Cyr61 is involved in neutrophil infiltration in joints by inducing IL-8 production by fibroblast-like synoviocytes in rheumatoid arthritis

Introduction

It is well known that neutrophils play very important roles in the development of rheumatoid arthritis (RA) and interleukin (IL)-8 is a critical chemokine in promoting neutrophil migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in RA promotes FLS proliferation and Th17 cell differentiation, thus Cyr61 is a pro-inflammatory factor in RA pathogenesis. In this study, we explored the role of Cyr61 in neutrophil migration to the joints of RA patients.

Methods

RA FLS were treated with Cyr61 and IL-8 expression was analyzed by real-time PCR and ELISA. The migration of neutrophils recruited by the culture supernatants was determined by the use of a chemotaxis assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as a control. Arthritis severity was determined by visual examination of the paws and joint destruction was determined by hematoxylin-eosin (H&E) staining. Signal transduction pathways in Cyr61-induced IL-8 production were investigated by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay or chromatin immunoprecipitation (ChIP) assay.

Results

We found that Cyr61 induced IL-8 production by RA FLS in an IL-1β and TNF-α independent pathway. Moreover, we identified that Cyr61-induced IL-8-mediated neutrophil migration in vitro. Using a CIA animal model, we found that treatment with anti-Cyr61 mAb led to a reduction in MIP-2 (a counterpart of human IL-8) expression and decrease in neutrophil infiltration, which is consistent with an attenuation of inflammation in vivo. Mechanistically, we showed that Cyr61 induced IL-8 production in FLS via AKT, JNK and ERK1/2-dependent AP-1, C/EBPβ and NF-κB signaling pathways.

Conclusions

Our results here reveal a novel role of Cyr61 in the pathogenesis of RA. It promotes neutrophil infiltration via up-regulation of IL-8 production in FLS. Taken together with our previous work, this study provides further evidence that Cyr61 plays a key role in the vicious cycle formed by the interaction between infiltrating neutrophils, proliferated FLS and activated Th17 cells in the development of RA.     

TL1A increased IL-6 production on fibroblast-like synoviocytes by preferentially activating TNF receptor 2 in rheumatoid arthritis

TNF-like protein 1A (TL1A), a member of tumor necrosis factor family, recognized as a ligand of death receptor 3 (DR3) and decoy receptor 3 (DcR3). The interaction of TL1A and DR3 may participate in the pathogenesis of some autoimmune diseases including rheumatoid arthritis (RA). Our previous results showed that high concentrations of TL1A could be found in synovial and serum in RA patients, and it was correlated with disease severity. In addition, TL1A could promote Th17 differentiation induced by TGF-β and IL-6 and increased the production of IL-17A. In the present study, we found that TL1A could promote the expression of IL-6 on fibroblast-like synoviocytes (FLS) of RA patients via NF-κB and JNK signaling pathway. TL1A-stimulated FLS increased the percentage of Th17 of peripheral blood mononuclear cells (PBMC) in RA via the production of IL-6, a critical cytokine involved in the differentiation of Th17. Moreover, the blocking of tumor necrosis factor receptor 2 (TNFR2) decreased TL1A-stimulated IL-6 production by RA FLS. Our results suggest that TL1A was capable of acting on RA FLS to elevate IL-6 expression, which promoted the production of Th17. More importantly, we showed that TL1A could influence RA FLS through binding to TNFR2 rather than DR3 on FLS, which indicated that the treatment of TNF inhibitors not only blocked the TNF but also suppressed the TL1A in RA patients.     

IL-17 receptor and its functional significance in psoriatic arthritis

To delineate the functional significance of IL-17 Receptor (IL-17RA) and characterize the IL-17 producing T cell (Th17) subpopulation in psoriatic arthritis (PsA). Mononuclear cells from blood and synovial fluid (SF) were obtained from PsA (n=20), rheumatoid arthritis (RA, n=20) and osteoarthritis (OA, n=20) patients. Synoviocytes (FLS) were isolated from the synovium of RA (n=5), PsA (n=5) and OA (n=5) patients. IL-17RA expression in FLS was identified by western blotting (WB) and flowcytometry. T lymphocytes derived from the SF of these patients were studied to identify and phenotype the Th17 cells. The functional significance of IL-17RA was determined by evaluating its regulatory role on the production of proinflammatory cytokines and endopeptidase. IL-17RA expression was found to be significantly higher in FLS of RA (15.7%±4.9) and PsA (4.5%±0.9) in comparison to OA (1.14%±0.9). Western blot analyses showed that the relative intensity (RI) of IL-17RA protein was higher in RA and PsA compared to OA (Fisher exact, P<0.01). A significant enrichment of IL-17-producing CD4+ T cells (7.9%±2.8) was observed in the SF of PsA patients compared to that of OA patients (P<.001). Compared to OA-FLS, recombinant IL-17 induced higher levels of IL-6, IL-8, and MMP-3 production in PsA-FLS. Blockage of IL-17RA with an anti-IL-17RA antibody inhibited the production of IL-6, IL-8, and MMP-3. This is the first report to demonstrate the functional significance of IL-17RA in PsA. Results of this study support the hypothesis that IL-17RA blocking antibodies have the potential to be a therapeutic option for psoriatic arthritis.     

IL-17/Th17 targeting: on the road to prevent chronic destructive arthritis?

Interleukin-17A (IL-17A) contributes to the pathogenesis of arthritis. Data from experimental arthritis indicate IL-17 receptor signaling as a critical pathway in turning an acute synovitis into a chronic destructive arthritis. The identification of six IL-17 family members (IL-17A-F) may extend the role of this novel cytokine family in the pathogenesis of chronic destructive joint inflammation. Whether the successful anti-IL-17A cytokine therapy in murine arthritis can be effectively translated to human arthritis need to be tested in clinical trials in humans. Interestingly, IL-17A and IL-17F are secreted by the novel T helper subset named Th17. This novel pathogenic T cell population induces autoimmune inflammation in mice and is far more efficient at inducing Th1-mediated autoimmune inflammation in mice than classical Th1 cells (IFN-gamma). In addition to IL-17A and IL-17F, Th17 cells are characterized by expression of IL-6, TNF, GM-CSF, IL-21, IL-22 and IL-26. Th17 cells have been established as a separate lineage of T helper cells in mice distinct from conventional Th1 and Th2 cells. Whether this also applies to human Th17 and whether RA is a Th1 or a Th17 mediated disease is still not clear. This review summarizes the findings about the role of IL-17 in arthritis and discusses the impact of the discovery of the novel Th17 cells for arthritis. Further studies are needed to unravel the role of Th17 cells and the interplay of IL-17 and other Th17 cytokines in the pathogenesis of arthritis and whether regulating Th17 cell activity will have additional value compared to neutralizing IL-17A activity alone. This might help to reach the ultimate goal not only to treat RA patients but to prevent the development of this crippling disease.     

Tacrolimus potently inhibits human osteoclastogenesis induced by IL-17 from human monocytes alone and suppresses human Th17 differentiation

Tacrolimus (FK506, Prograf?) is an orally available, T cell specific and anti-inflammatory agent that has been proposed as a therapeutic drug in rheumatoid arthritis (RA) patients. It has been known that T cells have a critical role in the pathogenesis of RA. Recent studies suggest that Th17 cells, which mainly produce IL-17, are involved in many autoimmune inflammatory disease including RA. The present study was undertaken to assess the effect of tacrolimus on IL-17-induced human osteoclastogenesis and human Th17 differentiation. Human CD14(+) monocytes were cultured in the presence of macrophage-colony stimulating factor (M-CSF) and IL-17. From day 4, tacrolimus was added to these cultures. Osteoclasts were immunohistologically stained for vitronectin receptor 10days later. IL-17 production from activated T cells stimulated with IL-23 was measured by enzyme-linked immunosorbent assay (ELISA). Th17 differentiation from na?ve T cells was assayed by flow cytometry. Tacrolimus potently inhibited IL-17-induced osteoclastogenesis from human monocytes and osteoclast activation. Addition of tacrolimus also reduced production of IL-17 in human activated T cells stimulated with IL-23. Interestingly, the population of human IL-17(+)IFN-γ(-) CD4 T cells or IL-17(+)TNF-α(+) CD4 T cells were decreased by adding of tacrolimus. The present study demonstrates that the inhibitory effect of tacrolimus on IL-17-induced osteoclastogenesis from human monocytes. Tacrolimus also inhibited expression of IL-17 or TNF-α by reducing the proportion of Th17, suggesting that therapeutic effect on Th17-associated disease such as RA, inflammatory bowel disease, multiple sclerosis, psoriasis, or allograft rejection.     

Local treatment with the selective IkappaB kinase beta inhibitor NEMO-binding domain peptide ameliorates synovial inflammation

Nuclear factor (NF)-kappaB is a key regulator of synovial inflammation. We investigated the effect of local NF-kappaB inhibition in rat adjuvant arthritis (AA), using the specific IkappaB kinase (IKK)-beta blocking NF-kappaB essential modulator-binding domain (NBD) peptide. The effects of the NBD peptide on human fibroblast-like synoviocytes (FLS) and macrophages, as well as rheumatoid arthritis (RA) whole-tissue biopsies, were also evaluated. First, we investigated the effects of the NBD peptide on RA FLS in vitro. Subsequently, NBD peptides were administered intra-articularly into the right ankle joint of rats at the onset of disease. The severity of arthritis was monitored over time, rats were sacrificed on day 20, and tissue specimens were collected for routine histology and x-rays of the ankle joints. Human macrophages or RA synovial tissues were cultured ex vivo in the presence or absence of NBD peptides, and cytokine production was measured in the supernatant by enzyme-linked immunosorbent assay. The NBD peptide blocked interleukin (IL)-1-beta-induced IkappaB alpha phosphorylation and IL-6 production in RA FLS. Intra-articular injection of the NBD peptide led to significantly reduced severity of arthritis (p < 0.0001) and reduced radiological damage (p = 0.04). This was associated with decreased synovial cellularity and reduced expression of tumor necrosis factor (TNF)-alpha and IL-1-beta in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-beta-induced TNF-alpha production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-alpha-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-kappaB blockade using a small peptide inhibitor of IKK-beta has anti-inflammatory effects in AA and human RA synovial tissue as well as in two important cell types in the pathogenesis of RA: macrophages and FLS. These results indicate that IKK-beta-targeted NF-kappaB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis.     

Activation of formyl peptide receptor 1 elicits therapeutic effects against collagen-induced arthritis

Rheumatoid arthritis (RA) is an autoimmune disorder which shows production of autoantibodies, inflammation, bone erosion, swelling and pain in joints. In this study, we examined the effects of an immune-modulating peptide, WKYMVm, that is an agonist for formyl peptide receptors (FPRs). Administration of WKYMVm into collagen-induced arthritis (CIA) mice, an animal model for RA, attenuated paw thickness, clinical scores, production of type II collagen-specific antibodies and inflammatory cytokines. WKYMVm treatment also decreased the numbers of T_H1 and T_H17 cells in the spleens of CIA mice. WKYMVm attenuated T_H1 and T_H17 differentiation in a dendritic cell (DC)-dependent manner. WKYMVm-induced beneficial effects against CIA and WKYMVm-attenuated T_H1 and T_H17 differentiation were reversed by cyclosporin H but not by WRW4, indicating a crucial role of FPR1. We also found that WKYMVm augmented IL-10 production from lipopolysaccharide-stimulated DCs and WKYMVm failed to suppress T_H1 and T_H17 differentiation in the presence of anti-IL-10 antibody. The therapeutic administration of WKYMVm also elicited beneficial outcome against CIA. Collectively, we demonstrate that WKYMVm stimulation of FPR1 in DCs suppresses the generation of T_H1 and T_H17 cells via IL-10 production, providing novel insight into the function of FPR1 in regulating CIA pathogenesis.     

Retinoic acid increases Foxp3+ regulatory T cells and inhibits development of Th17 cells by enhancing TGF-beta-driven Smad3 signaling and inhibiting IL-6 and IL-23 receptor expression

The de novo generation of Foxp3+ regulatory T (Treg) cells in the peripheral immune compartment and the differentiation of Th17 cells both require TGF-beta, and IL-6 and IL-21 are switch factors that drive the development of Th17 cells at the expense of Treg cell generation. The major vitamin A metabolite all-trans retinoic acid (RA) not only enforces the generation of Treg cells but also inhibits the differentiation of Th17 cells. Herein we show that RA enhances TGF-beta signaling by increasing the expression and phosphorylation of Smad3, and this results in increased Foxp3 expression even in the presence of IL-6 or IL-21. RA also inhibits the expression of IL-6Ralpha, IRF-4, and IL-23R and thus inhibits Th17 development. In vitro, RA significantly promotes Treg cell conversion, but in vivo during the development of experimental autoimmune encephalomyelitis it does not increase the frequency of Treg cells in the face of an ongoing inflammation. However, RA suppresses the disease very efficiently by inhibiting proinflammatory T cell responses, especially pathogenic Th17 responses. These data not only identify the signaling mechanisms by which RA can affect both Treg cell and Th17 differentiation, but they also highlight that in vivo during an autoimmune reaction, RA suppresses autoimmunity mainly by inhibiting the generation of effector Th17 cells.     

Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes

Chronic inflammatory diseases are characterized by local tissue injury caused by immunocompetent cells, in particular CD4(+) T lymphocytes, that are involved in the pathogenesis of these disorders via the production of distinctive sets of cytokines. Here, we have characterized single CD4(+) T cells that infiltrate inflamed tissue taken from patients with psoriasis, Crohn's disease, rheumatoid arthritis, or allergic asthma. Results from a cytokine production and gene profile analysis identified a population of in vivo differentiatedretinoid-related orphan receptor gamma-expressing T cells, producing high levels of IL-17, that can represent up to 30% of infiltrating T lymphocytes. Activated Th17 cells produced IL-26, TNF-alpha, lymphotoxin-beta, and IL-22. IL-17 and IL-22 concentrations secreted by tissue infiltrating Th17 cells could reach up to 100 nM and were inversely correlated with the production of Th1- and Th2-associated cytokines. In addition, tissue-infiltrating Th17 cells are also characterized by high cell surface expression of CCR6, a chemokine receptor that was not expressed by Th1 and Th2 cells, isolated from the same lesions, and by the production of CCL20/MIP3alpha, a CCR6 ligand, associated with tissue infiltration. Culture supernatants of activated Th17 cells, isolated from psoriatic lesions, induced the expression of gene products associated with inflammation and abnormal keratinocyte differentiation in an IL-17 and IL-22-dependent manner. These results show that tissue-infiltrating Th17 cells contribute to human chronic inflammatory disease via the production of several inflammatory cytokines and the creation of an environment contributing to their migration and sequestration at sites of inflammation.     

IL-27-producing CD14(+) cells infiltrate inflamed joints of rheumatoid arthritis and regulate inflammation and chemotactic migration

Interleukin (IL)-27, a heterodimeric cytokine, has been reported to be involved in the pathogenesis of autoimmune diseases through mediating differentiation of Th1 or Th17 cells and immune cell activity or survival. However, the origin and effects of IL-27 in joints of rheumatoid arthritis (RA) remain unclear. In this study, we investigated the distribution and anti-inflammatory roles of IL-27 in RA synovium. The IL-27 levels in plasma of RA patients, osteoarthritis (OA) patients, or healthy volunteers (n=15 per group) were equivalent and were at most 1 ng/ml, but the IL-27 level in synovial fluid of RA patients (n=15, mean 0.13 ng/ml; range 0.017-0.37 ng/ml) was significantly higher than that in synovial fluid of OA patients (n=15, mean 0.003 ng/ml; range 0-0.033 ng/ml) and potentially lower than in plasma. We analyzed the protein level of IL-27 produced by RA fibroblast-like synoviocytes (FLSs) or mononuclear cells (MNCs) from RA or OA synovial fluid or peripheral blood and showed that IL-27 in RA joints was derived from MNCs but not from FLSs. We also found by flow cytometry that IL-27-producing MNCs were CD14(+), and that these CD14(+)IL-27(+) cells were clearly detected in RA synovium but rarely in OA synovium by immunohistochemistry. Furthermore, we demonstrated that a relatively physiological concentration of IL-27 below 10 ng/ml suppressed the production of IL-6 and CCL20 from RA FLSs induced by proinflammatory cytokines through the IL-27/IL-27R axis. In the synovial fluid of RA, the IL-27 level interestingly had positive correlation with the IFN-γ level (r=0.56, p=0.03), but weak negative correlation with the IL-17A level (r=-0.30, p=0.27), implying that IL-27 in inflammatory joints of RA induces Th1 differentiation and suppresses the development or the migration of Th17 cells. These findings indicate that circulating IL-27-producing CD14(+) cells significantly infiltrate into inflamed regions such as RA synovium and have anti-inflammatory effects in several ways: both directly through the reduction of IL-6 production, and possibly through the induction of Th1 development and the suppression of Th17 development; and indirectly by regulation of recruitment of CCR6(+) cells, such as Th17 cells, through the suppression of CCL20 production. Our results suggest that such a serial negative feedback system could be applied to RA therapy.     

Hypoxia-Inducible Factor-2α Is an Essential Catabolic Regulator of Inflammatory Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that manifests as chronic inflammation and joint tissue destruction. However, the etiology and pathogenesis of RA have not been fully elucidated. Here, we explored the role of the hypoxia-inducible factors (HIFs), HIF-1α (encoded by HIF1A) and HIF-2α (encoded by EPAS1). HIF-2α was markedly up-regulated in the intimal lining of RA synovium, whereas HIF-1α was detected in a few cells in the sublining and deep layer of RA synovium. Overexpression of HIF-2α in joint tissues caused an RA-like phenotype, whereas HIF-1α did not affect joint architecture. Moreover, a HIF-2α deficiency in mice blunted the development of experimental RA. HIF-2α was expressed mainly in fibroblast-like synoviocytes (FLS) of RA synovium and regulated their proliferation, expression of RANKL (receptor activator of nuclear factor–κB ligand) and various catabolic factors, and osteoclastogenic potential. Moreover, HIF-2α–dependent up-regulation of interleukin (IL)-6 in FLS stimulated differentiation of T_H17 cells—crucial effectors of RA pathogenesis. Additionally, in the absence of IL-6 (Il6 ^−/− mice), overexpression of HIF-2α in joint tissues did not cause an RA phenotype. Thus, our results collectively suggest that HIF-2α plays a pivotal role in the pathogenesis of RA by regulating FLS functions, independent of HIF-1α.     

The role of interleukin-17 in mediating joint destruction in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, persistent inflammatory joint disease with systemic involvement that affects about 1% of the world’s population, that ultimately leads to the progressive destruction of joint. Effective medical treatment for joint destruction in RA is lacking because the knowledge about molecular mechanisms leading to joint destruction are incompletely understood. It has been confirmed that cytokine-mediated immunity plays a crucial role in the pathogenesis of various autoimmune diseases including RA. Recently, IL-17 was identified, which production by Th17 cells. IL-17 has proinflammatory properties and may promote bone and joint damage through induction of matrix metalloproteinases and osteoclasts. In mice, intra-articular injection of IL-17 into the knee joint results in joint inflammation and damage. In addition, it has been shown that blocking IL-17/IL-17R signaling is effective in the control of rheumatoid arthritis symptoms and in the prevention of joint destruction. In this article, we will briefly discuss the biological features of IL-17/IL-17R and summarize recent advances on the role of IL-17/IL-17R in the pathogenesis and treatment of joint destruction in RA.     

Altered memory T cell differentiation in patients with early rheumatoid arthritis.

The chronic immune response in rheumatoid arthritis (RA) might be driven by activated Th1 cells without sufficient Th2 cell differentiation to down-modulate inflammation. To test whether disordered memory T cell differentiation contributes to the typical Th1-dominated chronic inflammation in RA we investigated differentiation of resting CD4+ memory T cells in patients with early (6 wk to 12 mo) untreated RA and in age- and sex-matched healthy controls in vitro. No difference in cytokine secretion profiles of freshly isolated memory T cells was detected between patients and controls. A cell culture system was then employed that permitted the differentiation of Th effectors from resting memory T cells by short term priming. Marked differences were found in response to priming. Th2 cells could be induced in all healthy controls by priming with anti-CD28 in the absence of TCR ligation. By contrast, priming under those conditions resulted in Th2 differentiation in only 9 of 24 RA patients. Exogenous IL-4 could overcome the apparent Th2 differentiation defect in seven patients but was without effect in the remaining eight patients. In all patients a marked decrease in IL-2-producing cells and a significant increase in well-differentiated Th1 cells that produced IFN-gamma but not IL-2 were evident after priming with anti-CD3 and anti-CD28. The data suggest that CD4+ memory T cells from patients with early untreated RA manifest an intrinsic abnormality in their ability to differentiate into specific cytokine-producing effector cells that might contribute to the characteristic Th1-dominated chronic (auto)immune inflammation in RA.     

Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550)

Inhibitors of the JAK family of nonreceptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. In this study, we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4(+) T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-β. Moreover, CP-690,550 also prevented the activation of STAT1, induction of T-bet, and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with the inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving the inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells as well as innate immune cell signaling.     

Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce inflammatory cytokines and chemokines. The expressed chemokines are thought to be involved in the migration of inflammatory cells into the synovium. In this study we show that CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 enhanced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) from patients with RA, and their corresponding receptors, CCR2, CCR5, and CXCR4, respectively, were expressed by RA FLS. The chemokines stimulated RA FLS more effectively than skin fibroblasts. Culture with CCL2 enhanced phosphorylation of extracellular signal-related kinase 1 (ERK1) and ERK2, but not phosphorylation of p38 or Src. Moreover, activation of ERK1/2 was inhibited by pertussis toxin, a G(i)-coupled protein inhibitor, and RS-504393, CCR2 antagonist, suggesting that ERK1/2 was activated by CCL2 via CCR2 and G(i)-coupled protein. On the other hand, CCL2, CCL5, and CXCL12 were expressed on RA FLS, and their production was regulated by TNF-alpha, IL-1beta, and TGF-beta1. Our results indicate that the chemokines not only play a role in inflammatory cell migration, but are also involved in the activation of FLS in RA synovium, possibly in an autocrine or paracrine manner.