IL-12p40 Homodimer Ameliorates Experimental Autoimmune Arthritis.

IL-23 is the key cytokine that induces the expansion of Th17 cells. It is composed of p19 and p40 subunits of IL-12. The p40 subunit binds competitively to the receptor of IL-23 and blocks its activity. Our aim was to assess the preventive and therapeutic effect of the IL-12p40 homodimer (p40)2 subunit in autoimmune arthritis animal models. In the current study, using IL-1R antagonist-knockout mice and a collagen-induced arthritis model, we investigated the suppressive effect of (p40)2 on inflammatory arthritis. We demonstrated that the recombinant adenovirus-expressing mouse (p40)2 model prevented the development of arthritis when given before the onset of arthritis. It also decreased the arthritis index and joint erosions in the mouse model if transferred after arthritis was established. (p40)2 inhibited the production of inflammatory cytokines and Ag-specific T cell proliferation. It also induced CD4(+)CD25(+)Foxp3 regulatory T (Treg) cells in vitro and in vivo, whereas the generation of retinoic acid receptor-related organ receptor γt and Th17 cells was suppressed. The induction of Treg cells and the suppression of Th17 cells were mediated via activated STAT5 and suppressed STAT3. Our data suggest that (p40)2 suppressed inflammatory arthritis successfully. This could be a useful therapeutic approach in autoimmune arthritis to regulate the Th17/Treg balance and IL-23 signaling.

Amelioration of autoimmune arthritis by adoptive transfer of Foxp3-expressing regulatory B cells is associated with the Treg/Th17 cell balance.

BACKGROUND: Foxp3 is a key regulator of the development and function of regulatory T cells (Tregs), and its expression is thought to be T cell-restricted. We found that B cells in mice can express Foxp3 and B cells expressing Foxp3 may play a role in preventing the development of collagen-induced arthritis (CIA) in DBA/1J mice. METHODS: Foxp3 expression was modulated in CD19(+) B cells by transfection with shRNA or using an over-expression construct. In addition, Foxp3-transfected B cells were adoptively transferred to CIA mice. We found that LPS or anti-IgM stimulation induced Foxp3 expression in B cells. Foxp3-expressing B cells were found in the spleens of mice. RESULTS: Over-expression of Foxp3 conferred a contact-dependent suppressive ability on proliferation of responder T cells. Down-regulation of Foxp3 by shRNA caused a profound induction in proliferation of responder T cells. Adoptive transfer of Foxp3(+)CD19(+) B cells attenuated the clinical symptoms of CIA significantly with concomitant suppression of IL-17 production and enhancement of Foxp3 expression in CD4(+) T cells from splenocytes. CONCLUSION: Our data indicate that Foxp3 expression is not restricted to T cells. The expression of Foxp3 in B cells is critical for the immunoregulation of T cells and limits autoimmunity in a mouse model.

Retinoic acid attenuates rheumatoid inflammation in mice.

Retinoic acid is the active vitamin A derivative and is well-known to have diverse immunomodulatory actions. In this study, we investigated the impact of all-trans retinoic acid (ATRA), a biologic key metabolite of vitamin A, on the development of arthritis and the pathophysiologic mechanisms by which ATRA might have antiarthritic effects in animal model of rheumatoid arthritis (RA; collagen-induced arthritis [CIA] in DBA/1J mice). We showed that treatment with ATRA markedly suppressed the clinical and histologic signs of arthritis in the CIA mice. It reduced the expression of IL-17 in the arthritic joints. Interestingly, Foxp3(+) regulatory T cells were markedly increased and IL-17-producing CD4(+) T cells (Th17 cells) were decreased in the spleens of ATRA-treated mice. In vitro treatment with ATRA induced the expression of Foxp3 and repressed the IL-17 expression in the CD4(+) T cells in mice. ATRA suppressed the production of total IgG and IgG2a in splenocytes that were stimulated by LPS. It also reduced serum levels of total IgG and IgG2 anti-collagen Abs and germinal center formation in CIA mice. In addition, the ATRA-treated mice showed decreased osteoclast formation in arthritic joints. Moreover, ATRA downregulated the expression of receptor activator of NF-κB ligand, the leading player of osteoclastogenesis, in the CD4(+) T cells and fibroblast-like synoviocytes from patients with RA. Furthermore, ATRA prevented both human monocytes and mice bone marrow-derived monocytes/macrophage cells from differentiating into osteoclasts. These data suggest ATRA might be an effective treatment modality for RA patients.

Interleukin-22 promotes osteoclastogenesis in rheumatoid arthritis through induction of RANKL in human synovial fibroblasts.

OBJECTIVE: To examine the regulatory role of interleukin-22 (IL-22) in the expression of RANKL and induction of osteoclastogenesis in rheumatoid arthritis (RA). METHODS: Concentrations of IL-22 and RANKL in the serum and synovial fluid of RA patients were measured using enzyme-linked immunosorbent assay. RA synovial fibroblasts were treated with recombinant human IL-22 (rhIL-22), and the expression of RANKL messenger RNA (mRNA) and protein was measured using real-time polymerase chain reaction, Western blotting, and intracellular immunostaining. Human monocytes were cocultured with IL-22-prestimulated RA synovial fibroblasts and macrophage colony-stimulating factor, and osteoclastogenesis was assessed by counting the multinucleated cells (those staining positive for tartrate-resistant acid phosphatase). RESULTS: The IL-22 concentration in the synovial fluid was higher in RA patients than in patients with osteoarthritis (OA). The serum IL-22 concentration was also higher in RA patients than in OA patients and healthy volunteers, and this correlated with serum titers of rheumatoid factor and anti-cyclic citrullinated peptide antibodies. In RA synovial fibroblasts treated with rhIL-22, the expression of RANKL mRNA and protein was increased in a dose-dependent manner. IL-22-induced RANKL expression was down-regulated significantly by the inhibition of p38 MAPK/NF-κB or JAK-2/STAT-3 signaling. In human monocytes cocultured with IL-22-prestimulated RA synovial fibroblasts in the absence of exogenous RANKL, the monocytes differentiated into osteoclasts, but this osteoclastogenesis decreased after p38 MAPK/NF-κB or JAK-2/STAT-3 signaling was inhibited. CONCLUSION: These results show that IL-22 up-regulates RANKL expression in RA synovial fibroblasts and induces osteoclastogenesis. These effects are mediated by the p38 MAPK/NF-κB and JAK-2/STAT-3 signaling pathways.

Modulation of STAT-3 in rheumatoid synovial T cells suppresses Th17 differentiation and increases the proportion of Treg cells.

OBJECTIVE: To investigate the impact of STAT-3-mediated regulation on Th17 differentiation in patients with rheumatoid arthritis (RA). METHODS: CD4+ T cells isolated from peripheral blood (PB) and synovial fluid (SF) were stimulated to differentiate into Th17 cells or Treg cells. The activity of STAT-3 was knocked down by transfecting CD4+ T cells with small interfering RNA (siRNA). After 3 days in culture, the proportions of Th17 cells and Treg cells were measured by flow cytometry, and the production of interleukin-17 (IL-17) was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: The levels of IL-17, IL-6, IL-23, IL-1, and tumor necrosis factor α were significantly higher in RA SF and synovial tissue than in SF and synovial tissue from osteoarthritis patients. In RA synovial tissue, the expression of STAT-3 increased in proportion to the severity of synovitis, as shown by stromal cellularity, intimal hyperplasia, and inflammatory infiltration. The degree of Th17 differentiation was highest in RA SF, followed by RA PB, and lowest in normal subjects. In CD4+ T cells, transfection with STAT-3 siRNA prevented Th17 differentiation of mononuclear cells from RA PB and SF but increased the proportion of Treg cells. In contrast, inhibition of STAT-5, the transcription factor for Treg cells, increased the proportion of Th17 cells and reduced that of Treg cells. CONCLUSION: Our findings indicate that modulation of STAT-3 in CD4+ T cells affects the differentiation of Th17 cells and Treg cells in patients with RA. This role of STAT-3 in RA synovial T cells may provide a new therapeutic target for the management of RA.

Interleukin-2/anti-interleukin-2 monoclonal antibody immune complex suppresses collagen-induced arthritis in mice by fortifying interleukin-2/STAT5 signalling pathways.

In this study, we investigated the effects of administration of interleukin-2 (IL-2)/JES6-1 (anti-IL-2 monoclonal antibody) immune complexes on the expansion and activation of regulatory T (Treg) cells, the down-regulation of T helper type 17 (Th17) cells, and the control of the severity of collagen-induced arthritis (CIA). Wild-type and CIA-induced wild-type mice were injected intraperitoneally (i.p.) with IL-2 or IL-2/JES6-1 complex three times at 2-day intervals. Treg cell surface markers were analysed by flow cytometry. After injecting IL-2 or IL-2/JES6-1, the time kinetics of IL-2 signalling molecules was examined by FACS and Western blotting. Concentrations of IL-17 and IL-10 were measured by ELISA. Injection of IL-2/JES6-1 increased the proportion of Foxp3+ Treg cells among splenic CD4+ T cells, which reached the highest level on day 4 after injection. Up-regulation of CTLA4, GITR and glycoprotein-A repetitions predominant (GARP) was observed. Activation of p-signal transducer and activator of transcription 5 (STAT5) was apparent within 3 hr after injection of IL-2/JES6-1 complexes. Expression of IL-2 signalling molecules, including p-AKT and p-p38/mitogen-activated protein kinase, was also higher in splenocytes treated with IL-2/JES6-1 complexes. Injection of IL-2/JES6-1 complexes suppressed the induction of CIA and the production of IL-17 and inflammatory responses while increasing the level of IL-10 in the spleen. The expansion of Treg cells (via STAT5) and the concomitant increase in IL-2 signalling pathways by IL-2/JES6-1 complexes suggests their potential use as a novel therapeutic agent for the treatment of autoimmune arthritis.

TWEAK promotes the production of Interleukin-17 in rheumatoid arthritis.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is an inflammatory cytokine that modulates several biological responses by inducing chemokines and proinflammatory cytokines. We hypothesized that TWEAK could promote secretion of IL-17, an amplifier of inflammatory arthritis. To test this, we investigated the capacity of TWEAK to induce IL-17 production in T cells via the fibroblast growth factor-inducible gene 14 (Fn14, also known as TWEAK receptor) signal pathway in rheumatoid arthritis (RA). Fn14 and IL-17 were highly expressed in arthritic tissues of collagen-induced arthritis (CIA) mice. TWEAK induced production of IL-17 alone and synergistically with lipopolysaccharide. In naïve murine T cells, TWEAK promoted Th17 differentiation. The expression of Fn14 was predominant in Th17 cells. TWEAK and IL-17 concentrations were significantly higher in synovial fluid and serum in RA patients than OA patients. In addition, we identified CD4(+)IL-17(+)Fn14(+) cells in synovium from RA patients. TWEAK promoted IL-17 production synergistically with IL-23 or IL-21 and blockade of Fn14 with Fn14-Fc suppressed Th17 differentiation. Conversely, this treatment enhanced Treg differentiation. These results suggest that TWEAK induces IL-17 production and may be a therapeutic target in the treatment of RA.

Measurement of interleukin-33 (IL-33) and IL-33 receptors (sST2 and ST2L) in patients with rheumatoid arthritis.

The interleukin-33 (IL-33)/ST2 pathway has emerged as an intercellular signaling system that participates in antigen-allergen response, autoimmunity and fibrosis. It has been suggested that IL-33/ST2 signaling has been involved in the pathogenesis of rheumatoid arthritis (RA), because IL-33 and its receptor have been specifically mapped to RA synovium. The aim of this study was to determine the levels of IL-33 and sST2 in sera and synovial fluids in patients with RA. The serum level of IL-33 was significantly higher in patients with RA (294.9 ± 464.0 pg/mL) than in healthy controls (96.0 ± 236.9 pg/mL, P = 0.002). The synovial fluid level of IL-33 was significantly higher in RA patients than in osteoarthritis patients. The level of serum sST2 was higher in RA patients than in healthy controls (P = 0.042). A significant relationship was found between the levels of IL-33 and IL-1ß (r = 0.311, P = 0.005), and IL-33 and IL-6 (r = 0.264, P = 0.017) in 81 RA patients. The levels of IL-33, sST2 and C-reactive protein decreased after conventional disease-modifying antirheumatic drugs treatment in 10 patients with treatment-naïve RA. Conclusively, IL-33 is involved in the pathogenesis of RA and may reflect the degree of inflammation in patients with RA.

CTLA4-Ig modifies dendritic cells from mice with collagen-induced arthritis to increase the CD4+CD25+Foxp3+ regulatory T cell population.

Cytotoxic T lymphocyte antigen-4 (CTLA4) and IgG fusion protein, CTLA4-Ig, is a therapeutic agent used for rheumatoid arthritis. It binds B7 molecules on dendritic cells (DCs) and thereby blocks B7/CD28 costimulatory interaction and inhibits effective T cell proliferation. However, the effect of CTLA4-Ig on the regulatory T cell (Treg) is still not known. In this study, we investigated the influence of CTLA4-Ig on the CD4+CD25+Foxp3+ Treg population in collagen-induced arthritis (CIA) mouse model. CTLA4-Ig suppressed CIA and increased the CD4+CD25+Foxp3+ Treg population in joint and spleen. When CD11c + DCs and CD4+T cells from CIA mice were cultured with anti-CD3, CTLA4-Ig increased the CD4+CD25 + Foxp3+ Treg population in a TGF-beta-dependent manner. When CD11c + DCs from CIA mice were treated with CTLA4-Ig and adoptively transferred into CIA-induced mice, arthritis did not develop in association with the increase in CD4+CD25+Foxp3+ Treg population. However, in CTLA4-Ig-untreated DC-transferred CIA mice, arthritis developed and then rapidly progressed. Our study demonstrated that CTLA4-Ig suppressed CIA by modifying DCs from CIA mice into tolerogenic DCs to increase the CD4+CD25+Foxp3+ Treg population and this seems to be the new immune regulatory mechanism of CTLA4-Ig.

Epigallocatechin-3-gallate ameliorates autoimmune arthritis by reciprocal regulation of T helper-17 regulatory T cells and inhibition of osteoclastogenesis by inhibiting STAT3 signaling.

The green tea polyphenol epigallocatechin-3-gallate is a potent antioxidant. Here, we describe the effects of epigallocatechin-3-gallate on T cell differentiation and osteoclast differentiation in an animal model of arthritis. Mice with collagen-induced arthritis were injected intraperitoneally with epigallocatechin-3-gallate, 3 times/wk after the primary immunization. Surface markers of T helper 17 cells and regulatory T cells were analyzed by flow cytometry. Flow cytometry, Western blotting, and enzyme-linked immunosorbent assays were used to evaluate the effect of epigallocatechin-3-gallate on cell signaling in the collagen-induced arthritis model. Epigallocatechin-3-gallate decreased the arthritis index and showed protective effects against joint destruction in collagen-induced arthritis mice. The expression of cytokines, oxidative stress proteins, and phosphorylated-signal transducer and activator of transcription-3, 705 and 727, were significantly less in mice treated with epigallocatechin-3-gallate than it was in controls. Epigallocatechin-3-gallate reduced the expression of osteoclast markers in vitro and in vivo relative to the control, and the antiosteoclastic activity was observed in epigallocatechin-3-gallate-treated, interferon-γ knockout mice. The proportion of forkhead box protein 3-positive regulatory T cells was increased in the spleens of mice treated with epigallocatechin-3-gallate compared with control mice, whereas the proportion of T helper 17 cells was reduced. In vitro, the expression of nuclear respiratory factor 2, heme oxygenase-1, and extracellular signal-regulated kinase was increased significantly by epigallocatechin-3-gallate. We demonstrated that the administration of epigallocatechin-3-gallate attenuated the symptoms of arthritis, inhibited osteoclastogenesis and T helper 17 cell activation, and increased the number of regulatory T cells. At the molecular level, the antiarthritic effects of epigallocatechin-3-gallate may be due to induction of phosphorylated-extracellular signal-regulated kinase, nuclear respiratory factor 2, and heme oxygenase-1 and inhibition of signal transducer and activator of transcription-3 activation.

IL-17-mediated Bcl-2 expression regulates survival of fibroblast-like synoviocytes in rheumatoid arthritis through STAT3 activation.

INTRODUCTION: Fibroblast-like synoviocytes (FLSs) are a major cell population of the pannus that invades adjacent cartilage and bone in rheumatoid arthritis (RA). The study was undertaken to determine the effect of interleukin-17 (IL-17) on the survival and/or proliferation of FLSs from RA patients and to investigate whether signal tranducer and activator of transcription 3 (STAT3) is implicated in this process. METHODS: Bcl-2 and Bax expression in FLSs was determined using the real-time PCR and western blot analysis. The expression of Bcl-2 and phosphoSTAT3 in synovial tissues was investigated by confocal microscope. Apoptosis of FLSs was detected by Annexin V/propidium iodide staining and/or phase contrast microscopy. The proliferation of FLSs was determined by CCK-8 ELISA assay. RESULTS: The pro-apoptotic Bax is decreased and anti-apoptotic Bcl-2 is increased in FLSs from RA patients compared with those from patients with osteoarthritis (OA). IL-17 upregulated the expression of Bcl-2 in FLSs from RA patients, but not in FLSs from OA patients. STAT3 was found to mediate IL-17-induced Bcl-2 upregulation in FLSs from RA patients. Additionally, IL-17 promoted the survival and proliferation of FLSs from RA patients. Most importantly, treatment with STAT3 inhibitor reversed the protective effect of IL-17 on FLSs apoptosis induced by sodium nitroprusside (SNP). CONCLUSIONS: Our data demonstrate that STAT3 is critical in IL-17-induced survival of FLS from RA patients. Therefore, therapeutic strategies that target the IL-17/STAT3 pathway might be strong candidates for RA treatment modalities.

Adoptive transfer of all-trans-retinal-induced regulatory T cells ameliorates experimental autoimmune arthritis in an interferon-gamma knockout model.

Maintaining an appropriate balance between subsets of CD4(+) helper T cells and T regulatory cells (Tregs) is a critical process in immune homeostasis and a protective mechanism against autoimmunity and inflammation. To identify the role of vitamin A-related compounds, we investigated the regulation of interleukin (IL)-17-producing helper T cells (Th17 cells) and Tregs treated with all-trans-retinal (retinal). CD4(+)T cells or total cells from the spleens of C57BL/6 mice were stimulated under Treg-polarizing (anti-CD3/CD28 and TGF-ß) or Th17-polarizing (anti-CD3/CD28, TGF-ß, and IL-6) conditions in the presence or absence of retinal. To analyze their suppressive abilities, retinal-induced Tregs or TGF-ß-induced Tregs were co-cultured with responder T cells. Collagen-induced arthritis (CIA) was established in interferon (IFN)-γ knockout mice. On day 13, retinal-induced Tregs were adoptively transferred to mice with established CIA after second immunizations. Compared with TGF-ß-induced Treg cells, retinal-induced Tregs showed increased Foxp3 expression and mediated stronger suppressive activity. Under Th17-polarizing conditions, retinal inhibited the production of IL-17 and increased the expression of Foxp3.Retinal-induced Tregs showed therapeutic effects in IFN-γ knockout CIA mice. Thus, we demonstrated that retinal reciprocally regulates Foxp3(+) Tregs and Th17 cells. These findings suggest that retinal, a vitamin A metabolite, can regulate the balance between pro- and anti-inflammatory immunity. A better understanding of the manipulation of Foxp3 and Tregs may enable the application of this tremendous therapeutic potential in various autoimmune diseases.

Myeloid differentiation primary response protein 88 blockade upregulates indoleamine 2,3-dioxygenase expression in rheumatoid synovial fibroblasts.

Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and has been implicated in tumor tolerance, autoimmune disease and asthma. IDO was detected in the joint synovial tissue in the inflammatory microenvironment of rheumatoid arthritis (RA), but IDO expression in joint synovial tissue is not sufficient to overcome the inflamed synovial environment. This study aimed to unravel the mechanisms involving the failure to activate tolerogenic IDO in the inflamed joint. We demonstrate that both poly (I:C) and lipopolysaccharide (LPS) induce expression of IDO in synovial fibroblasts. However, inflammatory cytokines such as IL-17, TNF-alpha, IL-12, IL-23 and IL-16 did not induce IDO expression. Poly (I:C) appeared to induce higher IDO expression than did LPS. Surprisingly, toll-like receptor (TLR)4-mediated IDO expression was upregulated after depletion of myeloid differentiation primary response protein 88 (MyD88) in synovial fibroblasts using small interfering RNA (siRNA). IDO, TLR3 and TLR4 were highly expressed in synovial tissue of RA patients compared with that of osteoarthritis patients. In addition, RA patients with severe disease activity had higher levels of expression of IDO, TLR3 and TLR4 in the synovium than patients with mild disease activity. These data suggest that upregulation of IDO expression in synovial fibroblasts involves TLR3 and TLR4 activation by microbial constituents. We showed that the mechanisms responsible for IDO regulation primarily involve MyD88 signaling in synovial fibroblasts, as demonstrated by siRNAmediated knockdown of MyD88.

Macrophage migration inhibitory factor enhances osteoclastogenesis through upregulation of RANKL expression from fibroblast-like synoviocytes in patients with rheumatoid arthritis.

INTRODUCTION: Macrophage migration inhibitory factor (MIF) is one of key regulators in acute and chronic immune-inflammatory conditions including rheumatoid arthritis (RA). We examined the effect of MIF on osteoclastogenesis, which is known to play a crucial role in bone destruction in RA. METHODS: The concentration of MIF and receptor activator of nuclear factor-κB ligand (RANKL) in the synovial fluid was measured by ELISA. MIF-induced RANKL expression of RA synovial fibroblasts was determined by real-time PCR and western blot. Osteoclastogenesis was analyzed in culture of human peripheral blood mononuclear cells (PBMC) with MIF. Osteoclastogenesis was also determined after co-cultures of rhMIF-stimulated RA synovial fibroblasts with human PBMC. RESULTS: Synovial fluid MIF concentration in RA patients was significantly higher than in osteoarthritis (OA) patients. The concentration of RANKL correlated with that of MIF in RA synovial fluids (r = 0.6, P < 0.001). MIF stimulated the expression of RANKL mRNA and protein in RA synovial fibroblasts, which was partially reduced by blocking of interleukin (IL)-1ß. Osteoclasts were differentiated from PBMC cultures with MIF and M-CSF, even without RANKL. Osteoclastogenesis was increased after co-culture of MIF-stimulated RA synovial fibroblasts with PBMC and this effect was diminished by RANKL neutralization. Blocking of PI3 kinase, p38 MAP kinase, JAK-2, NF-κB, and AP-1 also led to a marked reduction in RANKL expression and osteoclastogenesis. CONCLUSIONS: The interactions among MIF, synovial fibroblasts, osteoclasts, RANKL, and IL-1ß have a close connection in osteoclastogenesis and they could be a potential gateway leading to new therapeutic approaches in treating bone destruction in RA.

Regulation of B cell activating factor (BAFF) receptor expression by NF-ΚB signaling in rheumatoid arthritis B cells.

B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). High levels of B cell activating factor (BAFF) are detected in autoimmune diseases. BAFF and BAFF receptor (BAFF-R) are expressed in B and T cells of RA synovium. The study was undertaken to identify the NF-ΚB signal pathway involved in the induction of BAFF-R in human B cells. Immunohistochemical staining of NF-ΚB p65, NF-ΚB p50, BAFF, and BAFF-R was performed on sections of synovium from severe and mild RA and osteoarthritis (OA) patients. Peripheral blood mononuclear cells (PBMCs) were isolated from control and RA patients and B cells were isolated from controls. BAFF-R was analyzed by flow cytometry, realtime PCR and confocal staining after treatment with NF-ΚB inhibitors. NF-ΚB p65, NF-ΚB p50, BAFF, and BAFF-R were highly expressed in severe RA synovium relative to mild RA synovium or OA synovium. BAFF-R expression was reduced by NF-ΚB inhibitors in PBMCs and B cells from normal controls. We also showed reduction in expression of BAFF-R via inhibition of the NF-ΚB pathway in PBMCs of RA patients. BAFF/BAFF-R signaling is an important mechanism of pathogenesis in RA and that BAFF-R reduction by NF-ΚB blocking therapy is another choice for controlling B cells in autoimmune diseases such as RA.

Grape seed proanthocyanidin extract (GSPE) differentially regulates Foxp3(+) regulatory and IL-17(+) pathogenic T cell in autoimmune arthritis.

Grape seed proanthocyanidin extract (GSPE), which is the antioxidant derived from grape seeds, has been reported to possess a variety of potent properties. We have previously shown that GSPE attenuates collagen-induced arthritis. However the mechanism by which GSPE regulates the immune response remains unclear, although it may involve effects on the regulation of pathogenic T cells in autoimmune arthritis. To clarify this issue, we have assessed the effects of GSPE on differential regulation of Th17 and regulatory T (Treg) cells subsets in vitro in mouse and human CD4(+) T cells. We observed that GSPE decreased the frequency of IL-17(+)CD4(+)Th17 cells and increased induction of CD4(+)CD25(+)forkhead box protein 3 (Foxp3)(+) Treg cells. In vivo, GSPE effectively attenuated clinical symptoms of established collagen-induced arthritis in mice with concomitant suppression of IL-17 production and enhancement of Foxp3 expression (type II collagen-reactive Treg cells) in CD4(+) T cells of joints and splenocytes. The presence of GSPE decreased the levels of IL-21, IL-22, IL-26 and IL-17 production by human CD4(+) T cells in a STAT3-dependent manner. In contrast, GSPE induces Foxp3(+) Treg cells in humans. Our results suggest that GSPE possesses a reciprocal control over IL-17 and Foxp3. By potently regulating inflammatory T cell differentiation, GSPE may serve as a possible novel therapeutic agent for inflammatory and autoimmune diseases, including rheumatoid arthritis.

IL-17 increases cadherin-11 expression in a model of autoimmune experimental arthritis and in rheumatoid arthritis.

IL-17 plays important roles in synovial inflammation and bone destruction in the mouse model of autoimmune arthritis and in rheumatoid arthritis (RA). Cadherin-11 determines the behavior of synovial cells in their proinflammatory and destructive tissue response in inflammatory arthritis, and promotes the invasive behavior of fibroblast-like synoviocytes (FLS). The purpose of this study was to examine the effect of IL-17 on the expression of cadherin-11 in autoimmune experimental arthritis and in RA synovium. The severity of synovial inflammation and bone destruction were examined in IL-17-injected knee joints of mice with collagen-induced arthritis (CIA). Cadherin-11 expression was examined in the synovium of mice with CIA, of IL-1 receptor antagonist (IL-1Ra)-deficient mice and of patients with RA and osteoarthritis (OA). Cadherin-11 expression was also examined in the synovium of IL-17 injected knee joints from CIA mice and in IL-17-stimulated FLS of CIA mice and RA patients. IL-17 aggravated synovial inflammation and bone destruction in CIA. By immunohistochemistry, cadherin-11 expression was increased in the synovium of mice with CIA and IL-1Ra-deficient mice and in patients with RA. Synovial cadherin-11 expression in IL-17-injected knee joints, measured by real-time RT-PCR, Western blot and immunohistochemistry, was increased in CIA. Cadherin-11 expression was significantly increased by IL-17 in cultured FLS of CIA mice and RA patients, and these increases were blocked by NF-κB inhibitors. IL-17 increased the expression of cadherin-11 in vivo and in vitro, which implies that an IL-17-induced increase of cadherin-11 is involved in IL-17-induced aggravation of joint destruction and inflammation.

The expression of the receptor for advanced glycation end-products (RAGE) in RA-FLS is induced by IL-17 via Act-1.

INTRODUCTION: The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of arthritis. We conducted this study to determine the effect of interleukin (IL)-17 on the expression and production of RAGE in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). The role of nuclear factor-κB (NF-κB) activator 1 (Act1) in IL-17-induced RAGE expression in RA-FLS was also evaluated. METHODS: RAGE expression in synovial tissues was assessed by immunohistochemical staining. RAGE mRNA production was determined by real-time polymerase chain reaction. Act-1 short hairpin RNA (shRNA) was produced and treated to evaluate the role of Act-1 on RAGE production. RESULTS: RAGE, IL-17, and Act-1 expression increased in RA synovium compared to osteoarthritis synovium. RAGE expression and production increased by IL-17 and IL-1ß (*P <0.05 vs. untreated cells) treatment but not by tumor necrosis factor (TNF)-α in RA-FLS. The combined stimuli of both IL-17 and IL-1ß significantly increased RAGE production compared to a single stimulus with IL-17 or IL-1ß alone (P <0.05 vs. 10 ng/ml IL-17). Act-1 shRNA added to the RA-FLS culture supernatant completely suppressed the enhanced production of RAGE induced by IL-17. CONCLUSIONS: RAGE was overexpressed in RA synovial tissues, and RAGE production was stimulated by IL-17 and IL-1ß. Act-1 contributed to the stimulatory effect of IL-17 on RAGE production, suggesting a possible inhibitory target for RA treatment.

Impact of interleukin-21 in the pathogenesis of primary Sjögren's syndrome: increased serum levels of interleukin-21 and its expression in the labial salivary glands.

INTRODUCTION: Interleukin (IL)-21 is a cytokine that controls the functional activity of effector T helper cells and the differentiation of Th17 cells, and promotes B-cell differentiation. To test whether IL-21 participates in the pathogenesis of primary Sjögren's syndrome (SS), serum IL-21 level was measured and IL-21 expression in the labial salivary glands (LSG) was examined. METHODS: Serum IL-21 levels in 40 primary SS, 40 rheumatoid arthritis (RA), and 38 systemic lupus erythematosus (SLE) patients and 20 healthy controls were measured. Serum IL-21 levels of SS patients were assessed for correlations with laboratory data, including anti-nuclear antibody, anti-Ro/La antibodies, globulin, immunoglobulin (Ig) class, and IgG subclass. LSGs from 16 primary SS and 4 controls with sicca symptoms were evaluated for IL-21 and IL-21 receptor (IL-21R) expression by immunohistochemistry. Confocal microscopy was performed to further characterize the IL-21 positive cells. RESULTS: Primary SS patients had significantly higher serum IL-21 levels than controls, and these increments correlated positively with levels of IgG, IgG1. Serum IgG1 levels correlated with anti-Ro antibody titers. Immunohistochemical analyses showed that lymphocytic foci and the periductal area of the LSGs from SS patients expressed high levels of IL-21 and lower levels of IL-21R, whereas the control LSGs showed minimal expression of both antigens. The more the lymphocyte infiltrated, IL-21 expression in LSGs showed a tendency to increase. Confocal microscopic analyses revealed that IL-21 expressing infiltrating lymphocytes in the LSGs of SS patients also expressed CXCR5. CONCLUSIONS: Primary SS is associated with high serum IL-21 levels that correlate positively with serum IgG, especially IgG1, levels. The expression of IL-21 is increased as more lymphocytes infiltrated in LSGs. These observations suggest that IL-21 may play an important role in primary SS pathogenesis.