So Youn Min

STAT3 and NF-κB 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-1β 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-κB, 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.

Type II collagen autoimmunity in a mouse model of human rheumatoid arthritis

Type II collagen (CII) is expressed exclusively in the joint articular. Although the relationship between anti-CII immunity and human rheumatoid arthritis (RA) has been studied for a long time, definitive conclusions have not been reached. CII, as an autoantigen, has been studied extensively in small animal models, such as mice, and the collagen-induced arthritis (CIA) model has increased our understanding of the pathogenesis of human RA. In the present report, we summarize the available information on anti-CII immunity and discuss recent updates regarding pathogenesis in the CIA model, including the role of Th17 cells.

CD40 Engagement on Synovial Fibroblast Up-Regulates Production of Vascular Endothelial Growth Factor

We tested the impact of CD40 engagement on the production of vascular endothelial growth factor (VEGF) from rheumatoid synovial fibroblasts. Fibroblast-like synovial cells (FLS) were prepared from the synovial tissues of rheumatoid arthritis patients and cultured in the presence of CD40 ligand-transfected (CD40L+) L cells. VEGF levels were determined in the culture supernatants by ELISA. Stimulation of FLS by CD40L+ L cells increased the production of VEGF by 4.1-fold over the constitutive levels of unstimulated FLS. The CD40L on activated T cells from rheumatoid synovial fluid also up-regulated VEGF production from FLS. Neither indomethacin nor Abs to IL-1β, TNF-α, and TGF-β did affect CD40L-induced VEGF production. Stimulation of FLS with TNF-α, IL-1β, and TGF-β increased VEGF production by 1.6-, 2.0-, and 5.2-fold, respectively, and displayed an additive effect on the production of VEGF by CD40L. VEGF mRNA expression was also up-regulated by the stimulation of FLS with membranes from the CD40L+ L cells. Dexamethasone completely abrogated CD40L-induced VEGF production. In addition, pyrrolidine dithiocarbamate partially down-regulated CD40L-induced VEGF production, showing that the NF-κB pathway was partly involved in the signaling of CD40L leading to VEGF production. Collectively, these results suggest that the interaction between CD40 on synovial fibroblasts and CD40L expressed on activated T lymphocytes may be directly involved in the neovascularization in rheumatoid synovitis by enhancing the production of VEGF.

The role of IL-12 in inflammatory activity of patients with rheumatoid arthritis (RA)

The aim of this study was to investigate the role of IL‐12 in patients with RA. IL‐12 (p70) and its associated cytokines were measured in sera and synovial fluid (SF) using an enzyme‐linked immunosorbent method. Seven American College of Rheumatology (ACR) core set measures as well as IL‐12 levels were sequentially monitored at the commencement and 4 months after treatment with a low‐dose steroid and disease‐modifying anti‐rheumatic drugs (DMARDs). In sera, 64 (42.2%) of 152 RA patients had detectable concentrations of IL‐12 (p70), whereas one (1.4%) of 69 osteoarthritis (OA) patients and five (10%) of 50 healthy controls had detectable IL‐12 (P < 0.001). The median level of circulating IL‐12 was also higher in RA patients (P < 0.001). In SF, the number of patients with detectable IL‐12 and the median IL‐12 levels were significantly higher in RA patients (n = 53) than in OA patients (n = 22). In paired samples (n = 53) of sera and SF from RA patients, IL‐12 levels were higher in the SF than in sera (P < 0.001). Patients with detectable IL‐12 (n = 51) in sera had higher tender joint scores (P = 0.003), swollen joint scores (P < 0.001) and C‐reactive protein (CRP; P = 0.036), than those without (n = 55). Four months after treatment with DMARDs, the improved group showed a larger IL‐12 decrease than the non‐improved group (P = 0.017). The levels of IL‐12 correlated positively with those of IL‐2, interferon‐gamma, IL‐6, and tumour necrosis factor‐alpha, but were correlated inversely with those of IL‐10. Our results demonstrate that IL‐12 levels reflect RA disease activity and that IL‐12 is involved in the production of proinflammatory cytokines. An IL‐12 blockade could be useful for the treatment of RA.

Transforming growth factor β-transduced mesenchymal stem cells ameliorate experimental autoimmune arthritis through reciprocal regulation of Treg/Th17 cells and osteoclastogenesis.

OBJECTIVE Bone marrow-derived mesenchymal stem cells (MSCs) can prevent various autoimmune diseases. We examined the therapeutic potential of transforming growth factor β (TGFβ)-transduced MSCs in experimental autoimmune arthritis, using an accepted animal model of collagen-induced arthritis (CIA). METHODS DBA/1J mice with CIA were treated with syngeneic TGFβ-induced MSCs, whereas control mice received either vehicle or MSCs alone. Arthritis severity was assessed by clinical and histologic scoring. TGFβ-transduced MSCs were tested for their immunosuppressive ability and differential regulation in mice with CIA. T cell responses to type II collagen were evaluated by determining proliferative capacity and cytokine levels. The effects of TGFβ-transduced MSCs on osteoclast formation were analyzed in vitro and in vivo. RESULTS Systemic infusion of syngeneic TGFβ-transduced MSCs prevented arthritis development and reduced bone erosion and cartilage destruction. Treatment with TGFβ-transduced MSCs potently suppressed type II collagen-specific T cell proliferation and down-regulated proinflammatory cytokine production. These therapeutic effects were associated with an increase in type II collagen-specific CD4+FoxP3+ Treg cells and inhibition of Th17 cell formation in the peritoneal cavity and spleen. Furthermore, TGFβ-transduced MSCs inhibited osteoclast differentiation. CONCLUSION TGFβ-transduced MSCs suppressed the development of autoimmune arthritis and joint inflammation. These data suggest that enhancing the immunomodulatory activity of MSCs and modulating T cell-mediated immunity using gene-modified MSCs may be a gateway for new therapeutic approaches to clinical rheumatoid arthritis.

IL-23 Induces Receptor Activator of NF-κB Ligand Expression on CD4+ T Cells and Promotes Osteoclastogenesis in an Autoimmune Arthritis Model

IL-23, a clinically novel cytokine, targets CD4+ T cells. Recent IL-1Ra−/− mouse studies have demonstrated that IL-23 indirectly stimulates the differentiation of osteoclast precursors by enhancing IL-17 release from CD4+ T cells. IL-17, in turn, stimulates osteoclastogenesis in osteoclast precursor cells. In this study, we found that IL-23 up-regulates receptor activator of NF-κB ligand expression by CD4+ T cells, and thus contributes to osteoclastogenesis. This indirect pathway is mediated by NF-κB and STAT3. We have also demonstrated that IL-23 can influence osteoclastogenesis positively under the special conditions in the IL-1-dominant milieu of IL-1Ra−/− mice. We propose that IL-23-enhanced osteoclastogenesis is mediated mainly by CD4+ T cells. The results of this study show that IL-23 is a promising therapeutic target for the treatment of arthritis-associated bone destruction.

Elevated matrix metalloproteinase-9 in patients with systemic sclerosis.

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of cancer, autoimmune disease, and various pathologic conditions characterized by excessive fibrosis. In this study, we investigated the expression of MMP-9 and its clinical significance in systemic sclerosis (SSc). The patients (n = 42) with SSc had higher concentrations of MMP-9 and of tissue inhibitor of metalloproteinase-1 (TIMP-1) and a higher ratio of MMP-9 to TIMP-1 in sera than healthy controls (n = 32). Serum MMP-9 concentrations were significantly higher in the diffuse type (n = 23) than the limited type of SSc (n = 19). Serum concentrations of MMP-9 correlated well with the degree of skin involvement, as determined by the Rodnan score and with serum concentrations of transforming growth factor β. Moreover, dermal fibroblasts from patients with SSc produced more MMP-9 than those from healthy controls when they were stimulated with IL-1β, tumor necrosis factor α, or transforming growth factor β. Such an increase in MMP-9 production was partially blocked by treatment with cyclosporin A. In summary, the serum MMP-9 concentrations were elevated in SSc patients and correlated well with skin scores. The increased MMP-9 concentrations may be attributable to overproduction by dermal fibroblasts in SSc. These findings suggest that the enhanced production of MMP-9 may contribute to fibrogenic remodeling during the progression of skin sclerosis in SSc.

Induction of IL-10-producing CD4+CD25+ T cells in animal model of collagen-induced arthritis by oral administration of type II collagen.

Induction of oral tolerance has long been considered a promising approach to the treatment of chronic autoimmune diseases, including rheumatoid arthritis (RA). Oral administration of type II collagen (CII) has been proven to improve signs and symptoms in RA patients without troublesome toxicity. To investigate the mechanism of immune suppression mediated by orally administered antigen, we examined changes in serum IgG subtypes and T-cell proliferative responses to CII, and generation of IL-10-producing CD4+CD25+ T-cell subsets in an animal model of collagen-induced arthritis (CIA). We found that joint inflammation in CIA mice peaked at 5 weeks after primary immunization with CII, which was significantly less in mice tolerized by repeated oral feeding of CII before CIA induction. Mice that had been fed with CII also exhibited increased serum IgG1 and decreased serum IgG2a as compared with nontolerized CIA animals. The T-cell proliferative response to CII was suppressed in lymph nodes of tolerized mice also. Production of IL-10 and of transforming growth factor-β from mononuclear lymphocytes was increased in the tolerized animals, and CD4+ T cells isolated from tolerized mice did not respond with induction of IFN-γ when stimulated in vitro with CII. We also observed greater induction of IL-10-producing CD4+CD25+ subsets among CII-stimulated splenic T cells from tolerized mice. These data suggest that when these IL-10-producing CD4+CD25+ T cells encounter CII antigen in affected joints they become activated to exert an anti-inflammatory effect.

Type II collagen autoimmunity in rheumatoid arthritis

&NA; This review summarizes the autoimmune reaction to type II collagen (CII) autoimmunity with regard not only to antibody response to CII but also to the clinical significance or biological characteristics of the CII‐reactive T cell, focusing on studies of human RA rather than on animal models. The authors investigated the effect of the interaction between CII‐reactive T cells and fibroblast‐like synoviocytes (FLSs) on the production of inflammatory cytokines. When the CII‐reactive T cells were co‐cultured with FLS, the production of interleukin‐15 and tumor necrosis factor‐alpha from FLSs were significantly increased, and this increase was clearly presented in accord with the expansion of CII‐reactive T cells. In addition, the production of interferon‐&ggr; and interleukin‐17, T cell–derived cytokines, was increased by the co‐incubation of CII‐reactive T cells with FLSs. When FLSs were co‐cultured with CII‐stimulated T cells, the production of interleukin‐8, monocyte chemoattractant protein‐1, and macrophage inflammatory protein‐1&agr; was significantly enhanced. The increased production of these chemokines was strongly correlated with an increase in T‐cell response to CII. Conclusively, high reactivity to CII was frequently found in RA patients. Enhanced T‐cell responses to CII were associated with increased production of proinflammatory cytokines and chemokines, which were critical for inflammatory responses in RA. Interaction of CII‐reactive T cells with FLS further augmented this phenomenon. Taken together, the authors’ recent studies have suggested that autoimmunity to CII could play a crucial role not only in the initiation but also in the amplification and perpetuation of the inflammatory process in RA.

Cyclosporine differentially regulates interleukin-10, interleukin-15, and tumor necrosis factor α production by rheumatoid synoviocytes

OBJECTIVE To determine the direct effect of cyclosporin A (CSA) on the production of cytokines by rheumatoid synovial fibroblasts. METHODS Fibroblast-like synoviocytes (FLS) were prepared from the synovial tissues of patients with rheumatoid arthritis and cultured in the presence of CSA. The production of interleukin-10 (IL-10), IL-15, and tumor necrosis factor a (TNFalpha) by FLS was measured in culture supernatants by enzyme-linked immunosorbent assay. The expression of IL-10, IL-15, and TNFalpha messenger RNA (mRNA) in FLS was determined by polymerase chain reaction (PCR). RESULTS CSA (1-1,000 ng/ml) increased the production of IL-10, but decreased in a dose-dependent manner the levels of IL-15 and TNFalpha that were spontaneously secreted from FLS. CSA also potently inhibited the production of IL-15 and TNFalpha stimulated with interferon-gamma, IL-1beta, or lipopolysaccharide. The inhibitory effect of CSA on IL-15 and TNFalpha production depended on the increase in IL-10, since neutralizing anti-IL-10 antibodies were able to partially reverse this inhibition. In a semiquantitative PCR, CSA increased IL-10 mRNA expression but strongly suppressed IL-1beta-induced IL-15 and TNFalpha mRNA expression, indicating that the production of these cytokines by CSA was regulated at the transcriptional level. Results with the calcineurin inhibitor FK-506, but not with the immunosuppressant rapamycin, were similar to those with CSA. Agonists of cAMP displayed an additive effect on the changes produced in the IL-10, IL-15, and TNFalpha levels by CSA, while a cAMP antagonist almost completely abrogated the effect of CSA, suggesting that cAMP is the major intracellular signal that mediates cytokine regulation by CSA. CONCLUSION These results suggest that CSA differentially regulates the production of cytokines by rheumatoid synoviocytes via a cAMP-dependent pathway.