover, administration of anti-IL-17 antibodies to these dual-antibody-treated mice completely prevented the development of histopathologic changes of the ankle joints, significantly reduced edema of the hind paws, and prevented the production of anti-outer surface protein A borreliacidal antibodies. These findings demonstrate a role for the combined effects of IL-17, IL-6, and TGF-beta in the adaptive immune events leading to the development of Borrelia-induced arthritis.     

The importance of IL-1 beta and TNF-alpha,and the noninvolvement of IL-6, in the development of monoclonal antibody-induced arthritis

Injection of anti-type II collagen Ab and LPS induces arthritis in mice. The levels of IL-1 beta, IL-6, and chemokines (macrophage inflammatory protein (MIP)-1 alpha, MIP-2, and monocyte chemoattractant protein-1) in the hind paws increased with the onset of arthritis and correlated highly with arthritis scores. The level of TNF-alpha was also elevated, but only transiently. Quantitative real-time PCR analysis revealed increases in cytokine and chemokine mRNA. To elucidate the contribution of inflammatory cytokines and chemokines in arthritis development more directly, recombinant proteins, neutralizing Abs, and knockout mice were used. The injection of rIL-1 beta or TNF-alpha, but not IL-6 or chemokines, induced arthritis when mice were i.v. preinjected with anti-type II collagen Ab. However, a single injection of recombinant cytokines or chemokines into the hind paws did not induce swelling. Arthritis development was inhibited by neutralizing Ab against IL-1 beta, TNF-alpha, or MIP-1 alpha. In contrast, the inhibitory effect by anti-MIP-2 Ab was partial and, surprisingly, Abs to IL-6 and monocyte chemoattractant protein-1 showed no inhibitory effect. Furthermore, arthritis development in IL-1R(-/-) mice and TNFR(-/-) mice was not observed at all, but severe arthritis was developed in IL-6(-/-) mice. These results suggest that IL-1 beta and TNF-alpha play more crucial roles than IL-6 or chemokines in this model. Because arthritis was also developed in SCID mice, the development of arthritis in the Ab-induced mice model is due to a mechanism that does not involve T or B cells.     

Genomic dissection and prioritizing of candidate genes of QTL for regulating spontaneous arthritis on chromosome 1 in mice deficient for interleukin-1 receptor antagonist

Rheumatoid arthritis is a heterogeneous disease with clinical and biological polymorphisms. IL-1RN is a protein that binds to interleukin-1 (IL-1) receptors and inhibits the binding of IL-1-alpha and IL-1-beta. IL-1RN levels are elevated in the blood of patients with a variety of infectious, immune, and traumatic conditions. Balb/c mice deficient in IL-1ra (mouse gene of IL-1RN) develop spontaneous autoimmune arthritis while DBA/1 mice deficient in IL-1ra do not. Previously, we identified a major QTL that regulates the susceptibility to arthritis in Balb/c mice with IL-1ra deficiency. In this study, we found that the QTL may contain two peaks that are regulated by two sets of candidate genes. By haplotype analysis, the total genomic regions of candidate genes were reduced from about 19 Mbp to approximately 9 Mbp. The total number of candidate genes was reduced from 208 to 21.     

Absence of endogeneous interleukin-10 enhances the evolution of murine type-II collagen-induced arthritis.

Interleukin-10 (IL-10) exerts a wide spectrum of regulatory activities in the immune and inflammatory response. The aim of this study was to investigate the role of endogenous IL-10 in the modulation of the inflammatory response in mice subjected to collagen-induced arthritis. Collagen-induced arthritis (CIA) was induced in mice lacking the gene for IL-10 (IL-10 "knock-out", IL-10KO) and in wild-type control (IL-10WT) mice by an intradermal injection of 100 mul of the emulsion (containing 100 mug of bovine type II collagen) (CII) and complete Freund's adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. IL-10 wild type (WT) mice developed an erosive, hind paw arthritis when immunised with CII in CFA. Macroscopic clinical evidence of CIA first appeared as peri-articular erythema and oedema in the hind paws. The incidence of CIA was 100% by day 27 in the CII-challenged IL-10WT. The severity of CIA progressed over a 35-day period, with radiographic evaluation revealing focal resorption of bone. The histopathology of CIA included erosion of the cartilage at the joint margins. IL-10KO mice experienced higher rates of clinical signs and more severe knee and paw injury as compared to IL-10WT. The degree of oxidative and nitrosative damage was significantly higher in IL-10KO mice than in wild-type littermates, as indicated by elevated malondialdehyde levels and formation of nitrotyrosine and poly (ADP-ribose) synthetase (PARS). Plasma levels of the proinflammatory cytokines, tumour necrosis factor, interleukin-1beta and interleukin-6 were also greatly enhanced in comparison to wild-type mice. These data demonstrate that endogenous IL-10 exerts an anti-inflammatory role during chronic inflammation and tissue damage associated with collagen-induced arthritis, possibly by regulating neutrophil recruitment, and the subsequent cytokine and oxidant generation.     

An IFN-gamma-independent proinflammatory role of IL-18 in murine streptococcal cell wall arthritis

IL-18 is a member of the IL-1 family of proteins that exerts proinflammatory effects. It was formally known as IFN-gamma-inducing factor and is a pivotal cytokine for the development of Th1 responses. Apart from Th1 immune-stimulatory activity, IL-18 induces the production of proinflammatory cytokines such as TNF-alpha and IL-1 in vitro. The goal was to investigate the role of endogenous IL-18 in murine streptococcal cell wall (SCW)-induced arthritis. Furthermore, we investigated whether IL-18 neutralization had an impact on local TNF and IL-1 production. C57BL/6, BALB/c, and IFN-gamma-deficient mice were injected with 2 mg of rabbit anti-murine IL-18 Abs shortly before induction of arthritis by intra-articular injection of 25 microg of SCW fragments into the right knee joint. Suppression of joint swelling was noted on days 1 and 2 of SCW arthritis after blockade of endogenous IL-18. Analysis of local cytokine concentrations showed that IL-18, TNF-alpha, and IL-1ss levels were decreased. Severe inhibition of chondrocyte proteoglycan synthesis was seen in the vehicle-treated control animals, whereas a reversal of the inhibition of chondrocyte proteoglycan synthesis was found in the anti-IL-18-exposed animals. Blockade of endogenous IL-18 in IFN-gamma-deficient mice showed results similar to those found in wild-type animals, identifying a role for IL-18 that is IFN-gamma independent. The present study indicates that IL-18 is a proinflammatory cytokine during the onset of murine SCW arthritis, and this inflammatory role of IL-18 is IFN-gamma independent.     

Roles of IL-1 in the development of rheumatoid arthritis: consideration from mouse models

Expression of inflammatory cytokines is augmented in the joints of patients with rheumatoid arthritis (RA). We found that cytokine levels are also elevated in the joints of a mouse arthritis model, human T-cell leukemia virus type I (HTLV-I) transgenic (Tg) mouse. Depletion of IL-1 by gene targeting greatly reduced the incidence of the disease, indicating the importance of this cytokine in the development of arthritis. Furthermore, IL-1 receptor antagonist (IL-1Ra)-deficient mice develop autoimmunity and arthritis spontaneously. These observations suggest that excess IL-1 signaling the causes autoimmunity. We show that IL-1 activates the immune system non-specifically by inducing CD40L and OX40 co-signaling molecules on T cells. In this review, the roles of IL-1 in the development of autoimmunity and arthritis in mouse models will be discussed.     

STAT3 and NF-kappaB signal pathway is required for IL-23-mediated IL-17 production in spontaneous arthritis animal model IL-1 receptor antagonist-deficient mice

IL-23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 has proinflammatory activity, inducing IL-17 secretion from activated CD4(+) T cells and stimulating the proliferation of memory CD4(+) T cells. We investigated the pathogenic role of IL-23 in CD4(+) T cells in mice lacking the IL-1R antagonist (IL-1Ra(-/-)), an animal model of spontaneous arthritis. IL-23 was strongly expressed in the inflamed joints of IL-1Ra(-/-) mice. Recombinant adenovirus expressing mouse IL-23 (rAd/mIL-23) significantly accelerated this joint inflammation and joint destruction. IL-1beta further increased the production of IL-23, which induced IL-17 production and OX40 expression in splenic CD4(+) T cells of IL-1Ra(-/-) mice. Blocking IL-23 with anti-p19 Ab abolished the IL-17 production induced by IL-1 in splenocyte cultures. The process of IL-23-induced IL-17 production in CD4(+) T cells was mediated via the activation of Jak2, PI3K/Akt, STAT3, and NF-kappaB, whereas p38 MAPK and AP-1 did not participate in the process. Our data suggest that IL-23 is a link between IL-1 and IL-17. IL-23 seems to be a central proinflammatory cytokine in the pathogenesis of this IL-1Ra(-/-) model of spontaneous arthritis. Its intracellular signaling pathway could be useful therapeutic targets in the treatment of autoimmune arthritis.     

ABIN-3: a molecular basis for species divergence in interleukin-10-induced anti-inflammatory actions

Whereas interleukin-10 (IL-10) is an anti-inflammatory cytokine known to regulate macrophage activation, its full mechanism of action remains incompletely defined. In a screen to identify novel IL-10-induced genes, we cloned the mouse ortholog of human ABIN-3 (also termed LIND). ABIN-3 expression was induced selectively by IL-10 in both mouse and human mononuclear phagocytes coordinately undergoing proinflammatory responses. In contrast to the previously characterized ABINs, mouse ABIN-3 was incapable of inhibiting NF-kappaB activation by proinflammatory stimuli. Generation and analysis of ABIN-3-null mice demonstrated that ABIN-3 is unnecessary for the anti-inflammatory effects of IL-10 as well as for proper negative regulation of NF-kappaB. Conversely, human ABIN-3 was capable of inhibiting NF-kappaB activation in response to signaling from Toll-like receptor, IL-1, and tumor necrosis factor. Enforced expression of human ABIN-3 in human monocytic cells suppressed the cytoplasmic degradation of IkappaBalpha, the activation of NF-kappaB, and the induction of proinflammatory genes. Comparative sequence analyses revealed that mouse ABIN-3 lacks a complete ABIN homology domain, which was required for the functional activity of human ABIN-3. ABIN-3 is, thus, an IL-10-induced gene product capable of attenuating NF-kappaB in human macrophages yet is inoperative in mice and represents a basis for species-specific differences in IL-10 actions.