Alternative complement pathway activation is essential for inflammation and joint destruction in the passive transfer model of collagen-induced arthritis

Activation of each complement initiation pathway (classical, alternative, and lectin) can lead to the generation of bioactive fragments with resulting inflammation in target organs. The objective of the current study was to determine the role of specific complement activation pathways in the pathogenesis of experimental anti-type II collagen mAb-passive transfer arthritis. C57BL/6 mice were used that were genetically deficient in either the alternative pathway protein factor B (Bf(-/-)) or in the classical pathway component C4 (C4(-/-)). Clinical disease activity was markedly decreased in Bf(-/-) compared with wild-type (WT) mice (0.5 +/- 0.22 (n = 6) in Bf(-/-) vs 8.83 +/- 0.41 (n = 6) in WT mice (p < 0.0001)). Disease activity scores were not different between C4(-/-) and WT mice. Analyses of joints showed that C3 deposition, inflammation, pannus, cartilage, and bone damage scores were all significantly less in Bf(-/-) as compared with WT mice. There were significant decreases in mRNA levels of C3, C4, CR2, CR3, C3aR, and C5aR in the knees of Bf(-/-) as compared with C4(-/-) and WT mice with arthritis; mRNA levels for complement regulatory proteins did not differ between the three strains. These results indicate that the alternative pathway is absolutely required for the induction of arthritis following injection of anti-collagen Abs. The mechanisms by which these target organ-specific mAbs bypass the requirements for engagement of the classical pathway remain to be defined but do not appear to involve a lack of alternative pathway regulatory proteins.     

IL-1-independent role of IL-17 in synovial inflammation and joint destruction during collagen-induced arthritis.

T cell IL-17 displays proinflammatory properties and is expressed in the synovium of patients with rheumatoid arthritis. Its contribution to the arthritic process has not been identified. Here, we show that blocking of endogenous IL-17 in the autoimmune collagen-induced arthritis model results in suppression of arthritis. Also, joint damage was significantly reduced. In contrast, overexpression of IL-17 enhanced collagen arthritis. Moreover, adenoviral IL-17 injected in the knee joint of type II collagen-immunized mice accelerated the onset and aggravated the synovial inflammation at the site. Radiographic and histologic analysis showed markedly increased joint destruction. Elevated levels of IL-1beta protein were found in synovial tissue. Intriguingly, blocking of IL-1alphabeta with neutralizing Abs had no effect on the IL-17-induced inflammation and joint damage in the knee joint, implying an IL-1 independent pathway. This direct potency of IL-17 was underscored in the unabated IL-17-induced exaggeration of bacterial cell wall-induced arthritis in IL-1beta(-/-) mice. In conclusion, this data shows that IL-17 contributes to joint destruction and identifies an IL-1-independent role of IL-17. These findings suggest IL-17 to be a novel target for the treatment of destructive arthritis and may have implications for tissue destruction in other autoimmune diseases.     

Treatment with recombinant interferon-beta reduces inflammation and slows cartilage destruction in the collagen-induced arthritis model of rheumatoid arthritis

We investigated the therapeutic potential and mechanism of action of IFN-beta protein for the treatment of rheumatoid arthritis (RA). Collagen-induced arthritis was induced in DBA/1 mice. At the first clinical sign of disease, mice were given daily injections of recombinant mouse IFN-beta or saline for 7 days. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were assessed histologically 8 days after the onset of arthritis. Proteoglycan depletion was determined by safranin O staining. Expression of cytokines, receptor activator of NF-kappaB ligand, and c-Fos was evaluated immunohistochemically. The IL-1-induced expression of IL-6, IL-8, and granulocyte/macrophage-colony-stimulating factor (GM-CSF) was studied by ELISA in supernatant of RA and osteoarthritis fibroblast-like synoviocytes incubated with IFN-beta. We also examined the effect of IFN-beta on NF-kappaB activity. IFN-beta, at 0.25 microg/injection and higher, significantly reduced disease severity in two experiments, each using 8-10 mice per treatment group. IFN-beta-treated animals displayed significantly less cartilage and bone destruction than controls, paralleled by a decreased number of positive cells of two gene products required for osteoclastogenesis, receptor activator of NF-kappaB ligand and c-Fos. Tumor necrosis factor alpha and IL-6 expression were significantly reduced, while IL-10 production was increased after IFN-beta treatment. IFN-beta reduced expression of IL-6, IL-8, and GM-CSF in RA and osteoarthritis fibroblast-like synoviocytes, correlating with reduced NF-kappaB activity. The data support the view that IFN-beta is a potential therapy for RA that might help to diminish both joint inflammation and destruction by cytokine modulation.     

IL-18 is redundant in T-cell responses and in joint inflammation in antigen-induced arthritis

IL-18 is an important cofactor in Th1 immune responses and it has additional roles in inflammation. Recent reports suggest the contribution of IL-18 to immune responses may vary between mouse strains and immune contexts. We investigated the contribution of IL-18 to T-cell activation and joint inflammation in Ag-induced arthritis (AIA) in C57Bl/6 mice. AIA and cutaneous delayed-type hypersensitivity (DTH) reactions were induced in wild-type (WT) and IL-18-/- C57Bl/6 mice, and Ag-specific T-cell proliferation and IFN-gamma and IL-4 production were measured. The humoral immune response was measured as serum antibody to the disease-initiating Ag, methylated BSA (mBSA). Splenocyte production of IL-6 was measured by ELISA. To confirm the dependence of this model on Th1-cell-mediated immunity, IL-12p40-/- mice were similarly studied. WT mice developed synovitis, joint effusion, cartilage destruction and bone damage associated with induction of DTH, and in vitro Ag-specific T-cell proliferation and IFN-gamma production. Unexpectedly, IL-18-/- mice developed AIA and indices of T-cell activation were similar to those of WT mice. In contrast, IL-12p40-/- mice did not develop AIA, DTH or T-cell activation. WT and IL-18-/- mice, but not IL-12p40-/- mice, developed significantly increased serum antibody to mBSA compared with naive controls. WT and IL-18-/- splenocytes produced high levels of IL-6, whereas IL-12p40-/- cells had significantly lower IL-6 production compared with both. In conclusion, IL-18 is redundant both as a Th1 response cofactor and inflammatory cytokine, whereas IL-12p40-/- is a key cytokine, in AIA in C57Bl/6 mice.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

Interleukin-10 regulates the inflammasome-driven augmentation of inflammatory arthritis and joint destruction

Introduction

Activation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology.

Methods

Antigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays.

Results

In AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1β. When compared to controls, IL-10KO synovium showed increased expression of Il1b, Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1. In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis.

Conclusions

These data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0419-y) contains supplementary material, which is available to authorized users.     

C17 prevents inflammatory arthritis and associated joint destruction in mice

C17 was first described about ten years ago as a gene expressed in CD34+ cells. A more recent study has suggested a role for C17 in chondrogenesis and development of cartilage. However, based on sequence analysis, we believe that C17 has homology to IL-2 and hence we present the hypothesis that C17 is a cytokine possessing immune-regulatory properties. We provide evidence that C17 is a secreted protein preferentially expressed in chondrocytes, hence in cartilage-rich tissues. Systemic expression of C17 in vivo reduces disease in a collagen antibody-induced arthritis model in mice (CAIA). Joint protection is evident by delayed disease onset, minimal edema, bone protection and absence of diverse histological features of disease. Expression of genes typically associated with acute joint inflammation and erosion of cartilage or bone is blunted in the presence of C17. Consistent with the observed reduction in bone erosion, we demonstrate reduced levels of RANKL in the paws and sera of mice over-expressing C17. Administration of C17 at the peak of disease, however, had no effect on disease progression, indicating that C17's immune-regulatory activity must be most prominent prior to or at the onset of severe joint inflammation. Based on this data we propose C17 as a cytokine that s contributes to immune homeostasis systemically or in a tissue-specific manner in the joint.     

IL-1 alpha beta blockade prevents cartilage and bone destruction in murine type II collagen-induced arthritis, whereas TNF-alpha blockade only ameliorates joint inflammation.

Anti-TNF-alpha treatment of rheumatoid arthritis patients markedly suppresses inflammatory disease activity, but so far no tissue-protective effects have been reported. In contrast, blockade of IL-1 in rheumatoid arthritis patients, by an IL-1 receptor antagonist, was only moderately effective in suppressing inflammatory symptoms but appeared to reduce the rate of progression of joint destruction. We therefore used an established collagen II murine arthritis model (collagen-induced arthritis(CIA)) to study effects on joint structures of neutralization of either TNF-alpha or IL-1. Both soluble TNF binding protein and anti-IL-1 treatment ameliorated disease activity when applied shortly after onset of CIA. Serum analysis revealed that early anti-TNF-alpha treatment of CIA did not decrease the process in the cartilage, as indicated by the elevated COMP levels. In contrast, anti-IL-1 treatment of established CIA normalized COMP levels, apparently alleviating the process in the tissue. Histology of knee and ankle joints corroborated the finding and showed that cartilage and joint destruction was significantly decreased after anti-IL-1 treatment but was hardly affected by anti-TNF-alpha treatment. Radiographic analysis of knee and ankle joints revealed that bone erosions were prevented by anti-IL-1 treatment, whereas the anti-TNF-alpha-treated animals exhibited changes comparable to the controls. In line with these findings, metalloproteinase activity, visualized by VDIPEN production, was almost absent throughout the cartilage layers in anti-IL-1-treated animals, whereas massive VDIPEN appearance was found in control and sTNFbp-treated mice. These results indicate that blocking of IL-1 is a cartilage- and bone-protective therapy in destructive arthritis, whereas the TNF-alpha antagonist has little effect on tissue destruction.     

Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction.

Rheumatoid arthritis is a chronic inflammatory joint disease, leading to cartilage and bone destruction. In this study, we investigated the effects of local IL-4 application, introduced by a recombinant human type 5 adenovirus vector, in the knee joint of mice with collagen-induced arthritis. One intraarticular injection with an IL-4-expressing virus caused overexpression of IL-4 in the mouse knee joint. Enhanced onset and aggravation of the synovial inflammation were found in the IL-4 group. However, despite ongoing inflammation, histologic analysis showed impressive prevention of chondrocyte death and cartilage erosion. In line with this, chondrocyte proteoglycan synthesis was enhanced in the articular cartilage. This was quantified with ex vivo 35S-sulfate incorporation in patellar cartilage and confirmed by autoradiography on whole knee joint sections. Reduction of cartilage erosion was further substantiated by lack of expression of the stromelysin-dependent cartilage proteoglycan breakdown neoepitope VDIPEN in the Ad5E1 mIL-4-treated knee joint. Reduced metalloproteinase activity was also supported by markedly diminished mRNA expression of stromelysin-3 in the synovial tissue. Histologic analysis revealed marked reduction of polymorphonuclear cells in the synovial joint space in the IL-4-treated joints. This was confirmed by immunolocalization studies on knee joint sections using NIMP-R14 staining and diminished mRNA expression of macrophage-inflammatory protein-2 in the synovium tissue. mRNA levels of TNF-alpha and IL-1beta were suppressed as well, and IL-1beta and nitric oxide production by arthritic synovial tissue were strongly reduced. Our data show an impressive cartilage-protective effect of local IL-4 and underline the feasibility of local gene therapy with this cytokine in arthritis.     

T-614, a novel immunomodulator, attenuates joint inflammation and articular damage in collagen-induced arthritis

Introduction

T-614 is a novel oral antirheumatic agent for the treatment of rheumatoid arthritis. Whether it has immunomodulatory or disease-modifying properties and its mechanism of action are largely undetermined.

Methods

Rats with collagen-induced arthritis (CIA) were treated with T-614 (5 and 20 mg/kg) daily. Animals receiving methotrexate (1 mg/kg every 3 days) and the nonsteroidal anti-inflammatory agent nimesulide (10 mg/kg per day) were used as controls. A combination therapy group was treated with both T-614(10 mg/kg per day) and methotrexate (1 mg/kg every 3 days). Hind paw swelling was evaluated and radiographic scores calculated. Serum cytokine levels were assessed by Bio-plex analysis. Quantitative PCR was used to evaluate expression of mRNA for interferon-γ, IL-4 and IL-17. Serum IL-17 and anti-type II collagen antibodies (total IgG, IgG₁, IgG_2a, IgG_2b and IgM) were measured using ELISA.

Results

Oral T-614 inhibited paw swelling and offered significant protection against arthritis-induced cartilage and bone erosion, comparable to the effects of methotrexate. CIA rats treated with T-614 exhibited decreases in both mRNA expression of IL-17 in peripheral blood mononuclear cells and lymph node cells, and circulating IL-17 in a dose-dependent manner. T-614 also reduced serum levels of tumor necrosis factor-α, IL-1β and IL-6. A synergistic effect was observed for the combination of methotrexate and T-614. In addition, T-614 (20 mg/kg per day) depressed production of anti-type II collagen antibodies and differentially affected levels of IgG_2a subclasses in vivo, whereas IgM level was decreased without any change in the IgG₁ level. Together, the findings presented here indicate that the novel agent T-614 has disease-modifying effects against experimental arthritis, as opposed to nimesulide.

Conclusions

Our data suggested that T-614 is an effective disease-modifying agent that can prevent bone/cartilage destruction and inflammation in in CIA rats. Combination with methotrexate markedly enhances the therapeutic effect of T-614.     

Interleukin-7 deficiency in rheumatoid arthritis

Interleukin-7 (IL-7) is a stromal factor that is crucial for the development of T lymphocytes in humans and mice, and also B lymphocytes in mice. IL-7 can act as a T cell growth factor as well as a critical anti-apoptotic survival factor. The essential non-redundant role of this cytokine for T cell development in vivo is indicated by the phenotype of murine knockout models as well as by humans with a T^-B⁺NK⁺ form of severe combined immunodeficiency (SCID) resulting from mutations in IL-7 receptor α chain. IL-7 deficiency has now been found in patients with rheumatoid arthritis, a finding that relates not only to the T-lymphocyte status in this disease but also to the ability of patients with rheumatoid arthritis to recover from therapy-induced lymphopenia.     

Interleukin-7 deficiency in rheumatoid arthritis

Interleukin-7 (IL-7) is a stromal factor that is crucial for the development of T lymphocytes in humans and mice, and also B lymphocytes in mice. IL-7 can act as a T cell growth factor as well as a critical anti-apoptotic survival factor. The essential non-redundant role of this cytokine for T cell development in vivo is indicated by the phenotype of murine knockout models as well as by humans with a T-B+NK+ form of severe combined immunodeficiency (SCID) resulting from mutations in IL-7 receptor alpha chain. IL-7 deficiency has now been found in patients with rheumatoid arthritis, a finding that relates not only to the T-lymphocyte status in this disease but also to the ability of patients with rheumatoid arthritis to recover from therapy-induced lymphopenia.     

Dynamic activation of bone morphogenetic protein signaling in collagen-induced arthritis supports their role in joint homeostasis and disease

Introduction  

Rheumatoid arthritis is a chronic systemic autoimmune disease affecting peripheral joints and leading to loss of joint function. The severity and outcome of disease are dependent on the balance between inflammatory/destructive and homeostatic or repair pathways. Increasing evidence suggests a role for bone morphogenetic protein (BMP) signaling in joint homeostasis and disease.     

Intra-articular injections of high-molecular-weight hyaluronic acid have biphasic effects on joint inflammation and destruction in rat antigen-induced arthritis

To assess the potential use of hyaluronic acid (HA) as adjuvant therapy in rheumatoid arthritis, the anti-inflammatory and chondroprotective effects of HA were analysed in experimental rat antigen-induced arthritis (AIA). Lewis rats with AIA were subjected to short-term (days 1 and 8, n = 10) or long-term (days 1, 8, 15 and 22, n = 10) intra-articular treatment with microbially manufactured, high-molecular-weight HA (molecular weight, 1.7 x 10(6) Da; 0.5 mg/dose). In both tests, 10 buffer-treated AIA rats served as arthritic controls and six healthy animals served as normal controls. Arthritis was monitored by weekly assessment of joint swelling and histological evaluation in the short-term test (day 8) and in the long-term test (day 29). Safranin O staining was employed to detect proteoglycan loss from the epiphyseal growth plate and the articular cartilage of the arthritic knee joint. Serum levels of IL-6, tumour necrosis factor alpha and glycosaminoglycans were measured by ELISA/kit systems (days 8 and 29). HA treatment did not significantly influence AIA in the short-term test (days 1 and 8) but did suppress early chronic AIA (day 15, P < 0.05); however, HA treatment tended to aggravate chronic AIA in the long-term test (day 29). HA completely prevented proteoglycan loss from the epiphyseal growth plate and articular cartilage on day 8, but induced proteoglycan loss from the epiphyseal growth plate on day 29. Similarly, HA inhibited the histological signs of acute inflammation and cartilage damage in the short-term test, but augmented acute and chronic inflammation as well as cartilage damage in the long-term test. Serum levels of IL-6, tumour necrosis factor alpha, and glycosaminoglycans were not influenced by HA. Local therapeutic effects of HA in AIA are clearly biphasic, with inhibition of inflammation and cartilage damage in the early chronic phase but with promotion of joint swelling, inflammation and cartilage damage in the late chronic phase.     

Studies of T-cell activation in chronic inflammation

The strong association between specific alleles encoded within the MHC class II region and the development of rheumatoid arthritis (RA) has provided the best evidence to date that CD4+ T cells play a role in the pathogenesis of this chronic inflammatory disease. However, the unusual phenotype of synovial T cells, including their profound proliferative hyporesponsiveness to TCR ligation, has challenged the notion that T-cell effector responses are driven by cognate cartilage antigens in inflamed synovial joints. The hierarchy of T-cell dysfunction from peripheral blood to inflamed joint suggests that these defects are acquired through prolonged exposure to proinflammatory cytokines such as tumour necrosis factor (TNF)-alpha. Indeed, there are now compelling data to suggest that chronic cytokine activation may contribute substantially to the phenotype and effector function of synovial T cells. Studies reveal that chronic exposure of T cells to TNF uncouples TCR signal transduction pathways by impairing the assembly and stability of the TCR/CD3 complex at the cell surface. Despite this membrane-proximal effect, TNF selectively uncouples downstream signalling pathways, as is shown by the dramatic suppression of calcium signalling responses, while Ras/ERK activation is spared. On the basis of these data, it is proposed that T-cell survival and effector responses are driven by antigen-independent, cytokine-dependent mechanisms, and that therapeutic strategies that seek to restore T-cell homeostasis rather than further depress T-cell function should be explored in the future.     

Inhibition of HDAC activity by ITF2357 ameliorates joint inflammation and prevents cartilage and bone destruction in experimental arthritis

Inhibition of histone deacetylases (HDAC) has been shown to modulate gene expression and cytokine production after stimulation with several stimuli. In the present study, the antiinflammatory effect of a potent HDACi, ITF2357, was explored in different experimental models of arthritis. In addition, the bone protective effect of ITF2357 was investigated in vitro. Treatment of acute arthritis (Streptococcus pyogenes cell wall [SCW] arthritis) with ITF2357 showed that joint swelling and cell influx into the joint cavity were reduced. Furthermore, the chondrocyte metabolic function was improved by treatment of ITF2357. The production of proinflammatory cytokines by synovial tissue was reduced after ITF2357 treatment. To examine the effect of HDAC inhibition on joint destruction, ITF2357 was applied to both rat adjuvant arthritis and mouse collagen type II arthritis. ITF2357 treatment both ameliorates the severity scores in arthritis models and prevents bone destruction. In an in vitro bone destruction assay, ITF2357 was highly effective at a dose of 100 nmol/L. In conclusion, inhibition of HDAC prevents joint inflammation and cartilage and bone destruction in experimental arthritis.