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)-alpha, IL-1, and receptor activator of NF-kappaB ligand (RANKL). Additive or even synergistic effects with IL-1 and TNF-alpha 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.     

Local treatment with the selective IκB kinase β inhibitor NEMO-binding domain peptide ameliorates synovial inflammation

Nuclear factor (NF)-κB is a key regulator of synovial inflammation. We investigated the effect of local NF-κB inhibition in rat adjuvant arthritis (AA), using the specific IκB kinase (IKK)-β blocking NF-κB 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-β-induced IκBα 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)-α and IL-1-β in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-β-induced TNF-α production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-α-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-κB blockade using a small peptide inhibitor of IKK-β 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-β-targeted NF-κB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis.     

Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFα). We examined the in vivo relationship between IL-32 and TNFα, and the pathologic role of IL-32 in the TNFα-related diseases – arthritis and colitis. We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly, TNFα reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover, IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFα, we prepared an overexpression model mouse of human IL-32β (BM-hIL-32) by bone marrow transplantation. Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFα, IL-1β, and IL-6 especially in response to lipopolysaccharide stimulation. Moreover, serum TNFα concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting analysis of splenocytes showed that the expression of TNFα was increased in resting F4/80⁺ macrophages, and the expression of TNFα, IL-1β and IL-6 was increased in lipopolysaccharide-stimulated F4/80⁺ macrophages and CD11c⁺ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic acid-induced colitis. In addition, the transfer of hIL-32β-producing CD4⁺ T cells significantly exacerbated collagen-induced arthritis, and a TNFα blockade cancelled the exacerbating effects of hIL-32β. We therefore conclude that IL-32 is closely associated with TNFα, and contributes to the exacerbation of TNFα-related inflammatory arthritis and colitis.     

Peripheral blood but not synovial fluid natural killer T cells are biased towards a Th1-like phenotype in rheumatoid arthritis

Natural killer T (NKT) cells have been implicated in the regulatory immune mechanisms that control autoimmunity. However, their precise role in the pathogenesis of rheumatoid arthritis (RA) remains unclear. The frequency, cytokine profile and heterogeneity of NKT cells were studied in peripheral blood mononuclear cells (PBMCs) from 23 RA patients and 22 healthy control individuals, including paired PBMC–synovial fluid samples from seven and paired PBMC–synovial tissue samples from four RA patients. Flow cytometry revealed a decreased frequency of NKT cells in PBMCs from RA patients. NKT cells were present in paired synovial fluid and synovial tissue samples. Based on the reactivity of PBMC-derived NKT cells toward α-galactosylceramide, RA patients could be divided into responders (53.8%) and nonresponders (46.2%). However, NKT cells isolated from synovial fluid from both responders and nonresponders expanded upon stimulation with α-galactosylceramide. Analysis of the cytokine profile of CD4⁺ and CD4^- PBMC derived NKT cell lines from RA patients revealed a significantly reduced number of IL-4 producing cells. In contrast, synovial fluid derived NKT cell lines exhibited a Th0-like phenotype, which was comparable to that in healthy control individuals. This suggests that synovial fluid NKT cells are functional, even in patients with nonresponding NKT cells in their blood. We conclude that, because the number of Vα24⁺Vβ11⁺CD3⁺ NKT cells is decreased and the cytokine profile of blood-derived NKT cells is biased toward a Th1-like phenotype in RA patients, NKT cells might be functionally related to resistance or progression of RA. Providing a local boost to the regulatory potential of NKT cells might represent a useful candidate therapy for 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.     

TH-17 cells in rheumatoid arthritis

Introduction  

The aim of this study was to quantify the number of T-helper (TH)-17 cells present in rheumatoid arthritis (RA) synovial fluid (SF) and to determine the level of interleukin (IL)-17 cytokine in RA, osteoarthritis (OA) and normal synovial tissue, as well as to examine SF macrophages for the presence of IL-23, IL-27 and interferon (IFN)-γ.     

Rheumatoid synovial CD4+ T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells

CD4⁺ memory T cells (Tm) from rheumatoid arthritis peripheral blood (RAPB) or peripheral blood from normal donors produced IL-2, whereas fewer cells secreted IFN-γ or IL-4 after a brief stimulation. RAPB Tm contained significantly more IFN-γ producers than normal cells. Many rheumatoid arthritis (RA) synovial Tm produced IFN-γ alone (40%) and fewer cells produced IL-2 or IL-4. An in vitro model was employed to generate polarized T-helper (Th) effectors. Normal and RAPB Tm differentiated into both IFN-γ- and IL-4-producing effectors. RA synovial fluid (RASF) Tm demonstrated defective responsiveness, exhibiting diminished differentiation of IL-4 effectors, whereas RA synovial tissue (RAST) Tm exhibited defective generation of IFN-γ and IL-4 producers.     

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.     

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.     

IL-17 derived from juxta-articular bone and synovium contributes to joint degradation in rheumatoid arthritis

The origin and role of IL-17, a T-cell derived cytokine, in cartilage and bone destruction during rheumatoid arthritis (RA) remain to be clarified. In human ex vivo models, addition of IL-17 enhanced IL-6 production and collagen destruction, and inhibited collagen synthesis by RA synovium explants. On mouse cartilage, IL-17 enhanced cartilage proteoglycan loss and inhibited its synthesis. On human RA bone explants, IL-17 also increased bone resorption and decreased formation. Addition of IL-1 in these conditions increased the effect of IL-17. Blocking of bone-derived endogenous IL-17 with specific inhibitors resulted in a protective inhibition of bone destruction. Conversely, intra-articular administration of IL-17 into a normal mouse joint induced cartilage degradation. In conclusion, the contribution of IL-17 derived from synovium and bone marrow T cells to joint destruction suggests the control of IL-17 for the treatment of RA.     

Cytokine, activation marker, and chemokine receptor expression by individual CD4+ memory T cells in rheumatoid arthritis synovium

IL-10, IL-13, IFN-γ, tumor necrosis factor (TNF)-α, LT-α, CD154, and TNF-related activation-induced cytokine (TRANCE) were expressed by 2-20% of rheumatoid arthritis (RA) synovial tissue CD4⁺ memory T cells, whereas CD4⁺ cells that produced IL-2, IL-4, or IL-6 were not detected. Expression of none of these molecules by individual CD4⁺ cells correlated with the exception of TRANCE and IL-10, and TRANCE and TNF-α. A correlation between expression of IL-10 and CCR7, LT-α and CCR6, IFN-γ and CCR5, and TRANCE and CXCR4 was also detected.     

Tumour necrosis factor activates the mitogen-activated protein kinases p38α and ERK in the synovial membrane in vivo

Tumour necrosis factor (TNF) is considered to be a major factor in chronic synovial inflammation and is an inducer of mitogen-activated protein kinase (MAPK) signalling. In the present study we investigated the ability of TNF to activate MAPKs in the synovial membrane in vivo. We studied human TNF transgenic mice – an in vivo model of TNF-induced arthritis – to examine phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38MAPKα in the inflamed joints by means of immunoblot and immunohistochemistry. In addition, the effects of systemic blockade of TNF, IL-1 and receptor activator of nuclear factor-κB (RANK) ligand on the activation of MAPKs were assessed. In vivo, overexpression of TNF induced activation of p38MAPKα and ERK in the synovial membrane, whereas activation of JNK was less pronounced and rarely observed on immunohistochemical analysis. Activated p38MAPKα was predominantly found in synovial macrophages, whereas ERK activation was present in both synovial macrophages and fibroblasts. T and B lymphocytes did not exhibit major activation of any of the three MAPKs. Systemic blockade of TNF reduced activation of p38MAPKα and ERK, whereas inhibition of IL-1 only affected p38MAPKα and blockade of RANK ligand did not result in any decrease in MAPK activation in the synovial membrane. These data indicate that TNF preferentially activates p38MAPKα and ERK in synovial membrane exposed to TNF. This not only suggests that targeted inhibition of p38MAPKα and ERK is a feasible strategy for blocking TNF-mediated effects on joints, but it also shows that even currently available methods to block TNF effectively reduce activation of these two MAPKs.     

Cell contact interactions in rheumatology, The Kennedy Institute for Rheumatology, London, UK, 1-2 June 2000

The intricate interactions that regulate relationships between endogenous tissue cells and infiltrating immune cells in the rheumatic joint, particularly in rheumatoid arthritis (RA), were the subject of the meeting. A better understanding of these interactions might help to define intervention points that could be used to develop specific therapies. The presentations and discussions highlighted the fact that, once chronic inflammation is established, several pro-inflammatory loops involving tumour necrosis factor (TNF)-α and interleukin (IL)-1β can be defined. Direct cellular contact with stimulated T lymphocytes induces TNF-α and IL-1β in monocytes which in turn induce functions in fibroblast-like synoviocytes. The latter include the production of stromal cell-derived factor-1α (SDF-1α) which enhances the expression of CD40L in T cells, which stimulates SDF-1α production in synoviocytes, which in turn protects T and B cells from apoptosis and enhances cell recruitment thus favoring inflammatory processes. IL-1β and TNF-α also induce IL-15 in fibroblast-like synoviocytes, which induces the production of IL-17 which in turn potentiates IL-1β and TNF-α production in monocyte-macrophages. This underlines the importance of TNF-α and IL-1β in RA pathogenesis, and helps explain the efficiency of agents blocking the activity of these cytokines in RA. Factors able to block the induction of cytokine production (such as apolipoprotein A-I [apo A-I] and interferon [IFN]-β) might interfere more distally in the inflammatory process. Furthermore, stimulated T lymphocytes produce osteoclast differentiation factor (ODF), which triggers erosive functions of osteoclasts. Therefore, factors capable of affecting the level of T lymphocyte activation, such as IFN-β, IL-15 antagonist, or SDF-1α antagonist, might be of interest in RA therapy.     

T cells as key players for bone destruction in gouty arthritis?

The deposition of monosodium urate (MSU) crystals in synovial fluid and tissue leads to gouty arthritis frequently associated with synovial inflammation and bone erosions. The cellular mechanism that links MSU crystals to an increased number of osteoclasts has not yet been fully understood. In a recent issue of Arthritis Research & Therapy Lee and colleagues proposed that bone destruction in chronic gouty arthritis is at least in part dependent on expression by T cells of receptor activator of NF-κB ligand (RANKL). The authors showed that pro-resorptive cytokines such as IL-1β, IL-6, and TNFα are expressed within tophi and stromal infiltrates. In vitro stimulation with MSU crystals revealed monocytes as a source for these cytokines, whereas T cells produce RANKL, the major trigger of osteoclastogenesis.     

Osteopontin inhibition of miR-129-3p enhances IL-17 expression and monocyte migration in rheumatoid arthritis

BackgroundOsteopontin (OPN) is an important proinflammatory cytokine in rheumatoid arthritis (RA). Levels of OPN have been shown to be significantly correlated with interleukin-17 (IL-17) production and expression of Th17 cells in the synovial fluid of RA patients. Here, we investigated the role of OPN in monocyte migration, IL-17 production and osteoblasts.MethodsOPN and IL-17 expression profiles in osteoarthritis (OA) and RA synovial fluid were determined by enzyme-linked immunosorbent assay (ELISA). The expression of the microRNA, miR-129-3p, in osteoblasts was analyzed by real-time quantitative polymerase chain reaction (qPCR). Immunoreactive proteins were spotted by Western blotting. We used the collagen-induced arthritis (CIA) mouse model to investigate the role of OPN in monocyte migration during RA.ResultsOPN and IL-17 expression were higher in RA synovial fluid as compared to OA samples. We also found that OPN promotes IL-17 expression in osteoblasts and thereby enhances monocyte migration via the Syk/PI3K/Akt signaling pathway. miR-129-3p expression was found to be negatively regulated by OPN via the Syk/PI3K/Akt signal cascade. In contrast, lentiviral vectors expressing short hairpin RNA inhibited OPN expression and ameliorated articular swelling, cartilage erosion and monocyte infiltration in the ankle joints of CIA mice.ConclusionTo our knowledge, our study is the first to describe how OPN promotes monocyte migration by upregulating IL-17 expression in osteoblasts in RA disease.SignificanceThese findings indicate that OPN could serve as a potential therapeutic target for the treatment of RA.     

Blockade of Toll-like receptor 2 prevents spontaneous cytokine release from rheumatoid arthritis ex vivo synovial explant cultures

Introduction

The aim of this study was to examine the effect of blocking Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA) synovial cells.

Methods

RA synovial tissue biopsies, obtained under direct visualization at arthroscopy, were established as synovial explant cultures ex vivo or snap frozen for immunohistology. Mononuclear cell cultures were isolated from peripheral blood and synovial fluid of RA patients. Cultures were incubated with the TLR1/2 ligand, Pam3CSK4 (200 ng, 1 and 10 μg/ml), an anti-TLR2 antibody (OPN301, 1 μg/ml) or an immunoglobulin G (IgG) (1 μg/ml) matched control. The comparative effect of OPN301 and adalimumab (anti-tumour necrosis factor alpha) on spontaneous release of proinflammatory cytokines from RA synovial explants was determined using quantitative cytokine MSD multiplex assays or ELISA. OPN301 penetration into RA synovial tissue explants cultures was assessed by immunohistology.

Results

Pam3CSK4 significantly upregulated interleukin (IL)-6 and IL-8 in RA peripheral blood mononuclear cells (PBMCs), RA synovial fluid mononuclear cells (SFMCs) and RA synovial explant cultures (P < 0.05). OPN301 significantly decreased Pam3CSK4-induced cytokine production of tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, interferon (IFN)-γ and IL-8 compared to IgG control in RA PBMCs and SFMCs cultures (all P < 0.05). OPN301 penetration of RA synovial tissue cultures was detected in the lining layer and perivascular regions. OPN301 significantly decreased spontaneous cytokine production of TNF-α, IL-1β, IFN-γ and IL-8 from RA synovial tissue explant cultures (all P < 0.05). Importantly, the inhibitory effect of OPN on spontaneous cytokine secretion was comparable to inhibition by anti-TNFα monoclonal antibody adalimumab.

Conclusions

These findings further support targeting TLR2 as a potential therapeutic agent for the treatment of RA.