E Lubberts

Blocking of Interleukin-17 during Reactivation of Experimental Arthritis Prevents Joint Inflammation and Bone Erosion by Decreasing RANKL and Interleukin-1

Rheumatoid arthritis is characterized by an intermittent course of disease with alternate periods of remission and relapse. T cells, and in particular the T-cell cytokine interleukin-17 (IL-17), are expected to be involved in arthritic flares. Here, we report that neutralizing endogenous IL-17 during reactivation of antigen-induced arthritis prevents joint inflammation and bone erosion. Synovial IL-17 mRNA expression was clearly up-regulated during primary arthritis and was further enhanced after antigen rechallenge. Neutralization of IL-17 significantly prevented joint swelling at day 1 of flare and significantly suppressed joint inflammation and cartilage proteoglycan depletion at day 4, as assessed by histology. Blocking IL-17 also clearly reduced bone erosions. Cathepsin K, a marker of osteoclast-like activity, and synovial RANKL mRNA expression were both suppressed. The degree of bone erosions strongly correlated with the severity of joint inflammation, suggesting that anti-IL-17 treatment reduced bone erosion by suppressing joint inflammation. Interestingly, blocking IL-17 suppressed synovial expression of both IL-1beta and tumor necrosis factor-alpha, whereas blocking IL-1 did not affect tumor necrosis factor-alpha levels. These data indicate that IL-17 is an important upstream mediator in joint pathology during flare-up of experimental arthritis.

Overexpression of IL-17 in the knee joint of collagen type II immunized mice promotes collagen arthritis and aggravates joint destruction

IL17 is a new cytokine secreted by CD4+ activated memory T cells which is produced in the RA synovium [1-3] . IL-17 stimulated the production o f ILl f l and T N F a by human macrophages [2] and increased the effect o f IL-1 and TNFot on synoviocytes [3], indicating that it may play an important role in the pro-inf lammatory response. The pattern o f cellular responses induced by IL-17 is similar to that o f lL-1 , suggesting that IL-17 may also contribute to joint destruction. The potency o f IL-17 as a stimulator o f osteoclastogenesis has been shown in vitro [4] and IL-17 expression was found in the synovium of collagen induced arthritis mice [5]. In addition, IL-17 inhibits the chondrocyte metabol ism in murine intact articular cartilage through an NO mediated pathway [6], indicating direct involvement o f this T cell derived cytokine in cartilage damage. Although IL-17 is expressed in the RA synovium its in vivo role is still not clear. In this study, we investigated the effect o f local IL17 application, introduced by a recombinant adenoviral vector, in the knee joint o f collagen type II immunized mice. The data we summarized here shows that local IL-17 overexpression promotes collagen arthritis and indicates that IL-17-induced inflammation and joint damage is independent o f IL1.

Interleukin-17 acts independently of TNF-alpha under arthritic conditions.

The proinflammatory T cell cytokine IL-17 is a potent inducer of other cytokines such as IL-1 and TNF-α. The contribution of TNF in IL-17-induced joint inflammation is unclear. In this work we demonstrate using TNF-α-deficient mice that TNF-α is required in IL-17-induced joint pathology under naive conditions in vivo. However, overexpression of IL-17 aggravated K/B×N serum transfer arthritis to a similar degree in TNF-α-deficient mice and their wild-type counterparts, indicating that the TNF dependency of IL-17-induced pathology is lost under arthritic conditions. Also, during the course of the streptococcal cell wall-induced arthritis model, IL-17 was able to enhance inflammation and cartilage damage in the absence of TNF. Additional blocking of IL-1 during IL-17-enhanced streptococcal cell wall-induced arthritis did not reduce joint pathology in TNF-deficient mice, indicating that IL-1 is not responsible for this loss of TNF dependency. These data provide further understanding of the cytokine interplay during inflammation and demonstrate that, despite a strong TNF dependency under naive conditions, IL-17 acts independently of TNF under arthritic conditions.

An IFN-gamma-independent proinflammatory role of IL-18 in murine Streptococcal cell wall arthritis

IL-18 is a member of the IL-1 family of proteins that exerts proinflammatory effects. It was formally known as IFN-γ-inducing factor and is a pivotal cytokine for the development of Th1 responses. Apart from Th1 immune-stimulatory activity, IL-18 induces the production of proinflammatory cytokines such as TNF-α and IL-1 in vitro. The goal was to investigate the role of endogenous IL-18 in murine streptococcal cell wall (SCW)-induced arthritis. Furthermore, we investigated whether IL-18 neutralization had an impact on local TNF and IL-1 production. C57BL/6, BALB/c, and IFN-γ-deficient mice were injected with 2 mg of rabbit anti-murine IL-18 Abs shortly before induction of arthritis by intra-articular injection of 25 μg of SCW fragments into the right knee joint. Suppression of joint swelling was noted on days 1 and 2 of SCW arthritis after blockade of endogenous IL-18. Analysis of local cytokine concentrations showed that IL-18, TNF-α, and IL-1β levels were decreased. Severe inhibition of chondrocyte proteoglycan synthesis was seen in the vehicle-treated control animals, whereas a reversal of the inhibition of chondrocyte proteoglycan synthesis was found in the anti-IL-18-exposed animals. Blockade of endogenous IL-18 in IFN-γ-deficient mice showed results similar to those found in wild-type animals, identifying a role for IL-18 that is IFN-γ independent. The present study indicates that IL-18 is a proinflammatory cytokine during the onset of murine SCW arthritis, and this inflammatory role of IL-18 is IFN-γ independent.

Intra-articular IL-10 gene transfer regulates the expression of collagen-induced arthritis (CIA) in the knee and ipsilateral paw

We studied the effects of local IL‐10 application, introduced by a recombinant human type 5 adenovirus vector, in the mouse knee joint during the early phase of CIA. One intra‐articular injection with the IL‐10‐expressing virus (Ad5E1mIL‐10) caused substantial over‐expression of IL‐10 in the mouse knee joint, using virus dosages which did not induce distracting inflammation. High expression of IL‐10 was noted for a few days, being maximal at day 1. One intra‐articular injection of Ad5E1mIL‐10 in the knee joints of collagen type II (CII)‐immunized mice, before onset of CIA was noted, reduced the incidence of collagen arthritis in that knee. Of high interest, the protective effect of local IL‐10 expression by Ad5E1mIL‐10 was not restricted to the knee joint alone. The arthritis incidence in the ipsilateral paw was highly suppressed. In contrast, local IL‐10 over‐expression was not effective when treatment was started after onset of CIA. Further analysis in the acute streptococcal cell wall‐induced arthritis model revealed that local IL‐10 over‐expression markedly suppressed the production of tumour necrosis factor‐alpha (TNF‐α) and IL‐1α, but had no significant effect on IL‐1β and IL‐12 production in the inflamed synovium. These data indicate that local over‐expression of IL‐10 in the knee joint of mice regulates the expression of collagen arthritis, probably through down‐regulation of TNF‐α.

Requirement of IL-17 Receptor Signaling in Radiation-Resistant Cells in the Joint for Full Progression of Destructive Synovitis

IL-17 is a proinflammatory cytokine suspected to be involved in inflammatory and autoimmune diseases such as rheumatoid arthritis. In the present study, we report that IL-17R signaling is required in radiation-resistant cells in the joint for full progression of chronic synovitis and bone erosion. Repeated injections of Gram-positive bacterial cell wall fragments (streptococcal cell wall) directly into the knee joint of naive IL-17R-deficient (IL-17R−/−) mice had no effect on the acute phase of arthritis but prevented progression to chronic destructive synovitis as was noted in wild-type (wt) mice. Microarray analysis revealed significant down-regulation of leukocyte-specific chemokines, selectins, cytokines, and collagenase-3 in the synovium of IL-17R−/− mice. Bone marrow (BM) chimeric mice revealed the need for IL-17R expression on radiation-resistant joint cells for destructive inflammation. Chimeric mice of host wt and donor IL-17R−/− BM cells developed destructive synovitis in this chronic reactivated streptococcal cell wall arthritis model similar to wt→wt chimeras. In contrast, chimeric mice of host IL-17R−/− and donor wt BM cells were protected from chronic destructive arthritis similar as IL-17R−/−→IL-17R−/− chimeras. These data strongly indicate that IL-17R signaling in radiation-resistant cells in the joint is required for turning an acute macrophage-mediated inflammation into a chronic destructive synovitis.

Role of Interleukin 17 in Arthritis Chronicity through Survival of Synoviocytes via Regulation of Synoviolin Expression

Background The use of TNF inhibitors has been a major progress in the treatment of chronic inflammation. However, not all patients respond. In addition, response will be often lost when treatment is stopped. These clinical aspects indicate that other cytokines might be involved and we focus here on the role of IL-17. In addition, the chronic nature of joint inflammation may contribute to reduced response and enhanced chronicity. Therefore we studied the capacity of IL-17 to regulate synoviolin, an E3 ubiquitin ligase implicated in synovial hyperplasia in human rheumatoid arthritis (RA) FLS and in chronic reactivated streptococcal cell wall (SCW)-induced arthritis. Methodology/Principal Findings Chronic reactivated SCW-induced arthritis was examined in IL-17R deficient and wild-type mice. Synoviolin expression was analysed by real-time RT-PCR, Western Blot or immunostaining in RA FLS and tissue, and p53 assessed by Western Blot. Apoptosis was detected by annexin V/propidium iodide staining, SS DNA apoptosis ELISA kit or TUNEL staining and proliferation by PCNA staining. IL-17 receptor A (IL-17RA), IL-17 receptor C (IL-17-RC) or synoviolin inhibition were achieved by small interfering RNA (siRNA) or neutralizing antibodies. IL-17 induced sustained synoviolin expression in RA FLS. Sodium nitroprusside (SNP)-induced RA FLS apoptosis was associated with reduced synoviolin expression and was rescued by IL-17 treatment with a corresponding increase in synoviolin expression. IL-17RC or IL-17RA RNA interference increased SNP-induced apoptosis, and decreased IL-17-induced synoviolin. IL-17 rescued RA FLS from apoptosis induced by synoviolin knockdown. IL-17 and TNF had additive effects on synoviolin expression and protection against apoptosis induced by synoviolin knowndown. In IL-17R deficient mice, a decrease in arthritis severity was characterized by increased synovial apoptosis, reduced proliferation and a marked reduction in synoviolin expression. A distinct absence of synoviolin expressing germinal centres in IL-17R deficient mice contrasted with synoviolin positive B cells and Th17 cells in synovial germinal centre-like structures. Conclusion/Significance IL-17 induction of synoviolin may contribute at least in part to RA chronicity by prolonging the survival of RA FLS and immune cells in germinal centre reactions. These results extend the role of IL-17 to synovial hyperplasia.

Requirement of local synovial IL-17 receptor signaling in the progression of chronic synovitis and bone erosion in arthritis

IL-17 is a proinflammatory cytokine that is suspected to be involved in development of rheumatoid arthritis (RA). However, the role of IL-17–IL-17 receptor signaling in the effector phase of arthritis, including requirement of this signaling pathway in synoviocytes, is still not fully elucidated. Here we demonstrate, using IL-17 receptor deficient (IL-17R-/-) mice, a requirement for IL-17R signaling in immune reactivity and progression of synovitis and bone erosion in a T cell mediated mBSA arthritis model and chronic streptococcal cell wall arthritis. Of great interest, chimeric mice of host wild-type (wt) and donor IL-17R-/- bone marrow (BM) cells developed chronic synovitis similar to that in wt/wt chimeras. In contrast, chimeric mice of host IL-17R-/- and donor wt BM cells were protected from progressive synovitis similar to IL-17R-/-/ IL-17R-/- chimeras, suggesting a crucial role of resident synovial cells. Weakened mobilization of neutrophils into the joint and downregulation of synovial mRNA expression for leukocyte specific chemokines and selectins, and MMP-1 was found in IL-17R-/- mice. These data suggest that, in addition to the role played by IL-17–IL-17R signaling in development of immunity, synovial IL-17–IL-17R signaling plays a critical role in the effector phase of arthritis. Activation of IL-17R on fibroblast-like synoviocytes appears pivotal.

Cytokine regulation of bone destruction

Bone is a dynamic tissue, composed of cells, collagenous matrix and inorganic elements. Per year, it has been estimated that 10% of the total bone mass in humans is being remodeled [1]. The growth, development and maintenance of bone is a highly regulated process [2]. Morphogenesis and remodeling of bone involve coordinate regulation of bone-forming cells (osteoblasts) and bone-resorbing cells (osteoclasts) [3,4]. Osteoblasts and osteoclasts arise from distinct cell lineages by physiologically controlled processes that involve cytokine, growth factors and hormones [4,5]. Osteoblasts arise from mesenchymal stem cells, whereas osteoclasts differentiate from hematopoietic monocyte/macrophage precursors. Osteoclasts are rarely seen under normal conditions. Imbalance between osteoclast and osteoblast activities can arise from a wide variety of hormonal changes or perturbations of inflammatory and growth factors, resulting in skeletal abnormalities characterized by decreased (osteoporosis) or increased (osteopetrosis) bone mass. Increased osteoclast activity is also seen in many pathological disorders, including Paget’s disease, lytic bone metastases, postmenopausal osteoporosis, or rheumatoid arthritis (RA), leading to net loss of bone. The discovery of the RANKL/RANK/OPG pathway was a major breakthrough in the understanding of the molecular mechanism of bone erosion. RANKL, RANK and OPG are three key molecules that regulate osteoclast recruitment and function (Fig. 1)

Genome-wide gene expression in experimental arthritis: defining new targets of chronic/destructive rheumatoid arthritis (RA)

Genome-wide expression analysis using microarrays enables us to visualize activation of complex signaling pathways in the total genome of an organism upon biological, pharmacological and toxicological stimulus or during pathological conditions. Rheumatoid Arthritis (RA) is a complex multigenic disease with yet unknown ethiology, and consequently, suitable target for genomics approach. We have used a high density DNA filter array, containing 25,142 DNA sequences, that represents a condensed mouse genome to analyze gene expression in animal models of RA. Welldefined animal models were chosen in order to investigate clear relationships between disease activity and gene expression. We have identified a number of genes whose targeted deletion or insertion results in modification of disease progression. Gene deletion of, e.g. IL-6 prevents development of sub-chronic inflammation without modifying the acute inflammation in zymosan-induced-arthritis (ZIA). Similarly, gene deletion of FcγR prevents some aspects of chronic inflammation in antigen-induced arthritis (AIA). On the other hand, adenoviral mediated gene transfer of IL-4 completely inhibits progression into the destructive phase in collagen-induced arthritis (CIA). Therefore we have analyzed gene-expression profiles in the following conditions: A) IL-6-/- vs. WT/ZIA, B) FcγR -/- vs. WT/AIA, C) AdIL-4 vs. AdC/CIA. Possible candidate genes for (sub)chronic inflammation or destructive arthritis were defined by two-parameter and cluster analysis of the expression profile. Seventy-seven common candidates, possibly involved in sub-chronic or destructive arthritis, were defined. Many of these are genes not yet inferred to be involved in inflammation. Selected ESTs were further analyzed and one candidate was cloned as a full length gene. Investigation into gene function is in progress. This approach, combining DNA array technology with cloning and functional characterization of candidate genes, proves highly effective in defining novel targets in inflammatory/autoimmune diseases such as RA.