Tendon degeneration is not mediated by regulation of Toll-like receptors 2 and 4 in human tenocytes.

We hypothesized that expression of Toll-like receptors (TLRs) 2 and 4 by tenocytes is involved in the catabolic processes of tendon degeneration. We investigated TLR2 and TLR4 expression by tenocytes in healthy and tendinotic Achilles tendons. We also investigated whether TLR2 and TLR4 could be upregulated in tendon explants using proinflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor alphpa (TNFalpha). Peroperatively harvested healthy (n = 5) and tendinotic (n = 13) Achilles tendon samples were examined by real-time RT-PCR and immunohistochemical staining for TLR2 and TLR4. In addition, the catabolic process in tendinopathy was analyzed by real-time RT-PCR for matrix metalloproteinases MMP1, MMP3, MMP9, and MMP13. Furthermore, healthy tendon explants were cultured in the presence of 20 ng/ml IL-1beta (n = 10) or 10 ng/mL TNFalpha (n = 8) for 4, 24, 48, and 72 h before analysis of TLR and MMP expression levels. Although mRNA levels for both TLR2 and TLR4 were detected in healthy and tendinotic Achilles tendons, we could not confirm expression of these receptors by immunohistochemical staining in either healthy or tendinotic tendon samples. Both receptors did not show significant transcriptional regulation in tendinopathy, although MMP3 was downregulated and MMP9 was upregulated in tendinopathy. In tendon explant cultures TLR2 mRNA was upregulated by TNFalpha (p < 0.05) and IL-1beta (not significant). TLR4 gene expression was not altered by addition of IL-1beta or TNFalpha. Tendon tissue can be stimulated to increase TLR2 gene expression by addition of catabolic factors TNFalpha or IL-1beta. However, the catabolic processes in Achilles tendinopathy cannot be attributed to regulation of TLR2 and TLR4 by tenocytes.

T cell dependence of chronic destructive murine arthritis induced by repeated local activation of Toll-like receptor-driven pathways: crucial role of both interleukin-1beta and interleukin-17.

OBJECTIVE: The pathogenesis of rheumatoid arthritis is often linked to bacterial infections. The present study was undertaken to develop a mouse model of chronic destructive arthritis induced by repeated intraarticular (IA) exposure to bacterial cell wall fragments and to investigate the cytokine dependence of this model. METHODS: Mice that were deficient in various cytokines were injected IA with cell wall fragments of Streptococcus pyogenes on days 0, 7, 14, and 21. The development of chronic destructive arthritis was compared between groups of mice lacking different cytokines, to assess which cytokines were crucial for development of chronic destructive arthritis. RESULTS: Repeated exposure of a joint to S pyogenes cell wall fragments resulted in the development of chronic destructive arthritis. In mice deficient in recombination-activating gene 2, streptococcal cell wall (SCW)-directed T cell reactivity was found and chronic arthritis did not develop, implicating T cells in the generation of chronic SCW-induced arthritis. Interleukin-17 (IL-17) receptor-deficient mice showed a reduction of joint destruction in the chronic stage, implicating a detrimental role of the recently discovered IL-17-producing T helper cells (Th17 cells). IL-23 expression was apparent during the late stages of arthritis. Joint swelling was no longer dependent on tumor necrosis factor alpha (TNFalpha) after the last flare, and pronounced cartilage damage was found after 28 days in TNFalpha-deficient mice. In contrast, IL-1beta-deficient mice were fully protected against joint swelling and cartilage and bone destruction during the late stages of disease. CONCLUSION: These findings indicate that the TNFalpha dependence of arthritis is lost during the erosive stage, when Th17 cells become crucial. IL-1beta dependence remains strong, consistent with its pivotal role in the generation of Th17 cells.

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-alpha. The contribution of TNF in IL-17-induced joint inflammation is unclear. In this work we demonstrate using TNF-alpha-deficient mice that TNF-alpha is required in IL-17-induced joint pathology under naive conditions in vivo. However, overexpression of IL-17 aggravated K/BxN serum transfer arthritis to a similar degree in TNF-alpha-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.

IL-32, a proinflammatory cytokine in rheumatoid arthritis.

IL-32 is a recently discovered cytokine that induces TNFalpha, IL-1beta, IL-6, and chemokines. We investigated whether IL-32 is expressed in the synovia of patients with rheumatoid arthritis (RA) and studied associations with disease severity and the presence of other cytokines. Immunohistochemistry revealed that IL-32 is highly expressed in RA synovial tissue biopsies, whereas IL-32 was not observed in synovial tissues from patients with osteoarthritis. Moreover, in synovial biopsies from 29 RA patients with active disease, the level of IL-32 staining correlated with erythrocyte sedimentation rate, a marker of systemic inflammation (R = 0.63 and P < 0.0003). Synovial staining of IL-32 also correlated with indices of synovial inflammation (R = 0.80 and P < 0.0001) as well as synovial presence of TNFalpha (R = 0.68 and P < 0.004), IL-1beta (R = 0.79 and P < 0.0001), and IL-18 (R = 0.82 and P < 0.001). IL-32 was a potent inducer of prostaglandin E(2) release in mouse macrophages and human blood monocytes, an important property for inflammation. After the injection of human IL-32gamma into the knee joints of naïve mice, joint swelling, with pronounced influx of inflammatory cells and cartilage damage, was observed. In TNFalpha-deficient mice, IL-32-driven joint swelling was absent and cell influx was markedly reduced, but loss of proteoglycan was unaffected, suggesting that IL-32 activity is, in part, TNFalpha-dependent. IL-32, strongly associated with TNFalpha, IL-1beta, and IL-18, appears to play a role in human RA and may be a novel target in autoimmune diseases.

Interleukin-17 receptor deficiency results in impaired synovial expression of interleukin-1 and matrix metalloproteinases 3, 9, and 13 and prevents cartilage destruction during chronic reactivated streptococcal cell wall-induced arthritis.

OBJECTIVE: To examine the role of interleukin-17 receptor (IL-17R) signaling in cartilage destruction and its interrelationship with synovial IL-1 expression during chronic reactivated streptococcal cell wall (SCW)-induced arthritis. METHODS: SCW arthritis was repeatedly induced in wild-type (WT) and IL-17R-deficient (IL-17R-/-) mice. At different time points, joint inflammation was assessed by using calipers to measure joint swelling. On day 42, mice were killed, and knee joints were removed for histologic analysis. Quantitative polymerase chain reaction (PCR) analyses for different proinflammatory mediators and matrix metalloproteinases (MMPs) were performed on inflamed synovium from WT and IL-17R-/- mice after 5 repeated injections of SCW fragments. RESULTS: IL-17R signaling did not play a significant role in acute joint swelling induced by a single injection of SCW fragments directly into the joint. However, repeated local injections of SCW fragments into the knee joints of IL-17R-/- mice resulted in fewer infiltrating cells in the joint compared with WT mice. Moreover, histologic analysis on day 42 revealed a significant suppression of the degree of chondrocyte death and an absence of cartilage surface erosion in IL-17R-/- mice. Quantitative PCR analysis revealed impaired synovial expression of IL-1, IL-6, cyclooxygenase 2, stromelysin (MMP-3), gelatinase B (MMP-9), and collagenase 3 (MMP-13) in IL-17R-/- mice. CONCLUSION: These data show a critical role of IL-17R signaling in driving the synovial expression of proinflammatory and catabolic mediators, such as IL-1 and different MMPs, during progression from an acute, macrophage-driven joint inflammation to a chronic, cartilage-destructive, T cell-mediated synovitis. Prevention of IL-17R signaling warrants consideration as a therapeutic target in chronic destructive arthritis.

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.

Induction of cartilage damage by overexpression of T cell interleukin-17A in experimental arthritis in mice deficient in interleukin-1.

OBJECTIVE: To examine the capacity of T cell interleukin-17A (IL-17A; referred to hereinafter as IL-17) to induce cartilage damage during experimental arthritis in the absence of IL-1. METHODS: Local IL-17 gene transfer was performed in the knee joint of IL-1-deficient mice and wild-type controls during streptococcal cell wall (SCW)-induced arthritis. Knee joints were isolated at various time points for histologic analysis of cartilage proteoglycan (PG) depletion. Expression of messenger RNA for inducible nitric oxide synthase, matrix metalloproteinases (MMPs) 3, 9, and 13, and ADAMTS-4 was determined by quantitative polymerase chain reaction analysis. VDIPEN staining was analyzed to study MMP-mediated cartilage damage. In addition, systemic anti-IL-1alpha/beta antibody treatment was performed in mice immunized with type II collagen and injected locally with an adenoviral vector expressing IL-17 or with control adenovirus. Knee joints were isolated and analyzed for cartilage PG depletion, chondrocyte death, and cartilage surface erosion. RESULTS: During SCW-induced arthritis, local T cell IL-17 gene transfer turned this acute, macrophage-driven joint inflammation into a severe, chronic arthritis accompanied by aggravated cartilage damage. Of high interest, the IL-1 dependency of cartilage PG depletion was fully abrogated when IL-17 was locally overexpressed in the joint. Moreover, local IL-17 gene transfer increased MMP expression without the need for IL-1, although IL-1 remained essential for part of the cartilage VDIPEN expression. Furthermore, when IL-17 was overexpressed in the knee joints of mice with collagen-induced arthritis, anti-IL-1 treatment did not reduce the degree of chondrocyte death or cartilage surface erosion. CONCLUSION: These data show the capacity of IL-17 to replace the catabolic function of IL-1 in cartilage damage during experimental arthritis.

Expression of toll-like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin-12 and interleukin-18 via interferon-gamma.

OBJECTIVE: To study the expression of Toll-like receptor 2 (TLR-2) and TLR-4 and its association with proinflammatory cytokines in synovial tissue from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals. METHODS: Synovial tissue specimens from 29 RA patients were stained for TLR-2, TLR-4, and proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-12, IL-17, IL-18, and tumor necrosis factor alpha [TNFalpha]). The expression of TLR-2, TLR-4, and cytokines as well as the degree of inflammation in synovial tissue were compared between patients with RA, patients with OA (n = 5), and healthy individuals (n = 3). Peripheral blood mononuclear cells (PBMCs) were incubated with IL-12 and IL-18, and TLR expression was assessed using fluorescence-activated cell sorter analysis. Production of TNFalpha and IL-6 was measured using Luminex bead array technology. RESULTS: In RA synovial tissue, the expression of TLR-2 was slightly higher than that of TLR-4. Interestingly, both TLR-2 and TLR-4 were expressed at higher levels in moderately inflamed synovium, as compared with synovial tissue with no or severe inflammation. TLR expression in both the lining and the sublining was associated with the presence of IL-12 and IL-18, but no other cytokines, in the lining. The expression of both TLRs was low in synovial tissue from OA patients and healthy donors. Stimulation of PBMCs with IL-12 and IL-18 resulted in increased expression of both TLR-2 and TLR-4; this could be blocked with anti-interferon-gamma (anti-IFNgamma) antibodies, suggesting a role for IFNgamma. Lipopolysaccharide- or lipoteichoic acid-mediated triggering of PBMCs incubated with IL-12/IL-18 or IFNgamma led to an increased production of both TNFalpha and IL-6, indicating the functionality of TLR-2 and TLR-4. CONCLUSION: TLR-2 and TLR-4 are expressed in synovial tissue of patients with clinically active disease and are associated with the levels of both IL-12 and IL-18. The synergistic effect of IL-12 and IL-18 on T cell IFNgamma production seems to regulate expression of TLR-2 and TLR-4 in the synovial tissue of RA patients.

Interleukin-18 promotes joint inflammation and induces interleukin-1-driven cartilage destruction.

Interleukin (IL)-18 is a member of the IL-1 family of proteins that exerts proinflammatory effects and is a pivotal cytokine for the development of Th1 responses. The goal of the present study was to investigate whether IL-18 induces joint inflammation and joint destruction directly or via induction of other cytokines such as IL-1 and tumor necrosis factor (TNF). To this end we performed both in vitro and in vivo kinetic studies. For in vivo IL-18 exposure studies C57BL/6, TNF-deficient, and IL-1-deficient mice were injected intra-articularly with 1.10(7) pfu mIL-18 adenovirus followed by histopathological examination. Local overexpression of IL-18 resulted in pronounced joint inflammation and cartilage proteoglycan loss in control mice. Of high interest, IL-18 gene transfer in IL-1-deficient mice did not show cartilage damage, although joint inflammation was similar to that in wild-type animals. Overexpression of IL-18 in TNF-deficient mice showed that TNF was partly involved in IL-18-induced joint swelling and influx of inflammatory cells, but cartilage proteoglycan loss occurred independent of TNF. In vitro cartilage degradation by IL-18 was found after a 72-hour culture period. Blocking of IL-1 with IL-1Ra or an ICE-inhibitor resulted in complete protection against IL-18-mediated cartilage degradation. The present study demonstrated that IL-18 induces joint inflammation independently of IL-1. In addition, we showed that IL-1beta generation, because of IL-18 exposure, was essential for marked cartilage degradation both in vitro and in vivo. These findings implicate that IL-18, in contrast to TNF, contributes through separate pathways to joint inflammation and cartilage destruction.

Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion.

OBJECTIVE: Interleukin-17 (IL-17) is a proinflammatory cytokine that is expressed in the synovium of rheumatoid arthritis (RA) patients. This T cell cytokine is implicated in the initiation phase of arthritis. However, the role of IL-17 during the effector phase of arthritis has still not been identified; this was the objective of the present study. METHODS: Mice with collagen-induced arthritis (CIA) were treated with polyclonal rabbit anti-murine IL-17 (anti-IL-17) antibody-positive serum or normal rabbit serum after the first signs of arthritis. In addition, during a later stage of CIA mice were selected and treated with anti-IL-17 antibody or control serum. Arthritis was monitored visually, and joint pathology was examined radiologically and histologically. Systemic IL-6 levels were measured by enzyme-linked immunosorbent assay, and local synovial IL-1 and receptor activator of NF-kappaB ligand (RANKL) expression was analyzed using specific immunohistochemistry. RESULTS: Treatment with a neutralizing anti-IL-17 antibody after the onset of CIA significantly reduced the severity of CIA. Radiographic analysis revealed marked suppression of joint damage in the knee and ankle joints. Histologic analysis confirmed the suppression of joint inflammation and showed prevention of cartilage and bone destruction after anti-IL-17 antibody therapy. Systemic IL-6 levels were significantly reduced after anti-IL-17 antibody treatment. Moreover, fewer IL-1beta-positive and RANKL-positive cells were detected in the synovium after treatment with neutralizing IL-17. Interestingly, initiation of anti-IL-17 antibody therapy during a later stage of CIA, using mice with higher clinical arthritis scores, still significantly slowed the progression of the disease. CONCLUSION: IL-17 plays a role in early stages of arthritis, but also later during disease progression. Systemic IL-6 was reduced and fewer synovial IL-1-positive and RANKL-positive cells were detected after neutralizing endogenous IL-17 treatment, suggesting both IL-1-dependent and IL-1-independent mechanisms of action. Our data strongly indicate that IL-17 neutralization could provide an additional therapeutic strategy for RA, particularly in situations in which elevated IL-17 may attenuate the response to anti-tumor necrosis factor/anti-IL-1 therapy.

IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kappa B ligand/osteoprotegerin balance.

IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-kappa B ligand (RANKL)/receptor activator of NF-kappa B/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-kappa B, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.

Increase in expression of receptor activator of nuclear factor kappaB at sites of bone erosion correlates with progression of inflammation in evolving collagen-induced arthritis.

OBJECTIVE: The receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL) pathway is critical in osteoclastogenesis and bone resorption and has been implicated in the process of focal bone erosion in arthritis. This study was undertaken to identify in vivo the hitherto-unknown origin and localization of RANK-expressing osteoclast precursor cells at sites of bone erosion in arthritis. METHODS: DBA-1 mice were immunized with bovine type II collagen/Freund's complete adjuvant and were given an intraperitoneal booster injection of type II collagen on day 21. Arthritis was monitored visually, and joint pathology was examined histologically. RANK and RANKL expression were analyzed using specific immunohistochemistry, and tartrate-resistant acid phosphatase (TRAP) staining was performed. In addition, TRAP and cathepsin K messenger RNA expression were analyzed by in situ hybridization. RESULTS: A marked increase in the number of cells expressing RANK correlated with the progression of synovial inflammation and clinical disease severity in evolving collagen-induced arthritis (CIA). Interestingly, RANK expression demonstrated a gradient pattern with increased numbers of RANK-positive cells within the synovial infiltrate in areas closer to periosteum and cortical bone. Cells expressing RANK included cells in synovial tissue, bone lining cells on the surface of trabecular bone at sites of erosion, and cells in periosteal areas adjacent to synovial inflammation. In areas where RANK-positive cells were abundant, TRAP-positive, multinucleated osteoclast-like cells were also present at sites of focal bone erosion, suggesting differentiation of synovially derived RANK-positive osteoclast precursor cells into osteoclasts. In addition, TRAP- and cathepsin K-double-positive osteoclast-like cells were detected on the synovial side of cortical bone at sites of early and advanced cortical bone erosion. Sites of RANK expression also correlated well with sites of RANKL expression, and there was a close correlation of the temporal expression of the receptor-ligand pair. CONCLUSION: Cells expressing RANK increased in abundance with the progression of arthritis in evolving CIA, and sites of RANK-expressing cells correlated with sites of TRAP-positive, multinucleated osteoclast-like cells as well as with sites of RANKL expression. These data support the hypothesis that the RANK/RANKL pathway plays an important role in the process of bone erosion in CIA.