The role of T cell interleukin-17 in conducting destructive arthritis: lessons from animal models

Interleukin-17 (IL-17) is a T cell cytokine spontaneously produced by cultures of rheumatoid arthritis (RA) synovial membranes. High levels have been detected in the synovial fluid of patients with RA. The trigger for IL-17 is not fully identified; however, IL-23 promotes the production of IL-17 and a strong correlation between IL-15 and IL-17 levels in synovial fluid has been observed. IL-17 is a potent inducer of various cytokines such as tumor necrosis factor (TNF)-α, IL-1, and receptor activator of NF-κB ligand (RANKL). Additive or even synergistic effects with IL-1 and TNF-α in inducing cytokine expression and joint damage have been shown in vitro and in vivo. This review describes the role of IL-17 in the pathogenesis of destructive arthritis with a major focus on studies in vivo in arthritis models. From these studies in vivo it can be concluded that IL-17 becomes significant when T cells are a major element of the arthritis process. Moreover, IL-17 has the capacity to induce joint destruction in an IL-1-independent manner and can bypass TNF-dependent arthritis. Anti-IL-17 cytokine therapy is of interest as an additional new anti-rheumatic strategy for RA, in particular in situations in which elevated IL-17 might attenuate the response to anti-TNF/anti-IL-1 therapy.     

Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin

Pathological processes involved in the initiation of rheumatoid synovitis remain unclear. We undertook the present study to identify immune and stromal processes that are present soon after the clinical onset of rheumatoid arthritis (RA) by assessing a panel of T cell, macrophage, and stromal cell related cytokines and chemokines in the synovial fluid of patients with early synovitis. Synovial fluid was aspirated from inflamed joints of patients with inflammatory arthritis of duration 3 months or less, whose outcomes were subsequently determined by follow up. For comparison, synovial fluid was aspirated from patients with acute crystal arthritis, established RA and osteoarthritis. Rheumatoid factor activity was blocked in the synovial fluid samples, and a panel of 23 cytokines and chemokines measured using a multiplex based system. Patients with early inflammatory arthritis who subsequently developed RA had a distinct but transient synovial fluid cytokine profile. The levels of a range of T cell, macrophage and stromal cell related cytokines (e.g. IL-2, IL-4, IL-13, IL-17, IL-15, basic fibroblast growth factor and epidermal growth factor) were significantly elevated in these patients within 3 months after symptom onset, as compared with early arthritis patients who did not develop RA. In addition, this profile was no longer present in established RA. In contrast, patients with non-rheumatoid persistent synovitis exhibited elevated levels of interferon-γ at initiation. Early synovitis destined to develop into RA is thus characterized by a distinct and transient synovial fluid cytokine profile. The cytokines present in the early rheumatoid lesion suggest that this response is likely to influence the microenvironment required for persistent RA.     

Rheumatoid synovial fluid interleukin-17-producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression,but little interleukin-22 and interleukin-23R expression

Introduction  

Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity.     

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.     

Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice

Interleukin-17 is a T cell-derived proinflammatory cytokine. This cytokine is suspected to be involved in the development of rheumatoid arthritis (RA) because this cytokine expression is augmented in synovial tissues of RA patients. The pathogenic roles of IL-17 in the development of RA, however, still remain to be elucidated. In this study, effects of IL-17 deficiency on collagen-induced arthritis (CIA) model were examined using IL-17-deficient mice (IL-17(-/-) mice). We found that CIA was markedly suppressed in IL-17(-/-) mice. IL-17 was responsible for the priming of collagen-specific T cells and collagen-specific IgG2a production. Thus, these observations suggest that IL-17 plays a crucial role in the development of CIA by activating autoantigen-specific cellular and humoral immune responses.     

Interleukin-17: the missing link between T-cell accumulation and effector cell actions in rheumatoid arthritis?

The prominence of T cells and monocyte/macrophages in rheumatoid synovium suggests T cells may localize and amplify the effector functions of monocyte/macrophages in rheumatoid disease. However, while T cells are abundant in rheumatoid joints, classic T-cell derived cytokines are scarce, especially when compared to the levels of monokines IL-1 beta and TNF-alpha. For this reason, it has been speculated that monocyte/macrophages may act independently of T cells in rheumatoid disease and that the role of T cells may be more or less irrelevant to core disease mechanisms. The question of T-cell influence requires re-evaluation in light of the characterization of IL-17, a T-cell derived cytokine that is abundant in rheumatoid synovium and synovial fluid. IL-17 has a number of pro-inflammatory effects, both directly and through amplification of the effects of IL-1 beta and TNF-alpha. IL-17 is able to induce expression of pro-inflammatory cytokines and stimulate release of eicosanoids by monocytes and synoviocytes. Furthermore, IL-17 has been implicated in the pathogenesis of inflammatory bone and joint damage through induction of matrix metalloproteinases and osteoclasts, as well as inhibition of proteoglycan synthesis. In animal models of arthritis, intra-articular injection of IL-17 results in joint inflammation and damage. The recognition of IL-17 as a pro-inflammatory T cell derived cytokine, and its abundance within rheumatoid joints, provides the strongest candidate mechanism to date through which T cells can capture and localize macrophage effector functions in rheumatoid arthritis. As such, IL-17 warrants consideration for its potential as a therapeutic target in rheumatoid arthritis.     

Interleukin-1 production by mononuclear cells from rheumatoid synovial effusions

The polypeptide interleukin-1 (IL-1) is a cytokine that may mediate inflammation and connective tissue damage in rheumatoid arthritis (RA). We examined cytokine production by normal blood and by rheumatoid synovial mononuclear cells with sensitive (picomolar) assays. The assays were immunolabeling and immunoblotting with rabbit anti-IL-1 beta sera, and proliferation of the murine D10 cell line to IL-1. Little or no cytokine was detected in rheumatoid joint fluid or in exudate mononuclear cells from patients with acute rheumatoid flares. The mononuclear cells could be induced to make IL-1 upon stimulation with lipopolysaccharide (LPS). The responsive cells were monocytes, since all could be double-labeled with anti-IL-1 and the monocyte-specific CD14 antibody. More than 80% of the synovial fluid monocytes made IL-1 beta after 24 hr in 2 ng/ml LPS. Other agents failed to induce IL-1 from enriched populations of monocytes including interferon gamma (IFN-gamma), poly (I/C), phorbol myristate acetate (PMA), concanavalin A (Con A), phytohemagglutinin (PHA), and anti-CD3 antibodies. Relatively high levels of dendritic cells (DC) were present in RA effusions, but these did not produce IL-1 in response to any of the above stimuli. Blood dendritic cells also did not make IL-1, whereas blood monocytes responded comparably to synovial exudate cells. The data indicate that rheumatoid exudate monocytes make very little IL-1 during acute flares of arthritis and that this cytokine is primarily a macrophage rather than a dendritic cell product.     

Kirenol exerts a potent anti-arthritic effect in collagen-induced arthritis by modifying the T cells balance

Rheumatoid arthritis is characterized by the imbalance of T cells, which leads to increased pro-inflammatory and reduced anti-inflammatory cytokines. Modulating the balance among T cells is crucial for the treatment of RA. Kirenol is a major diterpenoid components of Herba Siegesbeckiae, which has been applied for arthritic therapy for centuries. Since prior research showed Kirenol exhibited anti-inflammatory effect in rats, in this study we have evaluated the effect and mechanism of bioactive Kirenol in a rat model of collagen-induced arthritis (CIA) on modulation of T cells. After immunization with bovine type II collagen (CII), Wistar rats were orally administered saline (CIA group), 2 mg/kg Kirenol or 2 mg/kg prednisolone daily for 30 days. The severity of arthritis was clinically and histologically assessed. The numbers of CD4?CD25?Foxp3? T regulatory cells (Tregs) and IFNγ?CD4? and IL4?CD4? T cells were determined by flow cytometry, the mRNA expression level of Foxp3 was quantified by RT-PCR, cytokine levels were measured by ELISA and CII-induced cell proliferation was quantified in vitro. Kirenol significantly delayed the occurrence and reduced the disease severity of CIA. Histological analysis confirmed Kirenol suppressed joint inflammation and inhibited cartilage and bone destruction, compared to the CIA group. Kirenol also upregulated the mRNA expression of Foxp3, increased the numbers of CD4?CD25?Foxp3? and IL4?CD4? T cells, and reduced the number of IFNγ?CD4? T cells. Kirenol reduced the levels of TNF-α, IL-17A and IL-6 in synovial fluid and TNF-α, IL-17A and IFN-γ in serum, and increased the serum levels of IL-4, IL-10 and TGF-β1. In addition, Kirenol inhibited the ability of CII to induce splenocyte, PBMC and lymph node cell proliferation in vitro, compared to cells from CIA rats. In conclusion, these results suggest that Kirenol may be a potential immunosuppressant for the treatment for rheumatoid arthritis.     

Effector function of resting T cells: activation of synovial fibroblasts

Synovial tissue in rheumatoid arthritis is characterized by infiltration with large numbers of T lymphocytes and APCs as well as hyperplasia of synovial fibroblasts. Current understanding of the pathogenesis of RA includes the concept that synovial fibroblasts, which are essential to cartilage and bone destruction, are regulated by cytokines derived primarily from monocyte-macrophage cells. Recently it has been found that synovial fibroblasts can also function as accessory cells for T cell activation by superantigens and other stimuli. We have now found that highly purified resting T cells, even in the absence of T cell mitogens, induce activation of synovial fibroblasts when cocultured for 6-24 h. Such activation was evident by induction or augmentation of mRNA for stromelysin, IL-6, and IL-8, gene products important in joint inflammation and joint destruction. Furthermore, increased production of IL-6 and IL-8 was quantitated by intracellular cytokine staining and flow cytometry. This technique, previously used for analysis of T cell function, was readily adaptable for assays of synovial fibroblasts. Resting T cells also induced synovial fibroblasts to produce PGE(2), indicating activation of expression of the cyclooxygenase 2 gene. Synergy was observed between the effects of IL-17, a cytokine derived from stimulated T cells that activates fibroblasts, and resting T lymphocytes. Various subsets of T cells, CD4(+), CD8(+), CD45RO(+), and CD45RA(+) all had comparable ability to induce synovial fibroblast activation. These results establish an Ag-independent effector function for resting T cells that is likely to be important in inflammatory compartments in which large numbers of T lymphocytes and fibroblasts can come into direct contact with each other.     

Gene therapy for rheumatoid arthritis

Rheumatoid arthritis (RA) is a severe autoimmune systemic disease. Chronic synovial inflammation results in destruction of the joints. No conventional treatment is efficient in RA. Gene therapy of RA targets mainly the players of inflammation or articular destruction: TNF-alpha or IL-1 blocking agents (such as anti-TNF-alpha monoclonal antibodies, soluble TNF-alpha receptor, type II soluble receptor of IL-1, IL-1 receptor antagonist), antiinflammatory cytokines (such as IL-4, IL-10, IL-1), and growth factors. In this polyarticular disease, the vector expressing the therapeutic protein can be administered as a local (intra-articular injection) or a systemic treatment (extra-articular injection). All the main vectors have been used in experimental models, including the more recent lentivirus and adeno-associated virus. Ex vivo gene transfer was performed with synovial cells, fibroblasts, T cells, dendritic cells, and different cells from xenogeneic origin. In vivo gene therapy is simpler, although a less controlled method. Clinical trials in human RA have started with ex vivo retrovirus-expressing IL-1 receptor antagonists and have demonstrated the feasibility of the strategy of gene therapy. The best target remains to be determined and extensive research has to be conducted in preclinical studies.     

Dopamine induces IL-6-dependent IL-17 production via D1-like receptor on CD4 naive T cells and D1-like receptor antagonist SCH-23390 inhibits cartilage destruction in a human rheumatoid arthritis/SCID mouse chimera model

A major neurotransmitter dopamine transmits signals via five different seven-transmembrane G protein-coupled receptors termed D1-D5. Several studies have shown that dopamine not only mediates interactions into the nervous system, but can contribute to the modulation of immunity via receptors expressed on immune cells. We have previously shown an autocrine/paracrine release of dopamine by dendritic cells (DCs) during Ag presentation to naive CD4(+) T cells and found efficacious results of a D1-like receptor antagonist SCH-23390 in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis and in the NOD mouse model of type I diabetes, with inhibition of Th17 response. This study aimed to assess the role of dopaminergic signaling in Th17-mediated immune responses and in the pathogenesis of rheumatoid arthritis (RA). In human naive CD4(+) T cells, dopamine increased IL-6-dependent IL-17 production via D1-like receptors, in response to anti-CD3 plus anti-CD28 mAb. Furthermore, dopamine was localized with DCs in the synovial tissue of RA patients and significantly increased in RA synovial fluid. In the RA synovial/SCID mouse chimera model, although a selective D2-like receptor antagonist haloperidol significantly induced accumulation of IL-6(+) and IL-17(+) T cells with exacerbated cartilage destruction, SCH-23390 strongly suppressed these responses. Taken together, these findings indicate that dopamine released by DCs induces IL-6-Th17 axis and causes aggravation of synovial inflammation of RA, which is the first time, to our knowledge, that actual evidence has shown the pathological relevance of dopaminergic signaling with RA.     

IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

Interleukin-26 (IL-26), a member of the IL-10 cytokine family, induces the production of proinflammatory cytokines by epithelial cells. IL-26 has been also reported overexpressed in Crohn''s disease, suggesting that it may be involved in the physiopathology of chronic inflammatory disorders. Here, we have analyzed the expression and role of IL-26 in rheumatoid arthritis (RA), a chronic inflammatory disorder characterized by joint synovial inflammation. We report that the concentrations of IL-26 are higher in the serums of RA patients than of healthy subjects and dramatically elevated in RA synovial fluids compared to RA serums. Immunohistochemistry reveals that synoviolin⁺ fibroblast-like synoviocytes and CD68⁺ macrophage-like synoviocytes are the main IL-26-producing cells in RA joints. Fibroblast-like synoviocytes from RA patients constitutively produce IL-26 and this production is upregulated by IL-1-beta and IL-17A. We have therefore investigated the role of IL-26 in the inflammatory process. Results show that IL-26 induces the production of the proinflammatory cytokines IL-1-beta, IL-6, and tumor necrosis factor (TNF)-alpha by human monocytes and also upregulates the expression of numerous chemokines (mainly CCL20). Interestingly, IL-26-stimulated monocytes selectively promote the generation of RORgamma t⁺ Th17 cells, through IL-1-beta secretion by monocytes. More precisely, IL-26-stimulated monocytes switch non-Th17 committed (IL-23R⁻ or CCR6⁻ CD161⁻) CD4⁺ memory T cells into Th17 cells. Finally, synovial fluids from RA patients also induce Th17 cell generation and this effect is reduced after IL-26 depletion. These findings show that IL-26 is constitutively produced by RA synoviocytes, induces proinflammatory cytokine secretion by myeloid cells, and favors Th17 cell generation. IL-26 thereby appears as a novel proinflammatory cytokine, located upstream of the proinflammatory cascade, that may constitute a promising target to treat RA and chronic inflammatory disorders.     

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.     

Development of proteoglycan-induced arthritis is independent of IL-17

IL-17 is the hallmark cytokine for the newly identified subset of Th cells, Th17. Th17 cells are important instigators of inflammation in several models of autoimmune disease; in particular, collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE), which were previously characterized as Th1-mediated diseases. Although high levels of IFN-gamma are secreted in CIA and EAE, disease is exacerbated in IFN-gamma- or IFN-gamma receptor-deficient mice due to the ability of IFN-gamma to suppress IL-17 secretion. However, in proteoglycan-induced arthritis (PGIA), severe arthritis is dependent on the production of IFN-gamma. We were therefore interested in determining the role of IL-17 in PGIA. We assessed the progression of arthritis in IL-17-deficient (IL-17-/-) mice and found the onset and severity of arthritis were equivalent in wild-type (WT) and IL-17-/- mice. Despite evidence that IL-17 is involved in neutrophil recruitment, synovial fluid from arthritic joints showed a comparable proportion of Gr1+ neutrophils in WT and IL-17-/- mice. IL-17 is also implicated in bone destruction in autoimmune arthritis, however, histological analysis of the arthritic joints from WT and IL-17-/- mice revealed a similar extent of joint cellularity, cartilage destruction, and bone erosion despite significantly reduced RANKL (receptor activator of NK-kappaB ligand) expression. There were only subtle differences between WT and IL-17-/- mice in proinflammatory cytokine expression, T cell proliferation, and autoantibody production. These data demonstrate that IL-17 is not absolutely required for autoimmune arthritis and that the production of other proinflammatory mediators is sufficient to compensate for the loss of IL-17 in PGIA.     

Interleukin-34 as a promising clinical biomarker and therapeutic target for inflammatory arthritis

Interleukin-34 (IL-34), recently identified as a novel inflammatory cytokine and the second ligand for colony-stimulating factor-1 receptor, is known to play regulatory roles in the development, maintenance, and function of mononuclear phagocyte lineage cells – especially osteoclasts. Regarding its primary effect on osteoclasts, IL-34 has been shown to stimulate formation and activation of osteoclasts, which in turn magnifies osteoclasts-resorbing activity. In addition to its role in osteoclastogenesis, IL-34 has been implicated in inflammation of synovium via augmenting production of inflammatory mediators, in which altered IL-34 expression is regulated by pro-inflammatory cytokines responsible for cartilage degradation. Indeed, IL-34 has been documented to be highly expressed in inflamed synovium of rheumatoid arthritis (RA) and knee osteoarthritis (OA) patients, which are recognized as inflammatory arthritis. Furthermore, a number of clinical studies demonstrated that IL-34 levels were significantly increased in the circulation and synovial fluid of patients with RA and knee OA. Its levels were also found to be positively associated with disease severity – especially radiographic severity of both RA and knee OA patients. Interestingly, emerging evidence has accumulated that functional blockage of IL-34 with specific antibody can alleviate the severity of inflammatory arthritis. It is therefore reasonable to speculate that IL-34 may be developed as a potential biomarker and a new therapeutic candidate for inflammatory arthritis. To date, there are numerous studies showing IL-34 involvement and association with many aspects of inflammatory arthritis. Herein, this review aimed to summarize the recent findings regarding regulatory role of IL-34 in synovial inflammation-mediated cartilage destruction and update the current comprehensive knowledge on usefulness of IL-34-based treatment in inflammatory arthritis – particularly RA and knee OA.