ObjectiveTo investigate whether S100A8 is actively involved in matrix metalloproteinase (MMP)–mediated chondrocyte activation.MethodsS100A8 and S100A9 proteins were detected in inflamed knee joints from mice with various forms of murine... more
ObjectiveTo investigate whether S100A8 is actively involved in matrix metalloproteinase (MMP)–mediated chondrocyte activation.MethodsS100A8 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.ResultsS100A8 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 α, IL‐1β, IL‐17, and interferon‐γ 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 μg/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‐1β on MMP‐3, MMP‐13, and ADAMTS‐5. Mouse patellae incubated with both IL‐1β and S100A8 had elevated levels of NITEGE within the cartilage matrix when compared with patellae incubated with IL‐1β or S100A8 alone.ConclusionThese 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.
Research Interests: Immunology, Biology, Polysaccharides, Medicine, Cell line, and 15 moreHumans, Cartilage, Mice, Animals, Arthritis, Clinical Sciences, Public health systems and services research, Chondrocytes, Matrix Metalloproteinases, Knee Joint, Knockout Mice, Chondrocyte, Matrix Metalloproteinase, Interleukin, and Interferon gamma
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Cartilage destruction in murine antigen induced arthritis is characterized by enhanced degradation of proteoglycans and inhibition of chondrocyte proteoglycan synthesis. Within this model common NSAIDs only suppress joint swelling, and to... more
Cartilage destruction in murine antigen induced arthritis is characterized by enhanced degradation of proteoglycans and inhibition of chondrocyte proteoglycan synthesis. Within this model common NSAIDs only suppress joint swelling, and to some extent granulocyte infiltration, but leave the process of cartilage destruction undisturbed. Evidence is now accumulating that the vast amount of activated granulocytes in the joint space are of minor importance, and that interleukin-1 (IL-1) is the key mediator in this process. Treatment of mice with neutralizing antibodies against IL-1 resulted in relief of chondrocyte proteoglycan synthesis inhibition and prevented matrix destruction. This indicates that it makes sense to focus future therapy on elimination of IL-1.
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Although different models for rheumatoid arthritis have been studied, the pathogenesis in humans remains unknown. A possible mechanism is the crossreactivity between bacterial components and the target-tissue, the cartilage. The existence... more
Although different models for rheumatoid arthritis have been studied, the pathogenesis in humans remains unknown. A possible mechanism is the crossreactivity between bacterial components and the target-tissue, the cartilage. The existence of this crossreactivity is supported by various data from clinical and experimental studies. Here we provide direct evidence that priming in vivo with cell wall fragments of Streptococcus pyogenes or Escherichia coli can induce a cellular and humoral anti-cartilage response in Balb/c mice in vitro. T cells isolated from these mice can be stimulated in vitro to proliferate by a variety of antigens among which are the priming bacterium, an unrelated bacterium, small bacterial components and diverse antigens of cartilagenous origin. In bacterium-primed mice antibodies were also detected that displayed a reactivity to cartilage extract besides the reactivity to bacteria. A crossreactive response occurred in vivo in certain circumstances: a delayed type hypersensitivity reaction could be elicited in cell-wall-primed mice by challenge with cartilage extract. For the expression of this crossreactive response in vivo however, it was obligatory to attenuate the mouse's suppressor-circuit. In this paper we would suggest a mechanism for the pathology of chronic arthritis, based on repeated challenges with different bacterial stimuli.
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It was found that recovery of articular chondrocyte proteoglycan (PG) synthesis was retarded in old mice after in vivo exposure to both IL-1 or hydrogen peroxide. We examined whether this could be related to diminished serum levels of... more
It was found that recovery of articular chondrocyte proteoglycan (PG) synthesis was retarded in old mice after in vivo exposure to both IL-1 or hydrogen peroxide. We examined whether this could be related to diminished serum levels of insulin-like growth factor 1 (IGF-1), the main anabolic factor, or to changes in cartilage IGF responsiveness with age. A small decline of IGF-1 concentration was observed in serum of old mice, but the level still appeared to be supra-optimal to maintain normal cartilage PG synthesis over a culture period of 1 to 3 days. Moreover, PG synthesis was at least equally stimulated in patellar cartilage from 18-month-old mice compared to 3-month-old mice over a wide range of IGF-1 concentrations, and similar findings were obtained after stimulation with serum. In addition, we studied the capacity of IGF-1 or serum to induce recovery of PG synthesis in vitro after IL-1 exposure in vivo. In a 3-day culture period normal cartilage PG synthesis was stimulated to the same extent with serum or IGF-1, but recovery from IL-1 mediated suppression of PG synthesis was more pronounced with serum. This latter capacity was similar for serum of mice aged 3 or 18 months and was noted for both young and old cartilage. Our data show that retarded recovery of chondrocyte PG synthesis in old mice cannot be explained by age-related changes in IGF-1 availability and cartilage responses to IGF. They also indicate that serum factors other than IGF-1 are important for recovery, either alone or in combination with IGF-1.
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Research Interests: Rheumatology, Immunology, Cell Biology, Gene expression, Signal Transduction, and 15 moreMice, Female, Animals, Male, Mitogen Activated Protein Kinase, Clinical Sciences, Public health systems and services research, Analysis of Variance, Western blot, Transforming Growth Factor Beta, Sensitivity and Specificity, fibroblasts, Synovial membrane, reverse transcriptase polymerase chain reaction, and mRNA expression
ObjectiveMembers of the suppressor of cytokine signaling (SOCS) family are key negative intracellular regulators of cytokine and growth factor responses, including those that regulate immune responses in autoimmune disorders, such as... more
ObjectiveMembers of the suppressor of cytokine signaling (SOCS) family are key negative intracellular regulators of cytokine and growth factor responses, including those that regulate immune responses in autoimmune disorders, such as rheumatoid arthritis (RA). The aim of this study was to investigate modulation of T cell immunity for the treatment of experimental arthritis, via enhanced expression of SOCS‐3 in splenic antigen‐presenting cells (APCs) obtained after intravenous injection of adenovirus encoding SOCS‐3.MethodsDBA/1 mice were immunized with type II collagen, and adenovirus vectors were administered by intravenous injection before the clinical onset of collagen‐induced arthritis (CIA). Splenic cellular responses were analyzed by measuring cytokine production, using Luminex multi‐analyte technology. Th cell populations were analyzed by flow cytometry.ResultsSystemic delivery of adenovirus encoding SOCS‐3 resulted in enhanced transgene expression in splenic APCs, which led to decreased production of interleukin‐23 (IL‐23), IL‐6, and tumor necrosis factor α, but significantly higher production of antiinflammatory IL‐10, by these cells. Fluorescence‐activated cell sorting analysis showed increased numbers of splenic CD4+ T cells after SOCS‐3 treatment. In the presence of SOCS‐3–transduced APCs, however, purified splenic CD3+ T cells showed reduced antigen‐specific proliferation and a significant reduction in the production of interferon‐γ (−43%), IL‐4 (−41%), and IL‐17 (−70%). Interestingly, the altered splenic cellular responses were accompanied by a protective effect on CIA development, and histologic analysis of knee joints showed reduced joint inflammation and connective tissue destruction.ConclusionThis study demonstrates effective prevention of CIA after intravenously induced overexpression of SOCS‐3; this is probably caused by the generation of tolerogenic APCs, which have an inhibitory effect on Th1, Th2, and especially, Th17 cell activity.
Research Interests: Immunology, Immune response, Flow Cytometry, Medicine, Prevention, and 15 moreGene expression, Mice, Animals, Male, Connective tissue, Arthritis, Clinical Sciences, Cytokine, Immune system, Public health systems and services research, Growth Factor, Knee Joint, Interleukin, Interferon gamma, and Genetic Therapy
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We have used neutralizing antibodies raised against murine recombinant interleukin 1 (IL-1) to demonstrate a role for IL-1 in the cartilage destruction and inflammation of antigen induced arthritis. Ex vivo production of IL-1 was... more
We have used neutralizing antibodies raised against murine recombinant interleukin 1 (IL-1) to demonstrate a role for IL-1 in the cartilage destruction and inflammation of antigen induced arthritis. Ex vivo production of IL-1 was demonstrated in tissue cultures of joint cross sections shortly after arthritis induction. Neutralizing antimurine IL-1 antibodies identified the activity to be about 80% IL-1 alpha 24 h after onset of arthritis. In animals receiving a single injection of anti-IL-1 antisera at Day -3, cartilage proteoglycan synthesis suppression during the first 2 days of arthritis was prevented. Normal proteoglycan synthesis was maintained until Day 4 when anti-IL-1 antisera was given at Days -2, 0, and 2 or arthritis. Dose response experiments showed that the reduction in inflammation was insufficient to account for the clearcut reduction in cartilage proteoglycan synthesis inhibition. Our results demonstrate that IL-1 plays a role in cartilage pathology in murine antigen induced arthritis.
Research Interests: Rheumatology, Immunology, Medicine, Antibodies, Cartilage, and 15 moreMice, Pubmed, Animals, Immunotherapy, The, Arthritis, Clinical Sciences, Proteoglycans, Antibody, Public health systems and services research, Articular Cartilage Wear, Bovine Serum Albumin, Interleukin, Neutralization tests, and Synovial membrane
STAT proteins play an important role in cytokine signaling. Some investigators have reported preferential activation of STAT-1, and others have reported preferential activation of STAT-3, in response to endogenous interleukin-6 (IL-6), in... more
STAT proteins play an important role in cytokine signaling. Some investigators have reported preferential activation of STAT-1, and others have reported preferential activation of STAT-3, in response to endogenous interleukin-6 (IL-6), in patients with rheumatoid arthritis. The present study was undertaken to investigate synovial STAT-1 and STAT-3 activation in an experimental animal model of arthritis. Zymosan was injected intraarticularly into naive wild-type (WT), IL-6(-/-), and STAT-1(-/-) mice to induce arthritis. Western blots of synovial lysates were probed with phosphospecific antibodies to detect STAT-1/STAT-3 activation. Inflammation was assessed histologically. Synovial gene expression of the STAT-induced feedback inhibitors suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 in WT and STAT-1(-/-) mice was investigated by reverse transcriptase-polymerase chain reaction. STAT-3 was activated in inflamed synovium of WT mice throughout the course of disease, whereas activated STAT-1 was observed only during the chronic phase. In IL-6(-/-) mice, STAT activation was limited to STAT-3 on day 1. Although macrophage influx was not inhibited, disease went into remission after day 7 in IL-6(-/-) mice. STAT-1 deficiency resulted in exacerbation of chronic joint inflammation and granuloma formation. In STAT-1(-/-) mice, STAT-3 activation in the inflamed joints was unaltered as compared with WT mice. However, synovial SOCS-1, but not SOCS-3, gene expression was markedly reduced in STAT-1(-/-) mice. The results in the IL-6(-/-) mice suggest that STAT-3 is involved in the chronicity of ZIA. Exacerbation of arthritis in STAT-1(-/-) mice suggests an opposing effect of STAT-1, i.e., suppression of joint inflammation. The expression of SOCS-1 could be the underlying mechanism by which STAT-1 controls joint inflammation.
Research Interests: Immunology, Inflammation, Macrophages, Medicine, Gene expression, and 15 moreMice, Chronic Disease, Female, Animals, Male, Animal Model, Arthritis, Clinical Sciences, Cytokine, Neutrophils, DNA binding proteins, Interleukin, Acute Disease, Gene knockout, and reverse transcriptase polymerase chain reaction
Research Interests: Immunology, Inflammation, Image Analysis, Medicine, Osteoarthritis, and 15 moreCartilage, Mice, Animals, Male, Glycosaminoglycan, Clinical Sciences, Proteoglycans, Public health systems and services research, Articular Cartilage Wear, Age Factors, Knee Joint, Articular Cartilage, Interleukin, Patella, and Drug synergism
Research Interests: Endocrinology, Immunology, Biology, Medicine, Internal Medicine, and 15 moreCartilage, Mice, Animals, Clinical Sciences, In Vivo, Proteoglycans, Public health systems and services research, Articular Cartilage Wear, Growth Factor, Articular Cartilage, Insulin Like Growth Factor, Interleukin, Transforming Growth Factor, Drug synergism, and In Vitro Techniques
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Research Interests: Immunology, Flow Cytometry, Immunohistochemistry, Rheumatoid Arthritis, Medicine, and 15 moreGene expression, Mice, Animals, Male, Arthritis, Inflammatory disease, Clinical Sciences, mRna expression levels, Public health systems and services research, Gene Targeting, Knee Joint, Mononuclear Phagocyte System, Quantitative polymerase chain reaction, Genetic Therapy, and mRNA expression
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Research Interests: Inflammation, Medicine, Mice, Animals, Connective tissue, and 15 moreArthritis, NADPH oxidase, Articular Cartilage Wear, Matrix Metalloproteinases, Granuloma, Synovitis, Knee Joint, Knockout Mice, The American, Interleukin, Synovial membrane, Tissue inhibitor of metalloproteinases (TIMPs), nicotinamide adenine dinucleotide phosphate, Medical and Health Sciences, and Drug combinations
ABSTRACT Gene therapy applied in the joints of rheumatoid arthritis (RA) patients can provide a local production of biological drugs to prevent side effects that are encountered when administered systemically. Gene therapy using a... more
ABSTRACT Gene therapy applied in the joints of rheumatoid arthritis (RA) patients can provide a local production of biological drugs to prevent side effects that are encountered when administered systemically. Gene therapy using a promoter (the DNA region of the gene that controls the expression) that is disease-responsive can even confine the expression of the biological drug to episodes of disease flares. Disease-inducible gene therapy has already been successful in mouse models of RA (van de Loo et al. 2004). Recently, gene therapy was also applied successfully in a mouse model of osteoarthritis (OA) (Ruan et al. 2013). Our goal is to validate promoters that can be successful in human gene therapy for RA and OA. To identify genes with potential for human gene therapy, we analysed microarrays of synovial samples from 20 RA patients, 12 OA patients and 7 patients with no joint disease. The upregulation of the RA-associated genes was analysed in an in vitro model of inflammation, by stimulating human THP-1 monocytes with the TLR2 ligand Pam3Cys. Subsequently, the promoter regions of these genes were cloned in a lentiviral construct expressing the firefly luciferase reporter gene. THP-1 cells were transduced with the promoter constructs and stimulated for 6 hours with 100 ng/ml Pam3Cys or 10% serum from 9 RA patients and 4 healthy volunteers. The microarray analysis showed 8 genes that were upregulated at least 4-fold in RA synovium and 7 genes that were upregulated at least 2.5-fold in OA. The increased expression was confirmed by qPCR measurements. 6 out of the 8 RA genes were upregulated in the Pam3Cys-stimulated THP-1 cells. When THP-1 cells were transduced with the constructs containing the promoter regions of the selected genes expressing the luciferase reporter gene and stimulated with Pam3Cys, CXCL10 promoter activity increased 15.5-fold, the IL-8 promoter 26.5-fold, the PCDHB16 promoter 12-fold and the CXCL9 promoter 2.6-fold. After stimulation with human serum from RA patients or healthy volunteers, the promoters of CXCL9, IL-8, TSG-6, CXCL10 and ADAM28 showed a higher activity with RA patient serum. These results show that promoters of genes that are upregulated in RA and OA synovium are promising for developing disease-inducible gene therapy in human, which has already been shown to be successful in mice.
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Research Interests: Immunology, Inflammation, Medicine, Humans, Cartilage, and 15 moreCollagen, Mice, Animals, T cells, Cell Transplantation, Arthritis, Clinical Sciences, Public health systems and services research, Foot, Articular Cartilage Wear, Recombinant Proteins, Knee Joint, Chondrocyte, Interleukin, and collagen-induced arthritis
Background Cardiovascular risk is increased in rheumatoid arthritis (RA) patients. Algorithms for cardiovascular (CV) risk stratification as SCORE or Framingham’s score are used in the general population. Before they can be used for (CV)... more
Background Cardiovascular risk is increased in rheumatoid arthritis (RA) patients. Algorithms for cardiovascular (CV) risk stratification as SCORE or Framingham’s score are used in the general population. Before they can be used for (CV) risk assessment in RA, validation by calibration and updating of these algorithms, is needed. It would be an advantage if long lasting RA cohorts could be used for this purpose. However, it is unclear if cholesterol levels in frozen serum samples can be used. There is no data available regarding the validity of cholesterol measurements in samples after long-term storage (>10 years). Objectives To assess the stability of serum total cholesterol (TC), HDL- and LDL-cholesterol (HDL-c, LDL-c) over time, by estimating the effect of storage time on cholesterol levels in frozen serum samples of RA patients. Methods In the Nijmegen early RA cohort that started in 1985, non-fasting blood samples were taken annually during follow-up and stored at -20oC. After thawing in 2011, TC and HDL-c were measured enzymatically. LDL-c was calculated using the Friedewald formula. Serum samples from year 0, 1, 2, 3, 5, 7 and 10, during follow-up were used. The cohort was divided into 5 sub-cohorts, starting at 1985 up to 2009 covering 5 years of calendar time each. Per subcohort, 30 RA patients were randomly selected. The data were analyzed for differences between subcohorts in cholesterol levels using longitudinal regression (linear mixed models) while age, gender, smoking, BMI, blood pressure, statin use, DAS28 score, rheumatoid factor positivity at baseline, were considered potential confounders. Results In total, 1002 serum samples were used from 152 patients, evenly distributed across sub-cohorts, with a mean±SD age 54±13.8 years, 65% were female and 80% were rheumatoid factor positive. In all sub-cohorts, the course of cholesterol levels over time was non-linear. Age and gender were added to the regression model as covariates with BMI at baseline being the only confounder. There was a significant storage effect for LDL-c levels (p=0.047), but not for TC (fig. 1) and HDL-c (p=0.099 and p=0.175 respectively). The differences between the youngest and oldest subcohorts were -0.17 mmol/L/year for LDL-c, -0.17 mmol/L/year for TC, and -0.02 mmol/L/year for HDL-c. Conclusions There was no storage effect in frozen serum on HDL-c and TC, but there was a significant storage effect on LDL. Although the found effects per additional year of storage are small, over time this effect could accumulate to a clinically relevant change and a correction factor can adjust for this effect. Therefore, serum samples stored for longer periods of time under stable conditions can be considered suitable for cholesterol measurements. Disclosure of Interest None Declared
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Research Interests: Pharmacology, Technology, Computational Biology, Kinetics, Biology, and 15 moreGene Therapy, Rheumatoid Arthritis, Biological Sciences, Molecular, Disease, Promoter, Gene, Arthritis, Protein Expression, Side Effect, Disease Activity, Transgene Expression, Molecular Targeted Therapy, Matrix Metalloproteinase, and Medical and Health Sciences
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Research Interests: Immunology, Gene Therapy, Cytokines, Inflammation, Medicine, and 15 moreBiological Sciences, Genetic Enhancement, Mice, Animals, Gene transfer techniques, Male, Arthritis, Glycoproteins, Cytokine, Protein Binding, Knee Joint, Autoantibodies, Interleukin, Genetic Therapy, and Medical and Health Sciences
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A prominent role of Toll-like receptor 4 (TLR4) in arthritis is emerging. TLR4 is functional in immune cells and stromal cells. The aim was to investigate the involvement of TLR4 in bone marrow (BM)-derived and resident cells in... more
A prominent role of Toll-like receptor 4 (TLR4) in arthritis is emerging. TLR4 is functional in immune cells and stromal cells. The aim was to investigate the involvement of TLR4 in bone marrow (BM)-derived and resident cells in arthritis. Reciprocal sex-mismatched BM transplantation was performed between IL-1Ra(-/-)TLR4(+/+) and IL-1Ra(-/-)TLR4(-/-) double knockout animals in Balb/c background. Arthritis was assessed macroscopically and by histopathology. Immunity was evaluated by splenic cytokine production and flow cytometry in draining lymph node (DLN) cells. Arthritis progression was reduced to a similar extent in animals lacking TLR4 on BM-derived, resident cells or both. Histology revealed that joint inflammation was partially TLR4-dependent in either BM-derived or resident cells. TLR4 plays an additive role in BM-derived and resident cells in promoting cartilage erosion. By contrast, TLR4 was equally important in BM-derived and resident cells in mediating bone erosion. Systemically, TLR4 in both BM-derived and resident cells contributed to IL-17 production by splenic T-cells, whereas in the DLNs of arthritic joints this was not the case. Interestingly, in DLN, the dominant cells producing IL-17 were CD4 negative, and cell numbers were determined by TLR4 in the BM-derived cells. TLR4 is necessary in both BM-derived and resident cells for full-blown joint swelling, inflammation and bone erosion. Furthermore, TLR4 on BM-derived and tissue-resident cells show an additive effect in cartilage destruction. Interestingly, TLR4 on BM-derived and tissue-resident cells are both required for IL-17 production in spleen, but only in BM-derived cells in DLN.
Research Interests: Pathology, Immunology, Inflammation, Medicine, Toll like receptor signaling, and 15 moreSpleen, Mice, Female, Animals, Male, Bone marrow, Arthritis, Clinical Sciences, Bone Marrow Transplantation, Public health systems and services research, Articular Cartilage Wear, Bone Marrow Cells, Interleukin, Lymph nodes, and stifle
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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-18Rβ) with unknown function has recently been identified. This... more
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-18Rβ) with unknown function has recently been identified. This study examined the ability of sIL-18Rβ to inhibit IL-18 biological activities and to modulate immune responses during collagen-induced arthritis (CIA).Methods:Adenoviruses encoding sIL-18Rβ 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-18Rβ in collagen-immunised mice led to enhanced transgene expression in splenic antigen-presenting cells (APC). A co-culture of these sIL-18Rβ-transduced APC with purified splenic CD3+ T cells led to a marked inhibition of IL-18-induced IFNγ, IL-4 and IL-17 production by CD3+ T cells. Remarkably, systemic treatment with Ad5.sIL-18Rβ 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 IFNγ (−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-18Rβ as a novel IL-18 inhibitor, which promotes CIA after intravenous overexpression by affecting Treg levels and supporting a T helper type 17 response.
Research Interests: Immunology, Immune response, Flow Cytometry, Cytokines, Rheumatoid Arthritis, and 15 moreMedicine, Mice, Animals, Male, Immunomodulation, Arthritis, Clinical Sciences, Cytokine, Immune system, Public health systems and services research, Biological activity, Knee Joint, Interleukin, Interferon gamma, and Regulatory T cell
Research Interests: Immunology, Biology, Gene Therapy, Inflammation, Gene expression, and 15 moreBiological Sciences, Humans, Genetic Enhancement, Collagen, Animals, Arthritis, Gene Delivery, Lipopolysaccharides, Articular Cartilage Wear, Enhancer, Knee Joint, Gene Transfer, Interleukin, Genetic Therapy, and Medical and Health Sciences
Research Interests: Immunology, Medicine, Gene expression, Signal Transduction, Mice, and 15 moreAnimals, Male, NF-kappa B, Proteins, Arthritis, Solubility, Clinical Sciences, Molecular cloning, Fibroblast, Public health systems and services research, Tumor necrosis factor-alpha, Knee Joint, Interleukin, Genetic Therapy, and collagen-induced arthritis
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Research Interests: Immunology, Inflammation, Medicine, Immunity, Arthritis, and 3 moreCells, Synovitis, and Innate Immune System
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Research Interests: Immunology, Gene Therapy, Treatment, Comparative Study, Medicine, and 15 moreVirus, Mice, Animals, Male, Cytomegalovirus, Animal Model, Arthritis, VECTOR, Clinical Sciences, Public health systems and services research, Knee Joint, Interleukin, interleukin-1 receptor antagonist, Genetic Therapy, and collagen-induced arthritis
Research Interests: Gene Therapy, Medicine, Biological Sciences, Genetic Enhancement, Mice, and 13 moreAnimals, Male, Arthritis, Gene Delivery, Knee Joint, Tropism, Integrin, Synovial membrane, Genetic Therapy, reverse transcriptase polymerase chain reaction, collagen-induced arthritis, Viral Vector, and Medical and Health Sciences
The mechanism underlying the chronic and intermittent course of rheumatoid arthritis is not elucidated. In the present study, the role of interleukin 1 (IL-1) was investigated in exacerbations of antigen-induced arthritis in mice. A... more
The mechanism underlying the chronic and intermittent course of rheumatoid arthritis is not elucidated. In the present study, the role of interleukin 1 (IL-1) was investigated in exacerbations of antigen-induced arthritis in mice. A flare-up of smoldering inflammation (weeks 3 to 4 of antigen-induced arthritis) was inducible by injection of a small amount of methylated bovine serum albumin into the hypersensitive knee joint. Immunohistochemistry showed IL-1 expression in the synovial lining layer and in focal areas of the inflamed synovium during the flare-up. IL-1 was also measured in 1-hour culture supernatant of synovial tissue taken during the flare-up by a bioassay. The expression of both immunoreactive and bioactive IL-1 in the hypersensitive joint peaked around 6 hours after antigen (2 micrograms of methylated bovine serum albumin) injection and declined thereafter. Antigen rechallenge induced an acute joint swelling of the arthritic joint but not in the naive joint of the sensitized mouse, yet synovia of both joints produced IL-1 after antigen injection. Remarkably, a single intravenous injection of rabbit anti-IL-1 alpha and -beta antibodies 1 hour before antigen rechallenge neutralized IL-1 in the joint. Anti-IL-1 treatment significantly reduced the antigen-induced joint swelling (30 to 40%) but did not affect the profound influx of polymorphonuclear cells in the onset of the exacerbation. However, a profound relief of the inflammation (synovitis) was obtained by IL-1 blockade on day 4 of the exacerbation. Chondrocyte proteoglycan synthesis was markedly suppressed in the antigen-challenged naive knee joints suggesting that this was a direct IL-1 effect as the inflammation was insignificant. Anti-IL-1 treatment was able to maintain chondrocyte proteoglycan synthesis in the antigen-rechallenged joint, which was highly suppressed in the control group. Furthermore, the enhanced proteoglycan breakdown in the antigen-rechallenged joints was significantly decreased in the anti-IL-1 group. We concluded that IL-1 is an important mediator in exacerbations of murine arthritis, and amelioration of cartilage pathology was obtained with anti-IL-1 antibody treatment.
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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... more
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.
Research Interests: Biology, Gene Therapy, Cytokines, Medicine, Biological Sciences, and 15 moreGenetic Enhancement, Mice, Animals, Male, Promoter, Luciferase, Arthritis, Physiological Response, Lipopolysaccharides, Enhancer, Gene Expression Regulation, Interleukin, Gene knockout, Genetic Therapy, and Medical and Health Sciences
Background No disease-modifying drugs are available for osteoarthritis (OA). Although the aetiology is still unknown, inflammation is indicated to be involved in the disease pathogenesis in many patients. Gene therapy can enable... more
Background No disease-modifying drugs are available for osteoarthritis (OA). Although the aetiology is still unknown, inflammation is indicated to be involved in the disease pathogenesis in many patients. Gene therapy can enable transduced joint cells to express an anti-inflammatory biological drug with long-lasting bioavailability. In addition, off-target effects can be further reduced by regulating the expression using a disease-responsive promoter that is only active during disease flares. Clinical studies in chronic diseases with anti-inflammatory recombinant Interleukin-10 (IL-10) are impeded by its short half-life. Disease-inducible gene therapy might therefore be the ideal system to provide IL-10 in an OA joint in a regulated fashion. The objective of this study was to test the feasibility of this approach in a human in-vitro model of the synovial membrane. Methods Cell suspensions of digested joint biopsies were mixed with Matrigel to form micromasses, in which the cells migrate to the surface. The micromasses were transduced with a lentiviral vector containing the IL-10 gene under control of the inflammation-sensitive CXCL10 promoter (CXCL10p-IL10) or the constitutive active PGK promoter (PGK-IL10). Subsequently, micromasses were stimulated with inflammatory triggers Tumour Necrosis Factor-alpha (TNFα) and lipopolysaccharide (LPS). Gene expression effects were determined by qPCR and the production of cytokines by the synovial micromasses was measured by multiplex ELISA. Results In 1 week cultured micromasses, migrated cells formed a lining containing both synovial fibroblasts and macrophages similar to that seen in the synovium. This lining could be efficiently targeted by lentiviruses. After stimulation with LPS and TNFα, the production of CXCL10-controlled IL-10 expression increased by 9.7-fold and 7.2-fold respectively. The IL-10 production after transduction with PGK-IL10 was not influenced by proinflammatory stimulation. The induced levels of IL-10 were high enough to significantly reduce the production of IL-1β and IL-6 by the micromasses. The anti-inflammatory effect of CXCL10-controlled IL-10 might be mediated by SOCS3. Recombinant IL-10 increased SOCS3 expression by 3.0-fold 2 hour after stimulation. Conclusions This study on primary human synovial micromasses suggest that the CXCL10p-IL-10 gene therapeutic approach to suppress the innate cytokine response in the osteoarthritic joint might be a feasible approach for local treatment of OA.