RESUMO
OBJECTIVE: Increased Wisp1 expression was previously reported in experimental and human osteoarthritis (OA). Moreover, adenoviral overexpression of Wisp1 in naïve mouse knee joints resulted in early OA-like cartilage lesions. Here, we determined how the matricellular protein WISP1 is involved in the pathology that occurs in the complex osteoarthritic environment with aging and experimental OA in wild type (WT) and Wisp1-/- mice. METHODS: WT and Wisp1-/- mice were aged or experimental OA was induced with intraarticular collagenase injection, destabilization of the medial meniscus (DMM) or anterior cruciate ligament transection (ACLT). Joint pathology was assessed using histology and microCT. Protease expression was evaluated with qRT-PCR and activity was determined by immunohistochemical staining of the aggrecan neoepitope NITEGE. Protease expression in human end-stage OA synovial tissue was determined with qRT-PCR after stimulation with WISP1. RESULTS: With aging, spontaneous cartilage degeneration in Wisp1-/- was not decreased compared to their WT controls. However, we observed significantly decreased cartilage degeneration in Wisp1-/- mice after induction of three independent experimental OA models. While the degree of osteophyte formation was comparable between WT and Wisp1-/- mice, increased cortical thickness and reduced trabecular spacing was observed in Wisp1-/- mice. In addition, we observed decreased MMP3/9 and ADAMTS4/5 expression in Wisp1-/- mice, which was accompanied by decreased levels of NITEGE. In line with this, stimulation of human OA synovium with WISP1 increased the expression of various proteases. CONCLUSIONS: WISP1 plays an aggravating role in the development of post-traumatic experimental OA.
Assuntos
Artrite Experimental/genética , Proteínas de Sinalização Intercelular CCN/genética , Cartilagem Articular/metabolismo , Osteoartrite do Joelho/genética , Peptídeo Hidrolases/genética , Proteínas Proto-Oncogênicas/genética , Animais , Ligamento Cruzado Anterior/cirurgia , Artrite Experimental/diagnóstico por imagem , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/patologia , Colagenases , Modelos Animais de Doenças , Humanos , Injeções Intra-Articulares , Meniscos Tibiais/cirurgia , Camundongos , Camundongos Knockout , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Osteófito , Peptídeo Hidrolases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Membrana Sinovial/metabolismo , Via de Sinalização Wnt , Microtomografia por Raio-XRESUMO
OBJECTIVE: Low-density lipoproteins (LDL) in inflamed synovium is oxidized and taken-up by synoviocytes. In this study, we investigate whether direct injection of oxidized LDL (oxLDL) into a normal murine knee joint induces joint pathology and whether synovial macrophages are involved in that process. DESIGN: Synovium was obtained from end-stage osteoarthritis (OA) patients in order to analyze LDL-uptake. Murine knee joints were injected five consecutive days with oxLDL, LDL, or vehicle (phosphate buffered saline (PBS)). This procedure was repeated in mice depleted of synovial macrophages by intra-articular injection of clodronate liposomes 7 days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry, flow cytometry (FCM) and synovial RNA expression and protein production. RESULTS: Synovial tissue of OA patients showed extensive accumulation of apolipoprotein B. Multiple injections of oxLDL in murine knee joints significantly increased TGF-ß activity in synovial wash-outs, but did not induce catabolic or inflammatory processes. In contrast, repeated injections of oxLDL in macrophage-depleted knee joints led to increased synovial thickening in combination with significantly upregulated protein and RNA levels of CCL2 and CCL3. FCM-analyses revealed increased presence of monocytes and neutrophils in the synovium, which was confirmed by immunohistochemistry. Also protein levels of S100A8/A9 were significantly increased in synovial wash-outs of oxLDL-injected joints, as was expression of aggrecanase-induced neo-epitopes. Interestingly, no raise in TGF-ß concentrations was measured in macrophage-depleted joints. CONCLUSIONS: OxLDL can affect joint pathology, since synovial macrophages promote anabolic processes after oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity combined with increased influx of monocytes and neutrophils.
Assuntos
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/fisiologia , Líquido Sinovial/citologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Humanos , Injeções Intra-Articulares , Lipoproteínas LDL/administração & dosagem , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite/metabolismo , Líquido Sinovial/fisiologiaRESUMO
OBJECTIVE: Interleukin-1 (IL-1) is an alleged important cytokine in osteoarthritis (OA), although the exact contribution of IL-1 to joint destruction remains unclear. Here we investigated the involvement of IL-1α and IL-1ß in joint pathology during collagenase-induced OA (CiOA). METHODS: CiOA was induced in wild type (WT) and IL-1αß-/- mice. Additionally, IL-1 signaling was inhibited in WT mice with CiOA using osmotic pumps containing IL-1RA. Joint pathology was assessed using histology. Activity of cartilage-degrading enzymes was determined using antibodies against aggrecan neo-epitopes VDIPEN and NITEGE. Synovial gene expression was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Serum protein levels were measured with Luminex or enzyme-linked immunosorbent assay (ELISA). RESULTS: Synovial IL-1ß expression was strongly elevated 7 days after induction of CiOA in WT mice but decreased afterwards, whereas S100A8/A9, previously described to aggravate OA, remained elevated for 21 days. Remarkably, synovial inflammation was comparable between WT and IL-1αß-/- mice on day 7 of CiOA. In line, synovial mRNA expression of genes involved in IL-1 signaling and inflammatory mediators was comparable between WT and IL-1αß-/- mice, and serum levels for Keratinocyte Chemoattractant (KC)/IL-6/S100A8/S100A9/IL-10 were equal. Synovial matrix metalloproteinase (MMP)/aggrecanase expression and activity in cartilage was not different in WT and IL-1αß-/- mice on day 7 of CiOA. Cartilage destruction on day 42 was not different between WT and IL-1αß-/- mice, which was supported by our finding that IL-1RA treatment in WT mice with CiOA did not alter joint destruction. CONCLUSIONS: IL-1α and IL-1ß are not involved in synovial inflammation and cartilage destruction during CiOA, implicating that other mediators are responsible for the joint damage.
Assuntos
Cartilagem/patologia , Colagenases/metabolismo , Interleucina-1/metabolismo , Osteoartrite/metabolismo , Sinovite/metabolismo , Animais , Feminino , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoartrite/etiologia , Osteoartrite/patologia , Reação em Cadeia da Polimerase em Tempo Real , Membrana Sinovial/metabolismo , Sinovite/etiologia , Sinovite/patologia , TranscriptomaRESUMO
OBJECTIVE: Alarmins S100A8 and S100A9 are major products of activated macrophages regulating cartilage damage and synovial activation during murine and human osteoarthritis (OA). In the current study, we investigated whether S100A8 and S100A9 are involved in osteophyte formation during experimental OA and whether S100A8/A9 predicts osteophyte progression in early human OA. METHODS: OA was elicited in S100A9-/- mice in two experimental models that differ in degree of synovial activation. Osteophyte size, S100A8, S100A9 and VDIPEN neoepitope was measured histologically. Chondrogenesis was induced in murine mesenchymal stem cells in the presence of S100A8. Levels of S100A8/A9 were determined in plasma of early symptomatic OA participants of the Cohort Hip and Cohort Knee (CHECK) cohort study and osteophytes measured after 2 and 5 years. RESULTS: Osteophyte size was drastically reduced in S100A9-/- mice in ligaments and at medial femur and tibia on days 21 and 42 of collagenase-induced OA, in which synovial activation is high. In contrast, osteophyte size was not reduced in S100A9-/- mice during destabilised medial meniscus OA, in which synovial activation is scant. S100A8 increased expression and activation of matrix metalloproteinases during micromass chondrogenesis, thereby possibly increasing cartilage matrix remodelling allowing for larger osteophytes. Interestingly, early symptomatic OA participants of the CHECK study with osteophyte progression after 2 and 5 years had elevated S100A8/A9 plasma levels at baseline, while C-reactive protein, erythrocyte sedimentation rate and cartilage oligomeric matrix protein were not elevated at baseline. CONCLUSIONS: S100A8/A9 aggravate osteophyte formation in experimental OA with high synovial activation and may be used to predict osteophyte progression in early symptomatic human OA.
Assuntos
Artrite Experimental/metabolismo , Calgranulina A/fisiologia , Calgranulina B/fisiologia , Osteoartrite/metabolismo , Osteófito/metabolismo , Animais , Artrite Experimental/complicações , Artrite Experimental/patologia , Biomarcadores/metabolismo , Calgranulina A/deficiência , Cartilagem Articular/enzimologia , Cartilagem Articular/fisiopatologia , Condrogênese/fisiologia , Progressão da Doença , Feminino , Humanos , Masculino , Metaloproteinases da Matriz/biossíntese , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Osteoartrite/complicações , Osteoartrite/patologia , Osteófito/etiologia , Osteófito/patologia , Membrana Sinovial/metabolismo , Regulação para Cima/fisiologiaRESUMO
OBJECTIVE: A relation between osteoarthritis (OA) and increased cholesterol levels is apparent. In the present study we investigate OA pathology in apolipoprotein E (ApoE)(-)(/-) mice with and without a cholesterol-rich diet, a model for high systemic low density lipoprotein (LDL) cholesterol levels independent of weight. METHOD: Wild type (WT), Apoe(-)(/-), S100a9(-/-) and Apoe(-)(/-)S100a9(-/-) mice (C57BL/6 background) received a standard or cholesterol-rich diet. Experimental OA was induced by intra-articular injection of collagenase and animals were sacrificed at day 10 and day 36. RESULTS: Although minimal differences in cartilage damage were found between the WT and ApoE(-)(/-) mice, increased synovial thickening was found in the latter. Thirty-six days after OA-induction, ApoE(-)(/-) mice on a standard diet showed increased ectopic bone formation, particularly at the medial collateral ligament, compared with OA in WT mice. Furthermore, a significant increase in synovial gene expression of both S100a8 and S100a9 and S100A8/S100A9 protein levels was found in ApoE(-)(/-) mice, suggesting an activated inflammatory status of synovial cells. In both ApoE(-)(/-) and WT mice, addition of a cholesterol-rich diet resulted in excessive bone formation in the medial collateral ligament at late-time-point OA. Interestingly, at the early time point, proteoglycan deposition was already significantly increased in ApoE(-)(/-) mice compared with WT mice. Mice deficient for both ApoE and S100a9 also showed increased ectopic bone formation, but not synovial activation, suggesting a role for S100-proteins in cholesterol-mediated synovial activation. CONCLUSIONS: Increased cholesterol levels strongly elevate synovial activation and ectopic bone formation in early-stage collagenase-induced OA.
Assuntos
Artrite Experimental/sangue , LDL-Colesterol/sangue , Ossificação Heterotópica/sangue , Osteoartrite/sangue , Sinovite/sangue , Animais , Apolipoproteínas E/sangue , Apolipoproteínas E/deficiência , Artrite Experimental/complicações , Calgranulina A/fisiologia , Calgranulina B/fisiologia , Colesterol na Dieta/administração & dosagem , Dieta Hiperlipídica/efeitos adversos , Feminino , Erros Inatos do Metabolismo Lipídico/sangue , Erros Inatos do Metabolismo Lipídico/complicações , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ossificação Heterotópica/etiologia , Osteoartrite/complicações , Sinovite/etiologiaRESUMO
OBJECTIVES: In osteoarthritis (OA) chondrocytes surrounding lesions express elevated bone morphogenetic protein 2 (BMP2) levels. To investigate the functional consequence of chondrocyte-specific BMP2 expression, we made a collagen type II dependent, doxycycline (dox)-inducible BMP2 transgenic mouse and studied the effect of elevated BMP2 expression on healthy joints and joints with experimental OA. METHODS: We cloned a lentivirus with BMP2 controlled by a tet-responsive element and transfected embryos of mice containing a collagen type II driven cre-recombinase and floxed rtTA to gain a mouse expressing BMP2 solely in chondrocytes and only upon dox exposure (Col2-rtTA-TRE-BMP2). Mice were treated with dox to induce elevated BMP2 expression. In addition, experimental OA was induced (destabilisation of the medial meniscus model) with or without dox supplementation and knee joints were isolated for histology. RESULTS: Dox treatment resulted in chondrocyte-specific upregulation of BMP2 and severely aggravated formation of osteophytes in experimental OA but not in control mice. Moreover, elevated BMP2 levels did not result in alterations in articular cartilage of young healthy mice, although BMP2-exposure did increase VDIPEN expression in the articular cartilage. Strikingly, despite apparent changes in knee joint morphology due to formation of large osteophytes there were no detectible differences in articular cartilage: none with regard to structural damage nor in Safranin O staining intensity when comparing destabilisation of the medial meniscus with or without dox exposure. CONCLUSIONS: Our data show that chondrocyte-specific elevation of BMP2 levels does not alter the course of cartilage damage in an OA model in young mice but results in severe aggravation of osteophyte formation.
Assuntos
Artrite Experimental/genética , Proteína Morfogenética Óssea 2/genética , Cartilagem Articular/patologia , Condrócitos/metabolismo , Osteoartrite/genética , Osteófito/diagnóstico por imagem , RNA Mensageiro/metabolismo , Joelho de Quadrúpedes/diagnóstico por imagem , Animais , Artrite Experimental/diagnóstico por imagem , Artrite Experimental/patologia , Proteína Morfogenética Óssea 2/metabolismo , Meniscos Tibiais/cirurgia , Camundongos , Camundongos Transgênicos , Osteoartrite/diagnóstico por imagem , Osteoartrite/patologia , Radiografia , Joelho de Quadrúpedes/patologia , Regulação para CimaRESUMO
OBJECTIVES: Alarmins S100A8/A9 regulate pathology in experimental osteoarthritis (OA). Paquinimod is an immunomodulatory compound preventing S100A9 binding to TLR-4. We investigated the effect of paquinimod on experimental OA and human OA synovium. MATERIALS AND METHODS: Two OA mouse models differing in level of synovial activation were treated prophylactic with paquinimod. Synovial thickening, osteophyte size and cartilage damage were measured histologically, using an arbitrary score, adapted Pritzker OARSI score or imaging software, respectively. Human OA synovia were stimulated with S100A9, with or without paquinimod. RESULTS: Paquinimod treatment of collagenase-induced OA (CIOA) resulted in significantly reduced synovial thickening (57%), osteophyte size at the medial femur (66%) and cruciate ligaments (67%) and cartilage damage at the medial tibia (47%) and femur (75%; n=7, untreated n=6). In contrast, paquinimod did not reduce osteophyte size and reduced cartilage damage at one location only in destabilised medial meniscus, an OA model with considerably lower synovial activation compared with CIOA. In human OA synovium, paquinimod blocked proinflammatory (interleukin (IL)-6, IL-8, tumour necrosis factor-α) and catabolic (matrix metalloproteinases 1 and 3) factors induced by S100A9 (n=5). CONCLUSIONS: Prophylactic treatment of paquinimod reduces synovial activation, osteophyte formation and cartilage damage in experimental OA with high synovial activation (CIOA) and ameliorates pathological effects of S100A9 in OA synovium ex vivo.
Assuntos
Artrite Experimental/prevenção & controle , Calgranulina B/efeitos dos fármacos , Cartilagem Articular/patologia , Quinolinas/farmacologia , Membrana Sinovial/patologia , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Calgranulina B/metabolismo , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Colagenases/toxicidade , Modelos Animais de Doenças , Humanos , Imunossupressores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismoRESUMO
OBJECTIVE: Pain is the main problem for patients with osteoarthritis (OA). Pain is linked to inflammation, but in OA a subset of patients suffers from pain without inflammation, indicating an alternative source of pain. Nerve Growth Factor (NGF) inhibition is very efficient in blocking pain during OA, but the source of NGF is unclear. We hypothesize that damaged cartilage in OA releases Transforming Growth Factor-ß (TGF-ß), which in turn stimulates chondrocytes to produce NGF. DESIGN: Murine and human chondrocyte cell lines, primary bovine and human chondrocytes, and cartilage explants from bovine metacarpal joints and human OA joints were stimulated with TGF-ß1 and/or Interleukin-1 (IL-1)ß. We analyzed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1, Smad2/3 or Smad1/5/8 signaling to identify the TGF-ß pathway inducing NGF. RESULTS: NGF expression was consistently induced in higher levels by TGF-ß than IL-1 in all of our experiments: murine, bovine and human origin, in cell lines, primary chondrocytes and explants cultures. TAK1 inhibition consistently reduced TGF-ß-induced NGF whereas it fully blocked IL-1ß-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-ß-induced NGF expression. Despite the large variation in basal NGF in human OA samples (mRNA and histology), TGF-ß exposure led to a consistent high level of NGF induction. CONCLUSION: We show for the first time that TGF-ß induces NGF expression in chondrocytes, in a ALK5-Smad2/3 dependent manner. This reveals a potential alternative non-inflammatory source of pain in OA.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Interleucina-1beta/farmacologia , Fator de Crescimento Neural/efeitos dos fármacos , Osteoartrite/metabolismo , Dor/metabolismo , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Cartilagem Articular/metabolismo , Bovinos , Linhagem Celular , Condrócitos/metabolismo , Humanos , Camundongos , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Osteoartrite/complicações , Osteoartrite/genética , Dor/etiologia , Dor/genética , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/efeitos dos fármacos , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteína Smad2/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/efeitos dos fármacos , Proteína Smad3/genética , Proteína Smad3/metabolismoRESUMO
OBJECTIVES: To explore the association between S100A8/A9 serum levels with clinical and structural characteristics of patients with established knee, hip, or hand osteoarthritis (OA). METHOD: A cross-sectional exploratory study was conducted with 162 OA patients. Measures for pain, stiffness, and function included the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) questionnaire or the Australian Canadian Osteoarthritis Hand (AUSCAN) Index and for structural abnormalities, osteophytes and joint space narrowing grades. The association between S100A8/A9 and clinical or structural characteristics was analysed using linear regression or logistic regression where appropriate. RESULTS: The mean age of the OA patients was 56 years, 71% were female, and 61% had a Kellgren and Lawrence (K&L) score ≥ 2. The serum S100A8/A9 level did not differ between knee, hip, and hand OA patients and no association was found between serum S100A8/A9 and clinical characteristics. The serum S100A8/A9 level was negatively associated with the sum score of osteophytes after adjusting for sex and body mass index (BMI) [adjusted ß -0.015, 95% confidence interval (CI) -0.030 to 0.001, p = 0.062] and positively associated with erythrocyte sedimentation rate (ESR) > 12 mm/h (adjusted OR 1.002, 95% CI 1.000-1.004 p = 0.049) for each increase in S100A8/A9 of 1 ng/mL. For hand OA patients, a negative association of S100A8/A9 with sum score of joint space narrowing was found (adjusted ß -0.007, 95% CI -0.016 to 0.001, p = 0.099). CONCLUSIONS: The results from this cross-sectional exploratory study do not support an important role for serum S100A8/A9 levels as a biomarker for clinical and structural characteristics in established knee, hip, and hand OA patients. The inverse association with structural abnormalities and the positive association with ESR may reflect inflammatory synovial processes in patients with OA before structural abnormalities occur.
Assuntos
Calgranulina A/imunologia , Calgranulina B/imunologia , Osteoartrite do Quadril/imunologia , Osteoartrite do Joelho/imunologia , Biomarcadores/sangue , Calgranulina A/sangue , Calgranulina B/sangue , Estudos Transversais , Feminino , Articulação da Mão/imunologia , Articulação da Mão/metabolismo , Articulação da Mão/patologia , Articulação do Quadril/imunologia , Articulação do Quadril/metabolismo , Articulação do Quadril/patologia , Humanos , Articulação do Joelho/imunologia , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Masculino , Pessoa de Meia-Idade , Osteoartrite do Quadril/metabolismo , Osteoartrite do Quadril/patologia , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologiaRESUMO
OBJECTIVE: Synovitis is evident in a substantial subpopulation of patients with osteoarthritis (OA) and is associated with development of pathophysiology. Recently we have shown that adipose-derived stem cells (ASC) inhibit joint destruction in collagenase-induced experimental OA (CIOA). In the current study we explored the role of synovitis and alarmins S100A8/A9 in the immunomodulatory capacity of ASCs in experimental OA. METHOD: CIOA, characterized by synovitis, and surgical DMM (destabilization of medial meniscus) OA were treated locally with ASCs. Synovial activation, cartilage damage and osteophyte size were measured on histological sections. Cytokines in synovial washouts and serum were determined using Luminex or enzyme-linked immunosorbent assay (S100A8/A9), mRNA levels with reverse-transcriptase (RT)-qPCR. RESULTS: Local administration of ASCs at various time-points (days 7 or 14) after DMM induction had no effect on OA pathology. At day 7 of CIOA, already 6 h after ASC injection mRNA expression of pro-inflammatory mediators S100A8/A9, interleukin-1beta (IL-1ß) and KC was down-regulated in the synovium. IL-1ß protein, although low, was down-regulated by ASC-treatment of CIOA. S100A8/A9 protein levels were very high at 6 and 48 h and were decreased by ASC-treatment. The protective action of ASC treatment in CIOA was only found when high synovial inflammation was present at the time of deposition which was reflected by high serum S100A8/A9 levels. Finally, successful treatment resulted in significantly lower levels of serum S100A8/A9. CONCLUSION: Our study indicates that synovial activation rapidly drives anti-inflammatory and protective effects of intra-articularly deposited ASCs in experimental OA which is reflected by decreased S100A8/A9 levels.
Assuntos
Artrite Experimental/terapia , Calgranulina A/sangue , Calgranulina B/sangue , Meniscos Tibiais/cirurgia , Osteoartrite do Joelho/terapia , RNA Mensageiro/genética , Transplante de Células-Tronco/métodos , Membrana Sinovial/metabolismo , Tecido Adiposo/citologia , Animais , Calgranulina A/genética , Calgranulina B/genética , Cartilagem Articular/metabolismo , Colagenases/toxicidade , Modelos Animais de Doenças , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Camundongos , Osteoartrite do Joelho/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Sinovite/metabolismo , Sinovite/terapiaRESUMO
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.
Assuntos
Artrite Experimental/terapia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína S/genética , Proteínas Proto-Oncogênicas/agonistas , Receptores Proteína Tirosina Quinases/agonistas , Adenoviridae/genética , Animais , Artrite Experimental/genética , Artrite Experimental/patologia , Citocinas/genética , Citocinas/metabolismo , Terapia Genética/métodos , Injeções Intra-Articulares , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Proteína S/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais/imunologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Células Th1/imunologia , Células Th1/patologia , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
OBJECTIVE: Fibrosis is a major contributor to joint stiffness in osteoarthritis (OA). We investigated several factors associated with the persistence of transforming growth factor beta (TGF-ß)-induced fibrosis and whether these factors also play a role in OA-related fibrosis. DESIGN: Mice were injected intra-articularly (i.a.) with an adenovirus encoding either TGF-ß or connective tissue growth factor (CTGF). In addition, we induced OA by i.a. injection of bacterial collagenase into the right knee joint of C57BL/6 mice. mRNA was isolated from the synovium for Q-PCR analysis of the gene expression of various extracellular matrix (ECM) components, ECM degraders, growth factors and collagen cross-linking-related enzymes. Sections of murine knee joints injected with Ad-TGF-ß or Ad-CTGF or from experimental OA were stained for lysyl hydroxylase 2 (LH2). The number of pyridinoline cross-links per triple helix collagen in synovium biopsies was determined with high-performance liquid chromatography (HPLC). RESULTS: Expression of collagen alpha-1(I) chain precursor (Col1a1), tissue inhibitor of metalloproteinases 1 (TIMP1) and especially procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2b (Plod2b) were highly upregulated by TGF-ß but not by CTGF. Elevated expression of Plod2b mRNA was associated with high lysyl hydroxylase 2 (LH2) protein staining after TGF-ß overexpression and in experimental OA. Furthermore, in experimental OA the number of hydroxypyridinoline cross-links was significant increased compared to control knee joints. CONCLUSIONS: Our data show that elevated LH2b expression is associated with the persistent nature of TGF-ß-induced fibrosis. Also in experimental OA, LH2b expression as well as the number of hydroxypyridinoline cross-link were significantly upregulated. We propose that LH2b, and the subsequent increase in pyridinoline cross-links, is responsible for the persistent fibrosis in experimental OA.
Assuntos
Aminoácidos/metabolismo , Osteoartrite do Joelho/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Membrana Sinovial/patologia , Animais , Artrite Experimental , Cromatografia Líquida , Colágeno/genética , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Matriz Extracelular/genética , Fibrose , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estabilidade de RNA , Joelho de Quadrúpedes/patologia , Membrana Sinovial/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
OBJECTIVES: We investigated whether Abatacept might reduce proinflammatory cytokine production by macrophages upon contact with cytokine activated T cells and/or stimulation with TLR ligands. METHODS: Macrophages and cytokine stimulated T cells (Tck) were added together in the presence of Abatacept or a control Ig, with or without TLR ligands. The production of cytokines was determined by luminex. RESULTS: Abatacept reduced Tck-induced production of TNFa by macrophages. Tck and TLR ligands synergistically induced the production of proinflammatory cytokines by macrophages, especially IL-12p70. The production of IL-12p70 coincided with the production of IFNg, which were both reduced in the presence of Abatacept. CONCLUSIONS: Tck induce the production of TNFa by macrophages and facilitate the highly increased production of proinflammatory cytokines in the presence of TLR ligands. Abatacept was shown to potently suppress these pathways suggesting that its role may extend beyond antigen specific T cell mediated effector function.
Assuntos
Imunoconjugados/farmacologia , Imunossupressores/farmacologia , Macrófagos/efeitos dos fármacos , Linfócitos T/imunologia , Receptores Toll-Like/imunologia , Abatacepte , Comunicação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Citocinas/imunologia , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-12/biossíntese , Ligantes , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossínteseRESUMO
OBJECTIVE: To review the literature on the role and regulation of chondrocyte terminal differentiation (hypertrophy-like changes) in osteoarthritis (OA) and to integrate this in a conceptual model of primary OA development. METHODS: Papers investigating chondrocyte terminal differentiation in human OA cartilage and experimental models of OA were recapitulated and discussed. Focus has been on the occurrence of hypertrophy-like changes in chondrocytes and the factors described to play a role in regulation of chondrocyte hypertrophy-like changes in OA. RESULTS: Chondrocyte hypertrophy-like changes are reported in both human OA and experimental OA models by most investigators. These changes play a crucial part in the OA disease process by protease-mediated cartilage degradation. We propose that altered chondrocyte behavior and concomitant cartilage degradation result in a disease-amplifying loop, leading to a mixture of disease stages and cellular responses within an OA joint. CONCLUSION: Chondrocyte hypertrophy-like changes play a role in early and late stage OA. Since not all cells in an OA joint are synchronized, inhibition of hypertrophy-like changes might be a therapeutic target to slow down further OA progression.
Assuntos
Cartilagem Articular/patologia , Condrócitos/patologia , Osteoartrite/etiologia , Biomarcadores/metabolismo , Calcinose/complicações , Doenças das Cartilagens/complicações , Diferenciação Celular , Progressão da Doença , Humanos , Hipertrofia/complicaçõesRESUMO
OBJECTIVE: Several lines of evidence implicate cytosolic phospholipase A(2)α (cPLA(2)α) as a critical enzyme in inflammatory disorders, including rheumatoid arthritis. Since cells from the myeloid compartment regulate local and systemic disease pathogenesis, the present study was undertaken to examine the effect of cPLA(2)α inhibition in experimental arthritis, using a delivery system tailored to target monocyte functions by RNA interference (RNAi). METHODS: Mice with collagen-induced arthritis (CIA) were injected intravenously with an anti-cPLA(2)α small interfering RNA (siRNA) sequence (siPLA2) formulated as lipoplexes with the RPR209120/DOPE cationic liposome and a carrier DNA. The clinical course of joint inflammation was assessed, and the immunologic balance was analyzed by measuring T helper cell frequencies and cytokine expression. Biodistribution studies of siRNA were also performed. RESULTS: Weekly systemic injection of siPLA2 lipoplexes significantly reduced the incidence and severity of CIA, in both preventive and curative settings, as compared with findings in control animals. Histologic scores for inflammation and cartilage damage were reduced. The clinical effect was associated with local inhibition of tumor necrosis factor α secretion and lower cPLA(2)α expression and activity. The siPLA2 lipoplexes enabled triggering of in vivo RNAi-mediated gene silencing of cPLA(2)α in CD11b+ cells recovered from the spleen. While the treatment had no effect on anti-type II collagen (anti-CII) antibodies, CII-specific T helper cells producing interferon-γ, but not interleukin-17, in draining lymph node cells were decreased. CONCLUSION: Our findings indicate that systemic RNAi-mediated cPLA(2)α gene silencing in CD11b+ cells is effective in the treatment of CIA, and Th1 suppression is one of the potential underlying mechanisms, whereas Th17 suppression is not.
Assuntos
Artrite Experimental/imunologia , Artrite Experimental/terapia , Terapia Genética/métodos , Fosfolipases A2 do Grupo IV/genética , Células Th1/imunologia , Animais , Artrite Experimental/genética , Antígeno CD11b/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Citosol/enzimologia , Modelos Animais de Doenças , Fosfolipases A2 do Grupo IV/imunologia , Lipopeptídeos/genética , Lipopeptídeos/imunologia , Camundongos , Camundongos Endogâmicos DBA , Monócitos/citologia , Monócitos/imunologia , Células Mieloides/citologia , Células Mieloides/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Índice de Gravidade de Doença , Organismos Livres de Patógenos Específicos , Células Th1/citologiaRESUMO
Background: Post-bariatric body contouring surgery (BCS) treats redundant skin after massive weight loss; however, the complication risk is relatively high (23-70%). Most complications are wound-related, which may be partly due to a poor nutritional status after bariatric surgery. The objective of this observational study was to optimise nutrition preoperatively and assess the prevalence of wound-related complications after BCS. Methods: This prospective cohort study included 140 patients. Patients were treated according to the post-bariatric BCS guideline. Nutritional parameters were collected via pre- and peri-operative blood sampling; any deficiencies were treated. A protein-enriched diet was prescribed by a dietician 4 weeks preoperatively up until closure of all wounds. Complications were recorded using the Clavien-Dindo classification. Univariate and multivariate regression analyses were performed to identify variables associated with wound-related complications. Results: The overall wound-related complication rate was 51%. Most complications were minor, with only 4.3% was considered major. No significant differences in patient characteristics were found between patients with and without complications. Variables indicating an optimised nutritional state were not significantly associated with a decreased risk of complications; the most influential factor was a sufficient post-operative protein intake (OR 0.27, 95% CI 0.07 - 1.02, p = 0.05). Conclusion: The overall wound-related complication rate was in accordance with previous literature; however, major complications were few. This study showed a weak correlation between optimising nutritional state and better outcome after BCS, especially following a protein-enriched diet post-operatively. Therefore, we recommend continuing research on nutrition and wound-related complications, using homogeneous study populations and well-defined complications.
RESUMO
Osteoarthritis is characterized by progressive breakdown of articular cartilage. This review summarizes findings of the last year, which shed new light on mechanisms and factors involved in cartilage loss. Evidence is accumulating that the transcription factor hypoxia-inducible factor-2α (HIF-2α) is highly enhanced in OA cartilage and drives catabolic metalloproteinases, including the pivotal MMP-13. In addition, HIF-2α suppresses chondrocyte autophagy, herein promoting chondrocyte apoptosis. The crucial role of MMP-13 is further underlined by reduced OA pathology in MMP-13 deficient mice. An intriguing mechanism to drive MMP-13 production is activation of the chondrocyte discoidin domain receptor (DDR-2) receptor through interaction with denuded collagen type II. The latter might occur in a proteoglycan depleted peri-cellular matrix, where DDR-2 expression is enhanced in OA cartilage and transgenic suppression attenuates experimental OA. The initiating role of ADAMTS-5 in proteoglycan loss appears dependent on interaction with the transmembrane proteoglycan syndecan-4, since syndecan-4 deficient mice are less prone to experimental OA and display reduced ADAMTS-5 activity. Both aging and the osteoarthritis (OA) process itself induce deranged transforming growth factor-ß (TGFß)-receptor expression, causing a shift to dominant usage of the receptor ALK-1, in stead of ALK5 and resulting in a TGFß mediated catabolic pathway. ALK-1 rather than TGFß is a promising therapeutic target. Finally, the alarmins S100A8 and 9 have long been considered as markers of inflammatory joint destruction, but now appear to be catabolic mediators.
Assuntos
Osteoartrite/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Cartilagem Articular/metabolismo , Receptores com Domínio Discoidina , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Osteoartrite/fisiopatologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Mitogênicos/metabolismo , Sindecana-4/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
We report that streptococcal cell wall (SCW)-induced arthritis in rats, a T cell-dependent chronic, erosive polyarthritis, can be prevented by pretreatment of the rats with the mycobacterial 65-kD heat shock protein. This 65-kD protein shows extensive amino acid homology with prokaryotic and eukaryotic 65-kD heat shock proteins and is a ubiquitous bacterial common antigen. Both the clinical and histopathologic manifestations of the arthritis were prevented completely when rats were pretreated with 50 micrograms of 65-kD protein intraperitoneally at 35, 25, 15, or 5 d before administration of SCW. In such protected rats, SCW-specific T cell responses were suppressed, as compared with responses in arthritic rats. Pretreatment with 65-kD protein had no effect on the production of antibodies against SCW, on a nonspecific inflammatory reaction (zymosan-induced arthritis), or on general cellular immunity in vivo (delayed type hypersensitivity reaction to a nonrelated protein antigen). Furthermore, the protection against SCW arthritis was transferable by splenic T cells to naive recipients. Our data show that pretreatment with the 65-kD mycobacterial heat shock protein protects rats against a subsequent bacterium-induced arthritis. This protection is immunologically specific and resides in the lymphoid cell population.
Assuntos
Artrite/prevenção & controle , Proteínas de Choque Térmico/imunologia , Mycobacterium/imunologia , Streptococcus/imunologia , Animais , Artrite/imunologia , Artrite/patologia , Artrite Experimental/imunologia , Parede Celular/imunologia , Reações Cruzadas , Hipersensibilidade Tardia/imunologia , Imunização Passiva , Peso Molecular , Ratos , Ratos Endogâmicos Lew , Linfócitos T/imunologiaRESUMO
In this review the authors discuss the evidence for T helper type 17 (Th17) cells as pathogenic T cells in autoimmunity. Studies with cytokine-deficient mice or blocking of interleukin (IL)-17, IL-21 and IL-22 have resulted in a conflicting data set. Although in the experimental autoimmune encephalomyelitis model the role of Th17 cells remains a point of debate, this IL-17-producing T cell in experimental arthritis is clearly contributing to inflammation and destruction.
Assuntos
Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Interleucina-17/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Doenças Autoimunes/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , Camundongos , Camundongos Knockout , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
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.