Corrigendum: Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles.

This corrects the article DOI: 10.1038/srep43923.

Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles.

Stem cells possess significant age-dependent differences in their immune-response profile. These differences were analysed by Next-Generation Sequencing of six age groups from bone marrow mesenchymal stem cells. A total of 9,628 genes presenting differential expression between age groups were grouped into metabolic pathways. We focused our research on young, pre-pubertal and adult groups, which presented the highest amount of differentially expressed genes related to inflammation mediated by chemokine and cytokine signalling pathways compared with the newborn group, which was used as a control. Extracellular vesicles extracted from each group were characterized by nanoparticle tracking and flow cytometry analysis, and several micro-RNAs were verified by quantitative real-time polymerase chain reaction because of their relationship with the pathway of interest. Since miR-21-5p showed the highest statistically significant expression in extracellular vesicles from mesenchymal stem cells of the pre-pubertal group, we conducted a functional experiment inhibiting its expression and investigating the modulation of Toll-Like Receptor 4 and their link to damage-associated molecular patterns. Together, these results indicate for the first time that mesenchymal stem cell-derived extracellular vesicles have significant age-dependent differences in their immune profiles.

Therapeutic efficacy of Tyro3, Axl, and Mer tyrosine kinase agonists in collagen-induced arthritis.

OBJECTIVE: Hyperactivation of innate immunity by Toll-like receptors (TLRs) can contribute to the development of autoinflammatory or autoimmune diseases. This study evaluated the activation of Tyro3, Axl, Mer (TAM) receptors, physiologic negative regulators of TLRs, by their agonists, growth arrest-specific protein 6 (GAS-6) and protein S, in the prevention of collagen-induced arthritis (CIA). METHODS: Adenoviruses overexpressing GAS-6 and protein S were injected intravenously or intraarticularly into mice during CIA. Splenic T helper cell subsets from intravenously injected mice were studied by flow cytometry, and the knee joints of mice injected intravenously and intraarticularly were assessed histologically. Synovium from mice injected intraarticularly was evaluated for cytokine and suppressor of cytokine signaling (SOCS) expression. RESULTS: Protein S significantly reduced ankle joint swelling when overexpressed systemically. Further analysis of knee joints revealed a moderate reduction in pathologic changes in the joint and a significant reduction in the number of splenic Th1 cells when protein S was overexpressed systemically. Local overexpression of GAS-6 decreased joint inflammation and joint pathology. Protein S treatment showed a similar trend of protection. Consistently, GAS-6 and protein S reduced cytokine production in the synovium. Moreover, levels of messenger RNA for interleukin-12 (IL-12) and IL-23 were reduced by GAS-6 and protein S treatment, with a corresponding decrease in the production of interferon-γ and IL-17. TAM ligand overexpression was associated with an increase in SOCS-3 levels, which likely contributed to the amelioration of arthritis. CONCLUSION: This study provides the first evidence that TAM receptor stimulation by GAS-6 and protein S can be used to ameliorate arthritis when applied systemically or locally. TAM receptor stimulation limits proinflammatory signaling and adaptive immunity. This pathway provides a novel strategy by which to combat rheumatoid arthritis.

The natural soluble form of IL-18 receptor beta exacerbates collagen-induced arthritis via modulation of T-cell immune responses.

OBJECTIVE: IL-18 is a pluripotent cytokine that has been implicated in the development of rheumatoid arthritis. A soluble form of the IL-18 receptor accessory protein (sIL-18Rbeta) with unknown function has recently been identified. This study examined the ability of sIL-18Rbeta to inhibit IL-18 biological activities and to modulate immune responses during collagen-induced arthritis (CIA). METHODS: Adenoviruses encoding sIL-18Rbeta were administered intravenously in type II collagen-immunised DBA/1 mice. Humoral responses were analysed by determining anti-bovine collagen type II (BCII) antibody levels by ELISA. Cytokine production by splenic T cells and cytokine levels in serum were measured by Luminex multi-analyte technology. CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) were measured by flow cytometry. RESULTS: Intravenous delivery of Ad5.sIL-18Rbeta in collagen-immunised mice led to enhanced transgene expression in splenic antigen-presenting cells (APC). A co-culture of these sIL-18Rbeta-transduced APC with purified splenic CD3(+) T cells led to a marked inhibition of IL-18-induced IFNgamma, IL-4 and IL-17 production by CD3(+) T cells. Remarkably, systemic treatment with Ad5.sIL-18Rbeta caused an exacerbation of arthritis, and histological evaluation of knee joints showed increased cartilage and bone erosion. No significant differences were observed in anti-BCII antibodies, but the aggravation was accompanied by decreased IFNgamma (-30%) and IL-4 (-44%) and increased IL-17 (+84%) production by splenic CD3(+) T cells. In addition, reduced circulating levels of CD4(+)CD25(+)Foxp3(+) Treg and anti-inflammatory IL-10 were shown. CONCLUSION: This study identifies sIL-18Rbeta as a novel IL-18 inhibitor, which promotes CIA after intravenous overexpression by affecting Treg levels and supporting a T helper type 17 response.

Stimulation of chondrocyte-mediated cartilage destruction by S100A8 in experimental murine arthritis.

OBJECTIVE: To investigate whether S100A8 is actively involved in matrix metalloproteinase (MMP)-mediated chondrocyte activation. METHODS: S100A8 and S100A9 proteins were detected in inflamed knee joints from mice with various forms of murine arthritis, using immunolocalization. Murine chondrocyte cell line H4 was stimulated with proinflammatory cytokines or recombinant S100A8. Messenger RNA (mRNA) and protein levels were measured using reverse transcriptase-polymerase chain reaction and intracellular fluorescence-activated cell sorting (FACS). Breakdown of aggrecan on the pericellular surface of the chondrocytes was measured using VDIPEN and NITEGE antibodies and FACS, and breakdown in patellar cartilage was measured by immunolocalization. RESULTS: S100A8 and S100A9 proteins were abundantly expressed in and around chondrocytes in inflamed knee joints after induction of antigen-induced arthritis or onset of spontaneous arthritis in interleukin-1 (IL-1) receptor antagonist-knockout mice. Stimulation of chondrocytes by the proinflammatory cytokines tumor necrosis factor alpha, IL-1beta, IL-17, and interferon-gamma caused strong up-regulation of S100A8 mRNA and protein levels and up-regulation to a lesser extent of S100A9 levels. Stimulation of chondrocytes with S100A8 induced significant up-regulation of MMP-2, MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5 mRNA levels (up-regulated 4, 4, 3, 16, 8, and 4 times, respectively). VDIPEN and NITEGE neoepitopes were significantly elevated in a concentration-dependent manner in chondrocytes treated with 0.2, 1, or 5 microg/ml of S100A8. (VDIPEN levels were elevated 17%, 67%, and 108%, respectively, and NITEGE levels were elevated 8%, 33%, and 67%, respectively.) S100A8 significantly increased the effect of IL-1beta on MMP-3, MMP-13, and ADAMTS-5. Mouse patellae incubated with both IL-1beta and S100A8 had elevated levels of NITEGE within the cartilage matrix when compared with patellae incubated with IL-1beta or S100A8 alone. CONCLUSION: These findings indicate that S100A8 and S100A9 are found in and around chondrocytes in experimental arthritis. S100A8 up-regulates and activates MMPs and aggrecanase-mediated pericellular matrix degradation.

Application of a disease-regulated promoter is a safer mode of local IL-4 gene therapy for arthritis.

The application of disease-regulated promoters in local gene therapy for rheumatoid arthritis potentiates the development of a sophisticated treatment that relies on a restricted and fine-tuned supply of biologicals. Although several studies have investigated regulated promoters for achieving effective transgene expression during arthritis, none have explored their potential for minimizing deleterious effects arising from constitutive overexpression of transgenes under naive conditions. Using naive and collagen-induced arthritic mice, we examined the applicability of a hybrid interleukin-1 enhancer/interleukin-6 proximal promoter for achieving efficacious murine interleukin-4 gene therapy under arthritic conditions, while minimizing interleukin-4-induced inflammation under naive conditions. We found strong upregulation of transgene expression in virally transduced knee joints under arthritic conditions compared to levels in naive animals. Besides its responsiveness, the promoter strength proved sufficient for generating therapeutically efficacious levels interleukin-4, as demonstrated by the successful protection against cartilage erosion in collagen-induced arthritis. Most importantly, promoter-mediated restriction of the potent chemotactic interleukin-4 in naive animals strongly reduced the amounts of inflammatory cell influx. This study suggests the suitability of the interleukin-1 enhancer/interleukin-6 proximal promoter for the development of a local gene therapy strategy for rheumatoid arthritis that requires fine-tuned and restricted expression of transgenes with a pleiotrophic nature.

A novel role for suppressor of cytokine signaling 3 in cartilage destruction via induction of chondrocyte desensitization toward insulin-like growth factor.

OBJECTIVE: An important mechanism contributing to cartilage destruction in arthritis is chondrocyte desensitization toward its main anabolic factor, insulin-like growth factor 1 (IGF-1). In this study, we sought to determine the role of suppressor of cytokine signaling 3 (SOCS-3) in the induction of IGF-1 desensitization of murine chondrocytes. METHODS: Chondrocyte responsiveness to IGF-1 was assessed by 35S-sulfate incorporation into proteoglycans (PGs), via aggrecan messenger RNA expression, using quantitative real-time polymerase chain reaction or insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation (Western blot analysis). IGF-1 desensitization of patellar chondrocytes was studied in zymosan-induced arthritis. IGF-1 desensitization was induced in patellar cartilage explants or the H4 chondrocyte cell line, exposed to interleukin-1alpha (IL-1alpha). SOCS-3 protein expression was assessed by immunohistochemistry or by Western blot analysis of protein extracts. The role of SOCS-3 in IGF-1 signaling was elucidated by adenoviral overexpression. RESULTS: Exposure of murine articular cartilage to IL-1 caused a significant decrease in IGF-1-induced PG synthesis. This effect also occurred in inducible nitric oxide synthase-knockout mice, revealing the involvement of a secondary IL-1-induced factor other than nitric oxide. We showed that IL-1 significantly up-regulated SOCS-3 transcription and protein synthesis in H4 chondrocytes. In contrast, IL-18 was unable to induce SOCS-3 expression and failed to induce chondrocyte IGF-1 desensitization. Histologic analysis of samples from arthritic knee joints revealed high expression of SOCS-3 in chondrocytes. Through adenoviral overexpression of SOCS-3, we obtained direct evidence that SOCS-3 inhibits IGF-1-mediated cell signaling, since IRS-1 phosphorylation was reduced. CONCLUSION: This study demonstrates that IL-1-induced SOCS-3 expression is a novel mechanism of IGF-1 desensitization in chondrocytes; in conjunction with nitric oxide it can contribute to cartilage damage during arthritis.

Soluble interleukin-1 receptor accessory protein ameliorates collagen-induced arthritis by a different mode of action from that of interleukin-1 receptor antagonist.

OBJECTIVE: To discern the mode of interleukin-1 (IL-1) inhibition of soluble IL-1 receptor accessory protein (sIL-1RAcP) by comparison with IL-1 receptor antagonist (IL-1Ra) in arthritis. METHODS: Adenoviral vectors encoding either sIL-1RAcP or IL-1Ra were administered systemically before onset of collagen-induced arthritis in DBA/1 mice. Anti-bovine type II collagen IgG and IL-6 were quantified in serum. Proliferative response of splenic T cells was determined in the presence of sIL-1RAcP or IL-1Ra. The effect on IL-1 inhibition of recombinant sIL-1RAcP and IL-1Ra was further examined in vitro, using NF-kappaB luciferase reporter cell lines. Quantitative polymerase chain reaction was used to determine the relative messenger RNA expression of the IL-1 receptors. RESULTS: Adenoviral overexpression of both sIL-1RAcP and IL-1Ra resulted in amelioration of the collagen-induced arthritis. Both IL-1 antagonists reduced the circulating levels of antigen-specific IgG2a antibodies, but only IL-1Ra was able to inhibit lymphocyte proliferation. By using purified lymphocyte populations derived from NF-kappaB reporter mice, we showed that sIL-1RAcP inhibits IL-1-induced NF-kappaB activity in B cells but not T cells, whereas IL-1Ra inhibited IL-1 on both cell types. A study in a panel of NF-kappaB luciferase reporter cells showed that the sIL-1RAcP inhibits IL-1 signaling on cells expressing either low levels of membrane IL-1RAcP or high levels of IL-1RII. CONCLUSION: We show that the sIL-1RAcP ameliorated experimental arthritis without affecting T cell immunity, in contrast to IL-1Ra. Our results provide data in support of receptor competition by sIL-1RAcP as an explanation for the different mode of IL-1 antagonism in comparison with IL-1Ra.

An inflammation-inducible adenoviral expression system for local treatment of the arthritic joint.

To achieve a disease-regulated transgene expression for physiologically responsive gene therapy of arthritis, a hybrid promoter was constructed. The human IL-1 beta enhancer region (-3690 to -2720) upstream of the human IL-6 promoter region (-163 to +12) was essential in mounting a robust response in HIG-82 synovial fibroblasts and in RAW 264,7 macrophages. A replication-deficient adenovirus was engineered with luciferase (Luc) controlled by the IL-1/IL-6 promoter (Ad5.IL-1/IL-6-Luc). LPS caused a 23- and 4.6-fold induction of Luc. activity in RAW cells infected with Ad5.IL-1/IL-6-Luc or the conventional Ad5.CMV-Luc construct, respectively. Next, adenoviruses (10(6) ffu) were injected into the knees of C57Bl/6 mice. An intra-articular injection of zymosan, 3 days after Ad5.IL-1/IL-6-Luc, increased Luc. activity by 39-fold but had no effect in the Ad5.CMV-Luc joints. The constitutive CMV promoter was rapidly silenced and could not be reactivated in vivo. In contrast, the IL-1/IL-6 promoter could be reactivated by Streptococcal cell wall (SCW)-induced arthritis up to 21 days after infection. Next the IL-1/IL-6 promoter was compared to the C3-Tat/HIV-LTR two-component system in wild-type, IL-6(-/-) and IL-1(-/-) gene knockout mice. Both systems responded well to LPS-, zymosan- and SCW-induced arthritis. However, the basal activity of the IL-1/IL-6 promoter was lower and IL-6 independent. This study showed that the IL-1/IL-6 promoter is feasible to achieve disease-regulated transgene expression for treatment of arthritis.

Effectiveness of the soluble form of the interleukin-1 receptor accessory protein as an inhibitor of interleukin-1 in collagen-induced arthritis.

OBJECTIVE: To investigate whether the soluble form of interleukin-1 (IL-1) receptor accessory protein (sIL-1RAcP), whose physiologic function remains to be established, can serve as a specific inhibitor of IL-1 signaling in vitro, and to evaluate its applicability in collagen-induced arthritis (CIA). METHODS: Soluble IL-1RAcP was cloned from murine liver complementary DNA and expressed by the use of either an adenoviral vector (AdRGD) for sIL-1RAcP or a stable-transfected NIH3T3 fibroblast cell line. The ability of affinity-purified sIL-1RAcP to inhibit IL-1 signaling was tested on NF-kappaB luciferase reporter fibroblasts and quantified by luminometer. To investigate therapeutic efficacy, sIL-1RAcP was both locally (knee joint) and systemically overexpressed in collagen-immunized male DBA/1 mice. Severity of arthritis was monitored visually, and the pathologic process in the joint was examined histologically. Serum was obtained from mice to quantify IL-6 and anti-bovine type II collagen (BCII) antibody levels. RESULTS: Incubation of the NF-kappaB reporter fibroblast with purified sIL-1RAcP protein showed a marked reduction of IL-1-induced, but not tumor necrosis factor-induced, NF-kappaB activation. This showed a novel role for sIL-1RAcP as a specific inhibitor of IL-1 signaling. Local transplantation of sIL-1RAcP-producing NIH3T3 fibroblasts into the knee before onset of CIA had little or no effect on general disease severity in these mice. Histologic evaluation of the knee joints receiving sIL-1RAcP cell transplantation showed a marked reduction in both joint inflammation and bone and cartilage erosion. Local treatment with sIL-1RAcP had no profound effect on serum levels of IL-6 and anti-BCII antibodies, which is indicative of the ongoing presence of arthritis in distal joints. In contrast to local treatment, systemic treatment with the AdRGD for sIL-1RAcP markedly ameliorated CIA in all joints. CONCLUSION: In this study we demonstrated that sIL-1RAcP is a biologically active and innovative inhibitor of IL-1, and treatment of mice with sIL-1RAcP had a profound prophylactic effect on collagen-induced arthritis.

Adenoviral delivery of IL-18 binding protein C ameliorates collagen-induced arthritis in mice.

Elevated concentrations of interleukin-18 (IL-18) are found in both serum and synovial fluid of patients suffering from rheumatoid arthritis (RA) and this cytokine has recently been implicated in the development of experimental arthritis. In this present study, we developed an IL-18 neutralizing intervention and examined its efficacy for local intra-articular treatment of experimental arthritis. To this end we constructed an adenoviral vector containing the murine IL-18 binding protein isoform c gene (AdCMVIL-18BPc). The constructed adenoviral vector was validated on replication deficiency, transfection efficacy and ability to express biological functional IL-18BPc. Intra-articular overexpression of IL-18BPc significantly reduced incidence of collagen-induced arthritis (CIA) in treated kneejoints. Affected kneejoints of IL-18BPc-treated mice showed less severe arthritis, characterized by reduction of inflammation and destruction of bone and cartilage. Local intra-articular IL-1BPc treatment in both knees provided additional protection against CIA incidence and severity in distal paws. Measurement of serum levels of specific collagen type (CII) Abs revealed a moderate reduction of circulating IgG2a anti-CII Abs, while IgG1 anti-CII Abs remained at similar level. The present study underlines the involvement of IL-18 as an important proinflammatory cytokine in onset of experimental arthritis. Furthermore, it shows that endogenous IL-18 can be blocked efficiently through local adenoviral overexpression of IL-18BPc, indicating that treatment with IL-18BPc might contribute to joint protection in RA.

C3-Tat/HIV-regulated intraarticular human interleukin-1 receptor antagonist gene therapy results in efficient inhibition of collagen-induced arthritis superior to cytomegalovirus-regulated expression of the same transgene.

OBJECTIVE: To achieve disease-inducible expression of recombinant antiinflammatory proteins in order to allow autoregulation of drug dose by natural homeostatic mechanisms. METHODS: We compared the inducible 2-component expression system (C3-human immunodeficiency virus/transactivator of transcription [C3-Tat/HIV]) with the constitutive cytomegalovirus (CMV) promoter in the polyarticular collagen-induced arthritis (CIA) model in mice. DBA/1 mice were immunized with bovine type II collagen and were given boosters on day 21. On day 22, mice were injected intraarticularly with the adenoviral vectors AdCMVLuc, AdCMVhIL-1Ra, AdC3-Tat/HIV-Luc, or AdC3-Tat/HIV-hIL-1Ra. The injected knee joints and hind paws were then scored for signs of arthritis, and knee joint histology was compared. RESULTS: The CMV-driven interleukin-1 receptor antagonist (IL-1Ra) expression resulted in a high constitutive expression and amelioration of CIA. C3-Tat/HIV-driven IL-1Ra expression could be detected only on days 24, 29, and 35. Fourteen days after injection of the vectors, CIA was significantly better inhibited by the C3-Tat/HIV-driven IL-1Ra expression compared with the CMV-driven IL-1Ra expression. Moreover, prevention of CIA in the knee joints also prevented CIA in the untreated hind paws. CONCLUSION: Our data demonstrate for the first time the feasibility of an inducible expression system for local production of IL-1Ra for treatment of arthritis in the CIA model.

A tropism-modified adenoviral vector increased the effectiveness of gene therapy for arthritis.

Adenoviral vectors (AdV) are used for anti-inflammatory cytokine therapy in experimental arthritis. Cell entry of AdV is dependent on the initial recognition of the coxsackie-adenovirus receptor (CAR) on cells. Recently, an Arg-Gly-Asp (RGD) motif was introduced in the HI loop of the fiber knob, this enables the adenovirus to bypass CAR and mediate cell entry via RGD binding integrins. In this study, we explored the transduction efficiency of the RGD-modified adenovirus in synovium and compared the RGD-modified with the conventional adenoviral vector for their effectiveness to modulate the murine collagen-induced arthritis (CIA) model when used to overexpress mIL-1Ra in the knee joint. Twenty-four hours after intra-articular injection of 10(7) fluorescent forming units (ffu) virus, luciferase (luc) activity in Ad5LucRGD-injected joints was up to 38 times higher than in AdCMVLuc-injected joints, and in arthritic joints the transduction efficiency was up to 69 times higher for the Ad5LucRGD viruses. Transduction of the synovial lining by the RGD-modified adenovirus containing the mIL-1Ra transgene, markedly improved the inhibition of CIA compared with the conventional virus in both a prophylactic and therapeutic treatment protocol. These results show that targeting integrins with the RGD-modified AdV improved the outcome of gene therapy for arthritis.

Involvement of IL-6, apart from its role in immunity, in mediating a chronic response during experimental arthritis.

Interleukin-6 (IL-6) is highly produced during arthritis but its exact function is still unknown. In this study we examined if IL-6, apart from its role in immunity, was involved in the local inflammatory response in experimental arthritis. IL-6 deficient (IL-6(-/-)) and wild-type mice were first compared in the antigen-induced arthritis model. IL-6 deficiency resulted in a mild, transient inflammation whereas wild-type mice developed a chronic, destructive synovitis. Wild-type mice immunized with one-tenth of the normal antigen dose still developed chronic arthritis despite low antibody levels, excluding reduced humoral immunity in IL-6(-/-) mice as a crucial phenomenon. In addition, passive immune-complex-induced arthritis did not differ between wild-type and IL-6(-/-) mice. Another option is reduced levels of Th1 cells in IL-6(-/-) mice. However, transfer of antigen-specific wild-type lymph node cells to IL-6(-/-) mice enhanced acute joint inflammation and increased cartilage damage but still could not sustain chronic inflammation, suggesting involvement of nonimmune elements of IL-6 activity in chronicity. In line with this, nonimmunologically mediated zymosan-induced arthritis developed similarly in the first week, but only wild-type mice developed chronic synovitis. These results indicate an important role for IL-6 in propagation of joint inflammation, potentially independent of its role in immunity.

Animal models of arthritis in NOS2-deficient mice.

OBJECTIVE: To study the role of nitric oxide (NO) in cartilage destruction in murine models of arthritis and osteoarthritis. METHODS: Joint inflammation was induced in the knee joint by intraarticular injection of Zymosan. Osteoarthritis was induced by local injection of bacterial collagenase, causing joint instability. The effect of NO deficiency was studied by comparing the effects in normal mice and mice with genetically disrupted NOS2 (inducible NO synthase). Impact on articular cartilage was evaluated by histology and measurement of chondrocyte 35S-proteoglycan synthesis. RESULTS: NOS2 deficiency prevented chondrocyte proteoglycan synthesis inhibition in the arthritic cartilage and restored normal responsiveness to IGF-1. Net cartilage proteoglycan depletion was markedly reduced in the absence of NOS2, although inflammation was hardly affected. Osteoarthritic joint pathology was also significantly reduced, including diminished cartilage lesions and osteophyte formation. CONCLUSION: NO plays a major role in cartilage damage in both arthritic and osteoarthritic conditions.

Different roles of tumour necrosis factor alpha and interleukin 1 in murine streptococcal cell wall arthritis.

In this study two different aspects of tumour necrosis factor alpha (TNF-alpha) and interleukin 1 (IL-1) in locally induced murine streptococcal cell wall arthritis (SCW) were investigated. First, the kinetics and interdependence of TNF-alpha and IL-1 release; and second; their involvement in inflammation and cartilage destruction. Kinetic studies showed that the TNF-alpha peak level preceded the IL-1 peak level. However, in vivo neutralization of TNF-alpha did not result in decreased IL-1 bioactivity or immunoreactivity, suggesting that there is no dominant TNF-alpha-dependent IL-1 release in this model. Inflammation was studied by measuring knee joint swelling and inflammatory cell influx. Impact on cartilage was studied by measuring chondrocyte proteoglycan synthesis and cartilage proteoglycan depletion. The role of TNF-alpha in these phenomena was investigated using anti-TNF-alpha antibodies and tumour necrosis factor binding protein (TNFbp). Similarly, the role of IL-1 was studied using anti-IL-1 antibodies or IL-1 receptor antagonist (IL-1Ra). Anti-TNF-alpha treatment significantly reduced joint swelling, whereas this effect was not found by using anti-IL-1 or IL-1Ra. In contrast, neutralization of IL-1, but not TNF-alpha, resulted in a significant decrease of chondrocyte proteoglycan synthesis inhibition. Moreover, histology revealed that anti-IL-1 treatment reduced cartilage proteoglycan depletion and inflammatory cell influx. Combined anti-TNF-alpha/anti-IL-1 treatment significantly suppressed both inflammation and cartilage damage. However, the impact on these separate parameters did not exceed the effects of either anti-TNF-alpha or anti-TNF-1. It can be concluded that both TNF-alpha and IL-1 exert specific activities in SCW arthritis. The involvement of TNF-alpha in this model is limited to joint swelling, whereas IL-1 plays a dominant role in cartilage destruction and inflammatory cell influx.

Reduced cartilage proteoglycan loss during zymosan-induced gonarthritis in NOS2-deficient mice and in anti-interleukin-1-treated wild-type mice with unabated joint inflammation.

OBJECTIVE: To investigate the role of nitric oxide (NO) and interleukin-1 in (IL-1) joint inflammation and cartilage destruction during zymosan-induced gonarthritis (ZIA). METHODS: Monarticular arthritis was elicited by intraarticular injection of zymosan. The effect of NO deficiency on arthritis was studied in mice with genetically disrupted NOS2. The role of IL-1 was examined by treating wild-type mice with neutralizing anti-murine IL-1(alpha+beta) antibodies. Joint swelling was measured externally by the increased uptake of circulating 99mtechnetium pertechnetate. Proteoglycan (PG) synthesis was assessed using 35S-sulfate incorporation into patellae ex vivo. Histology evaluated exudation and infiltration of leukocytes and the extent of cartilage destruction. RESULTS: The proinflammatory mediators NO, IL-1, and IL-6 were released by the articular tissues during the first hours of inflammation. Interestingly, anti-IL-1 treatment moderately reduced, and NOS2 deficiency moderately enhanced, joint swelling. However, the influx of neutrophils into the joint occurred independently of IL-1 and NOS2 activities. In the first week of inflammation, chondrocyte PG synthesis was significantly suppressed and chondrocytes became unresponsive to their essential anabolic factor, insulin-like growth factor 1 (IGF-1). Anti-IL-1 treatment or NOS2 deficiency prevented the inhibition of PG synthesis, and the chondrocytes remained IGF-1 responsive. Intraarticular injections of IL-1alpha into NOS2-deficient mice did not affect PG synthesis, thus proving that NO mediated this IL-1 effect in vivo. Furthermore, histology showed that cartilage PG loss was markedly ameliorated in NOS2-deficient and anti-IL-1-treated mice. Intermediate cartilage pathology was found in mice that were heterozygous for disrupted NOS2. CONCLUSION: IL-1 and NO play a minor role in edema and neutrophil influx, but a major role in cartilage destruction of ZIA. In this model of murine arthritis, cartilage destruction was, for the most part, caused by pronounced suppression of PG synthesis and IGF-1 unresponsiveness of the chondrocytes, which were induced by de novo-synthesized IL-1 and were mediated by NOS2 activation.

Effect of interleukin 1 and leukaemia inhibitory factor on chondrocyte metabolism in articular cartilage from normal and interleukin-6-deficient mice: role of nitric oxide and IL-6 in the suppression of proteoglycan synthesis.

We studied the role of IL-6 and nitric oxide (NO) in IL-1 and leukaemia inhibitory factor (LIF) induced suppression of proteoglycan synthesis. Cartilage explants of patellae and femoral heads were incubated with IL-1 or LIF. Conditioned media were analysed for IL-6 activity (B9-assay) and NO content (Griess). Proteoglycan synthesis was assessed using [35S]sulfate incorporation. IL-1 dose dependently induced IL-6 synthesis and neutralizing IL-6 with antibodies did not reduce proteoglycan synthesis suppression, neither in explants nor in isolated chondrocytes. IL-6 independence was confirmed using cartilage from IL-6 deficient mice. IL-1 significantly increased NO release in normal and IL-6 deficient chondrocytes and addition of the NO synthase inhibitor, N(G)-monomethyl-L-arginine markedly alleviated proteoglycan synthesis suppression. LIF also induced proteoglycan synthesis suppression in cartilage from normal and IL-6 deficient mice, but the suppression was neither accompanied by nor dependent on NO release. Furthermore, proteoglycan synthesis suppression during experimental arthritis was similar in both normal and IL-6 deficient mice. We concluded that IL-6 is not a necessary cofactor in IL-1 and LIF induced suppression of proteoglycan synthesis. Furthermore, only the IL-1 induced suppression was mediated by NO, suggesting that inhibition of proteoglycan synthesis may occur through different pathways.

Interleukin-6 reduces cartilage destruction during experimental arthritis. A study in interleukin-6-deficient mice.

Using interleukin (IL)-6-deficient (IL-6(0/0) mice or wild-type mice, we investigated the controversial role of IL-6 in joint inflammation and cartilage pathology during zymosan-induced arthritis (ZIA). Monoarticular arthritis was elicited by injection of zymosan into the right knee joint cavity. Production of IL-1, tumor necrosis factor (TNF), IL-6, and nitric oxide by the inflamed knee was assessed in washouts of joint capsule specimens. Plasma corticosterone was measured using a radioimmunoassay. Proteoglycan synthesis was assessed using [35S]sulfate incorporation into patellas ex vivo. Joint swelling was quantified by joint uptake of circulating 99mTechnetium pertechnetate. Histology was taken to evaluate cellular infiltration and cartilage damage. Zymosan caused a rapid increase in articular IL-1, IL-6, TNF, and NO levels. Except for IL-6, the released amounts and time course of these mediators were comparable in the IL-6-deficient mice and the wild-type mice. Elevated plasma corticosterone levels were measured during the first day of arthritis in both strains. At day 2 of ZIA, joint inflammation (joint swelling and cell exudate) in IL-6-deficient mice was comparable with that in the wild-type mice. The marked suppression of chondrocyte proteoglycan synthesis and proteoglycan degradation were on the average higher in the IL-6-deficient mice. Together this resulted in a more pronounced proteoglycan depletion in the IL-6-deficient mice as compared with the wild-type mice during the first week of arthritis. Injection of recombinant IL-6 into the joint cavity corrected the IL-6 deficiency and significantly reduced cartilage destruction. Inflammation was more chronic in the wild-type mice, and these mice also showed a higher prevalence for osteophyte formation. In ZIA, IL-6 plays a dual role in connective tissue pathology, reducing proteoglycan loss in the acute phase and enhancing osteophyte formation in the chronic phase. The latter could be related to the more severe joint inflammation as seen in the normal (IL-6-producing) animals during the chronic phase of arthritis.

Prevention of murine collagen-induced arthritis in the knee and ipsilateral paw by local expression of human interleukin-1 receptor antagonist protein in the knee.

OBJECTIVE: To determine the efficacy of local human interleukin-1 receptor antagonist (HuIL-1Ra) gene therapy in murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were immunized against bovine type II collagen. Before the onset of arthritis, NIH/3T3 fibroblasts transfected with pMFG-IRAP were transplanted into the knee cavity. Normal NIH/3T3 cells served as controls. Paws were evaluated macroscopically for redness, swelling, and deformities during the course of arthritis. Swelling of the knee joints was measured by external gamma counting of 99mtechnetium accumulation in the joint. Paws and knee joints were dissected and processed for histologic studies to evaluate inflammation and cartilage destruction. RESULTS: The NIH/3T3 fibroblasts survived in the joint cavity of DBA mice for at least 7 days. The transduced cells expressed immunoreactive and bioactive HuIL-1Ra in the knee joint, and produced sufficient amounts to block the effect of 1 ng of recombinant murine IL-1alpha on chondrocyte proteoglycan synthesis. The onset of CIA was almost completely prevented in knee joints containing HuIL-1Ra-producing cells, whereas joints containing normal cells showed severe inflammation and destruction of cartilage. Moreover, onset of CIA in the draining joints (ipsilateral paws) of the HuIL-1Ra gene-bearing knees was also prevented. CONCLUSION: Local production of HuIL-1Ra in the knee was able to ameliorate the effects of IL-1 on cartilage and could prevent the onset of CIA not only in that knee, but also in the "draining" paw. This indicates the feasibility of gene transfer as a therapeutic approach to modulating arthritis.