RESUMEN
OBJECTIVES: To explore the impact of disease-modifying osteoarthritis drug (DMOAD) treatment on biomarker levels and their correlation with cartilage volume loss and disease symptoms in a 2-year phase III clinical trial in patients with knee OA. METHODS: 161 patients with knee OA (according-to-protocol population) were selected from a 2-year DMOAD trial studying the effect of licofelone (200 mg twice daily) versus naproxen (500 mg twice daily). Clinical evaluation of patients was carried out using the Western Ontario and McMaster Universities (WOMAC) questionnaire. Biomarker measurements of matrix metalloproteinase (MMP)-1, MMP-3, interleukin (IL)-6, C reactive protein (CRP), cartilage oligomeric matrix protein (COMP) and type I collagen C-terminal telopeptide (CTX-I) in serum, type II collagen C-terminal telopeptide (CTX-II) in urine, and knee MRI were performed at baseline and 2 years. RESULTS: Over time an increase occurred in all biomarker levels with the exception of IL-6, CRP and CTX-II which decreased. The increase in MMP-1 and MMP-3 was significantly less (p = 0.05; p < 0.01, respectively) in the licofelone group. The baseline MMP-1 level was significantly but inversely predictive of cartilage volume loss for the medial compartment in both univariate (p = 0.04) and multivariate (p ≤ 0.04) regression analyses, and COMP, a predictor for the lateral compartment, in both univariate and multivariate models (p < 0.01). Baseline levels of IL-6 and CRP also showed a significant relationship with volume loss for the medial compartment (univariate analysis, p = 0.04 and p = 0.01, respectively; multivariate analysis, p = 0.03, p = 0.01). A significant association (univariate) was observed between the change in the levels of MMP-1 (p = 0.03) and MMP-3 (p = 0.02) and cartilage volume loss (lateral compartment) over 2 years. Baseline levels of CTX-I correlated (p = 0.02) with an increase in the size of the bone marrow lesion in the medial compartment. The baseline CRP levels correlated with worsening of symptoms: WOMAC total index (p < 0.01), pain (p < 0.01) and function (p < 0.01). CONCLUSION: Higher baseline values of IL-6, CRP and COMP are predictive of greater risk of cartilage loss in OA. However, over time a reduction in MMP-1 and MMP-3 levels correlated best with reduction in cartilage volume loss and the effect of drug treatment. Baseline CRP was found to be a good predictor of the symptomatic response to treatment.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Metaloproteinasas de la Matriz/sangre , Osteoartritis de la Rodilla/tratamiento farmacológico , Anciano , Biomarcadores/sangre , Proteína C-Reactiva/metabolismo , Cartílago Articular/patología , Monitoreo de Drogas/métodos , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Naproxeno/uso terapéutico , Osteoartritis de la Rodilla/enzimología , Osteoartritis de la Rodilla/patología , Pirroles/uso terapéutico , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
OBJECTIVE: The primary objective of this study was to evaluate the ex vivo therapeutic efficacy of diacerein and its active metabolite, rhein, on osteoarthritic (OA) cartilage chondrocyte DNA fragmentation and death in the experimental canine model of OA. The study also aimed to explore the effect of the drug on the level of important factors involved in this phenomenon, i.e., caspase-3 and inducible nitric oxide synthase (iNOS). METHODS: OA knee cartilage was obtained from dogs that had received surgical sectioning of the anterior cruciate ligament (ACL) and were sacrificed 12 weeks after surgery. Cartilage explants were cultured in the presence or absence of therapeutic concentrations of diacerein (20 micrograms/ml) or rhein (20 micrograms/ml). Cartilage specimens were stained for TUNEL reaction and immunostained using specific antibodies for active caspase-3 and iNOS. Morphometric analyses were also performed. RESULTS: In OA cartilage specimens, a large number of chondrocytes in the superficial layers stained positive for TUNEL reaction. Treatment with therapeutic concentrations of diacerein (20 micrograms/ml) or rhein (20 micrograms/ml) significantly reduced the level of chondrocyte DNA fragmentation to about the same extent in both treatment groups (P < 0.006, P < 0.002, respectively). The levels of caspase-3 and iNOS in cartilage explants were also significantly decreased (caspase-3, diacerein P < 0.04; caspase-3, rhein P < 0.0003; and iNOS, rhein P < 0.009, respectively) when compared to the control group. CONCLUSIONS: This study shows that diacerein/rhein can effectively reduce the level of OA chondrocyte DNA fragmentation and death under the present experimental conditions. This effect is mediated by a decrease in the level of caspase-3 expression, which could possibly be related in part to the reduced level of iNOS and secondarily to NO production. These findings provide additional new information about the mechanisms of action of diacerein on the progression of OA.
Asunto(s)
Antraquinonas/farmacología , Antiinflamatorios no Esteroideos/farmacología , Inhibidores de Caspasas , Condrocitos/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Óxido Nítrico Sintasa/antagonistas & inhibidores , Osteoartritis de la Rodilla/tratamiento farmacológico , Animales , Cartílago/efectos de los fármacos , Caspasa 3 , Caspasas/inmunología , Células Cultivadas , Perros , Inhibidores Enzimáticos/farmacología , Etiquetado Corte-Fin in Situ , Oxigenasas de Función Mixta/farmacología , Óxido Nítrico Sintasa/inmunología , Óxido Nítrico Sintasa de Tipo II , Osteoartritis de la Rodilla/inmunología , Osteoartritis de la Rodilla/fisiopatologíaRESUMEN
The PGD(2) metabolite 15-deoxy-delta12,14 PGJ(2) (15d-PGJ(2)), a potent peroxisome proliferator-activated receptor gamma (PPARgamma) activator, has recently received attention for its potential antiinflammatory effects, but its effect on the cyclooxygenase-2 (COX-2) production is still under debate. We investigated the effect of 15d-PGJ(2) on COX-2 and prostaglandin E(2) (PGE(2)) production in the absence or the presence of interleukin-1beta (IL-1beta) in human osteoarthritic chondrocytes.Data showed that, as expected, IL-1beta induced both COX-2 and PGE(2) production. The addition of 15d-PGJ(2) completely blocked (by 93%) the IL-1beta-induced PGE(2) synthesis, whereas COX-2 level was only partially reduced (by 72%). Interestingly in the absence of any COX-2 inducer, 15d-PGJ(2) up-regulated COX-2 expression without concomitant elevation of PGE(2) synthesis. This study showed that the PPARgamma agonist, 15d-PGJ(2), exerts a dual effect on COX-2 production. The mechanisms by which 15d-PGJ(2) favors COX-2 production will be discussed.
Asunto(s)
Dinoprostona/biosíntesis , Factores Inmunológicos/farmacología , Interleucina-1/metabolismo , Isoenzimas/biosíntesis , Osteoartritis de la Rodilla/metabolismo , Peroxidasas/biosíntesis , Prostaglandina D2/farmacología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Ciclooxigenasa 2 , Humanos , Proteínas de la Membrana , Prostaglandina D2/análogos & derivados , Regulación hacia ArribaRESUMEN
OBJECTIVE: Although cartilage degradation characterizes osteoarthritis (OA), there is evidence that remodeling of subchondral bone in this disease is a contributing factor. Therapeutic strategies to modify the metabolism of subchondral bone osteoblasts may be indicated to treat OA. We studied the effects of diacerein and rhein on the metabolic and inflammatory variables of OA subchondral osteoblasts. METHODS: Human OA primary subchondral osteoblast cells were used. The effect of diacerein and rhein at therapeutic concentrations (5-20 microg/ml) was determined by osteoblast phenotypic factors, alkaline phosphatase, osteocalcin, and cAMP; on metabolic agents urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and insulin-like growth factor-1 (IGF-1); and on inflammatory mediators interleukin 6 (IL-6), prostaglandin E2 (PGE2), and cyclooxygenase-2 (COX-2). RESULTS: Diacerein and rhein did not affect either basal and 1,25(OH)2D3 induced alkaline phosphatase or parathyroid hormone (PTH) stimulated cAMP formation. Conversely, they dose dependently and statistically inhibited 1,25(OH)2D3 induced osteocalcin release, a situation explained by a reduction of mRNA levels for osteocalcin. Of the metabolic factors, they inhibited the production of uPA, with rhein showing slightly more potency; inhibitions of 69% and 57% were reached at the highest concentration (20 microg/ml) of rhein and diacerein, respectively. Both drugs also inhibited the PAI-1 level, albeit at a much lower level than for uPA. Interestingly, determination of the uPA/PAI1 ratio revealed that both drugs inhibited it about 55%, suggesting a decrease in uPA activity. In contrast, IGF-1 levels only increased slightly when cells were treated with rhein but not with diacerein. A transient dose dependent effect was found on IL-6 production; an inhibition was noted at low drug concentrations, which returned to basal levels at the highest concentration tested. PGE2 levels increased exponentially and were related to a concomitant increase in COX-2 levels in response to both drugs. CONCLUSION: Our data indicate that diacerein and rhein do not appear to affect OA subchondral bone cells' basal cellular metabolism, yet both agents reveal a direct effect at reducing the synthetic activities of osteoblasts, which could be responsible for abnormal subchondral bone remodeling occurring during the course of OA.
Asunto(s)
Antraquinonas/farmacología , Antiinflamatorios no Esteroideos/farmacología , Remodelación Ósea/fisiología , Huesos/fisiopatología , Osteoartritis/fisiopatología , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Anciano , Anciano de 80 o más Años , Huesos/patología , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoartritis/patología , Osteoblastos/metabolismoRESUMEN
OBJECTIVE: To determine the effects of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on interleukin-1 (IL-1) induction of nitric oxide (NO) and matrix metalloproteinase 13 (MMP-13) in human chondrocytes. METHODS: PPARgamma expression and synthesis in human chondrocytes were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Chondrocytes were cultured with IL-1beta, tumor necrosis factor alpha (TNFalpha), and IL-17 in the presence or absence of PPARgamma agonists, and NO and MMP-13 synthesis and expression levels were measured. Transient transfection experiments were performed with the 7-kb inducible NO synthase (iNOS) and 1.6-kb MMP-13 human promoters, as well as with the PPARgamma expression vector and the activator protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) reporter constructs. RESULTS: RT-PCR and immunohistochemical analysis revealed that human chondrocytes expressed and produced PPARgamma. Treatment of chondrocytes with PPARgamma ligands BRL 49653 and 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2), but not with PPARalpha ligand Wy 14643, decreased IL-1beta-induced NO and MMP-13 production in a dose-dependent manner. In addition, both iNOS and MMP-13 messenger RNA were inhibited in the presence of 15d-PGJ2. The inhibitory effect of PPARgamma activation was not restricted to IL-1beta, since TNFalpha- and IL-17-induced NO and MMP-13 production were also inhibited by 15d-PGJ2. In transient transfection experiments, we showed that a constitutively active form of mitogen-activated protein kinase kinase kinase 1 (AMEKK-1) induced the MMP-13 and iNOS human promoter activity. This process was reduced by 15d-PGJ2 and further inhibited by cotransfection with a PPARgamma expression vector. Similarly, in a PPARgamma-dependent manner, 15d-PGJ2 inhibited deltaMEKK-1-induced AP-1- and NF-kappaB-luciferase reporter plasmid activation. CONCLUSION: The findings of this study demonstrate that PPARgamma agonists inhibit IL-1beta induction of both NO and MMP-13 in human chondrocytes. The inhibition occurs at least at the transcriptional level through a PPARgamma-dependent pathway, probably by interfering with the activation of AP-1 and NF-kappaB.
Asunto(s)
Condrocitos/metabolismo , Colagenasas/biosíntesis , Interleucina-1/antagonistas & inhibidores , Quinasa 1 de Quinasa de Quinasa MAP , Óxido Nítrico/metabolismo , Prostaglandina D2/análogos & derivados , Receptores Citoplasmáticos y Nucleares/agonistas , Factores de Transcripción/agonistas , Activación Enzimática/genética , Humanos , Metaloproteinasa 13 de la Matriz , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Regiones Promotoras Genéticas/efectos de los fármacos , Prostaglandina D2/farmacología , Proteínas Serina-Treonina Quinasas/fisiología , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Factor de Transcripción AP-1/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
OBJECTIVE: Treatment of normal cartilage with transforming growth factor beta (TGFbeta) can increase the synthesis of collagenase 3 by chondrocytes and mimic the in situ distribution of this enzyme in osteoarthritic (OA) cartilage, which occurs predominantly in the deep zone. In this study, we examined the elements of the TGFbeta system that are potentially relevant to this effect. METHODS: TGFbeta1 and TGFbeta2 levels in cultured cartilage explants were determined by enzyme-linked immunosorbent assay (ELISA). OA cartilage explants were treated with small latent TGFbeta1 complex in the presence of various inhibitors, and collagenase 3 levels were determined by ELISA. The inhibitors were against serine proteases, plasmin, cathepsins, furin, and a neutralizing antibody against the mannose-6 phosphate/ insulin-like growth factor 2 receptor (M6P/IGF-2R). Small latent TGFbeta1, TGFbeta receptor types I, II, and III (TGFbetaRI, RII, and RIII), M6P/IGF-2R, and furin were immunolocalized in cartilage. RESULTS: Our data showed that latent TGFbeta1 is the major isoform that is synthesized; levels of 17.2 +/-1.7 pg/mg and 1.1 +/- 0.3 pg/mg tissue wet weight (mean +/- SEM) were found for total TGFbeta1 and TGFbeta2, respectively, in OA cartilage. A general serine protease inhibitor abrogated activation of both endogenous and exogenous small latent TGFbeta1. Plasmin and furin inhibitors and anti-M6P/IGF-2R reduced the levels of exogenous small latent TGFbeta1 complex-induced collagenase 3 by 33%, 95%, and 76%, respectively, but the cathepsin inhibitor had no effect. Immunolocalization of the small latent TGFbeta1 complex as well as of TGFbetaRI and RII revealed a statistically significant increase in the chondrocyte score in only the deep zone of OA cartilage. The M6P/IGF-2R level was significantly higher in OA cartilage in both the superficial and deep zones. Furin was found in normal cartilage exclusively in the superficial zone, whereas in OA cartilage, a level similar to that in normal cartilage was found in the superficial zone, but a significantly higher cell score (mean +/- SEM 23.6 +/- 4.7%) was registered in the deep zone. CONCLUSION: The mechanisms of TGFbeta activation/ activity with regard to collagenase 3 modulation in cartilage appear to be controlled by furin convertase with or without M6P/IGF-2R. These factors and the small latent TGFbeta complex are increased in the deep zone of OA cartilage, corresponding to the preferential site of collagenase 3 production.
Asunto(s)
Cartílago Articular/química , Colagenasas/efectos de los fármacos , Osteoartritis de la Rodilla/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Anciano , Colagenasas/metabolismo , Femenino , Furina , Humanos , Masculino , Metaloproteinasa 13 de la Matriz , Subtilisinas/fisiologíaRESUMEN
There is increasing evidence suggesting that chondrocyte death may contribute to the progression of osteoarthritis (OA). This study focused on the characterization of signaling cascade during NO-induced cell death in human OA chondrocytes. The NO generator, sodium nitroprusside (SNP), promoted chondrocyte death in association with DNA fragmentation, caspase-3 activation, and down-regulation of Bcl-2. Both caspase-3 inhibitor Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-CH2F and caspase-9 inhibitor Z-Leu-Glu(OCH3)-His-Asp(OCH3)-CH2F prevented the chondrocyte death. Blocking the mitogen-activated protein kinase pathway by the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or p38 kinase inhibitor SB202190 also inhibited the SNP-mediated cell death, suggesting possible requirements of both extracellular signal-related protein kinase 1/2 and p38 kinase for the NO-induced cell death. Furthermore, the selective inhibition of cyclooxygenase (COX)-2 by NS-398 or the inhibition of COX-1/COX-2 by indomethacin blocked the SNP-induced cell death. The chondrocyte death induced by SNP was associated with an overexpression of COX-2 protein (as determined by Western blotting) and an increase in PGE2 release. PD98059 and SB202190, but neither Z-DEVD FMK nor Z-LEHD FMK completely inhibited the SNP-mediated PGE2 production. Analysis of interactions between PGE2 and the cell death showed that PGE2 enhanced the SNP-mediated cell death, whereas PGE2 alone did not induce the chondrocyte death. These data indicate that NO-induced chondrocyte death signaling includes PGE2 production via COX-2 induction and suggest that both extracellular signal-related protein kinase 1/2 and p38 kinase pathways are upstream signaling of the PGE2 production. The results also demonstrate that exogenous PGE2 may sensitize human OA chondrocytes to the cell death induced by NO.
Asunto(s)
Condrocitos/metabolismo , Condrocitos/patología , Dinoprostona/biosíntesis , Isoenzimas/biosíntesis , Óxido Nítrico/fisiología , Osteoartritis/metabolismo , Osteoartritis/patología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Anciano , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/enzimología , Ciclooxigenasa 2 , Dinoprostona/fisiología , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Isoenzimas/antagonistas & inhibidores , Masculino , Proteínas de la Membrana , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Nitroprusiato/farmacología , Osteoartritis/enzimología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidoresRESUMEN
OBJECTIVE: To examine the cellular mechanisms by which the proinflammatory cytokine interleukin-17 (IL-17) induces the synthesis of 92-kd gelatinase (matrix metalloproteinase 9 [MMP-9]) by human monocyte/ macrophages in primary culture. METHODS: IL-17-stimulated human monocytes isolated from the peripheral blood of healthy donors were cultured in the presence of antiinflammatory cytokines, neutralizing antibodies against IL-1beta, tumor necrosis factor alpha (TNFalpha), or IL-1 receptor antagonist, and with protein kinase inhibitors of diverse specificity. MMP measurements were performed using specific enzyme-linked immunosorbent assays, while the expression of specific messenger RNA was determined by Northern blotting. Detection of phosphorylated proteins and specific transcriptional factors was performed by Western blotting and by gel retardation experiments, respectively. RESULTS: Biologically active IL-17 was detected in the synovial fluid of patients with rheumatoid arthritis. IL-17-induced MMP-9 production in human monocyte/ macrophages was dependent on endogenous prostaglandin E2 synthesis and related to autocrine stimulation by TNFalpha, but was IL-1beta independent. This activation involves both p42/44 and p38 kinases and nuclear factor kappaB. IL-17-inducible activator protein 1 and signal transducer and activator of transcription 1/3 may transactivate the MMP-9 promoter. CONCLUSION: IL-17 may contribute to an unbalanced production of proinflammatory cytokines and MMP-9 in diseased articular joint tissues by interacting with the macrophages in the rheumatoid synovium.
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Interleucina-17/farmacología , Macrófagos/metabolismo , Metaloproteinasa 9 de la Matriz/biosíntesis , Monocitos/metabolismo , Adulto , Anciano , Artritis Reumatoide/sangre , Artritis Reumatoide/fisiopatología , Ciclooxigenasa 2 , Proteínas de Unión al ADN/efectos de los fármacos , Dinoprostona/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Interleucina-1/antagonistas & inhibidores , Interleucina-17/análisis , Isoenzimas/biosíntesis , Isoenzimas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Proteínas de la Membrana , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Inhibidores de Proteínas Quinasas , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transducción de Señal/fisiología , Líquido Sinovial/química , Transactivadores/efectos de los fármacos , Factor de Transcripción AP-1/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
OBJECTIVE: To explore the signaling pathways by which the proinflammatory cytokine interleukin-17 (IL-17) may contribute to cartilage catabolism in osteoarthritis (OA) by inducing inducible nitric oxide synthase (iNOS) expression in chondrocytes. METHODS: We examined the IL-17-induced NO production in human OA chondrocytes, in combination with the proinflammatory cytokines IL-1beta, tumor necrosis factor alpha (TNF alpha), and leukemia inhibitory factor (LIF); the antiinflammatory cytokines IL-4, IL-10, and IL-13; and IL-1 receptor antagonist (IL-1Ra). Further, we explored the major intracellular signaling pathways through which IL-17 induced iNOS expression and NO production. RESULTS: Treatment with IL-17 induced a dose-dependent increase in the level of NO. When IL-17 was combined with the above factors, it resulted in a synergistic effect with TNF alpha, an additive effect with LIF, and no further effect than when used alone with IL-1beta. IL-4, IL-10, IL-13, and IL-1Ra had no true effect on IL-17-induced NO production. The cAMP mimetics, 3-isobutyl-1-methyl xanthine plus forskolin, completely blocked IL-17-induced NO production. KT-5720, genistein, and Calphostin C, inhibitors of protein kinase A (PKA), tyrosine kinase, and protein kinase C, respectively, reduced the IL-17-induced NO production by 72%, 56%, and 42%, respectively. Within minutes, IL-17 induced the phosphorylation of mitogen-activated protein kinase kinase-1/2 (MEK-1/2), -3/6 (MKK-3/6), p44/42, p38, and inhibitor of nuclear factor kappaB (I kappaB)-alpha, as well as the activation of mitogen-activated protein kinase-activated protein kinase-1 and -2 (MAPKAPK-1 and -2). Interestingly, IL-17 induced phosphorylation of the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) (p54/46) only when PKA was inhibited. Specific protein kinase inhibitors for MEK-1/2 (PD98059), p38 (SB202190), and nuclear factor kappaB (NF-kappaB) (pyrrolidine dithiocarbamate) each markedly decreased the IL-17-increased iNOS level and NO production. Inhibiting MAPK, including MEK-1/2 and p38, had no effect on the IL-17-induced activation of IkappaB-alpha, but reversed the IL-17 activation of MAPKAPK-1 and -2, respectively. CONCLUSION: These findings show that the stimulation of NO production by IL-17 is mediated mainly by a complex activation of kinases, especially PKA, NF-kappaB, and MAPK. NF-kappaB appears to require MAPK activation, with downstream activation of MAPKAPK probably acting as a transactivating factor, to induce iNOS expression.
Asunto(s)
Condrocitos/metabolismo , Interleucina-17/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Osteoartritis/metabolismo , Anciano , Células Cultivadas , Condrocitos/enzimología , Activación Enzimática , Femenino , Humanos , Masculino , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Osteoartritis/patología , Proteínas Quinasas S6 Ribosómicas/fisiología , Proteínas Quinasas S6 Ribosómicas 90-kDa , Transducción de Señal , Activación TranscripcionalRESUMEN
OBJECTIVE: To evaluate the in vitro effects of diacerhein, a new drug for the treatment of osteoarthritis (OA), and its active metabolite, rhein, on the production of nitric oxide (NO), prostaglandin (PGE2), cyclooxygenase-2 (COX-2), as well as the production and expression of the inducible nitric oxide synthase (iNOS) in human OA chondrocytes. These results were compared to those of the nonsteroidal antiinflammatory drug (NSAID) naproxen. METHODS: Human OA chondrocytes were incubated in the presence or absence of 25 units/ml recombinant human interleukin-1beta (rhIL-1beta) with or without therapeutic concentrations of diacerhein and rhein at 5, 10, and 20 microg/ml and naproxen at 30 and 90 microg/ml. Effect of the drugs was also tested on both OA chondrocytes and cartilage explants on increasing IL-1beta concentration (0-100 units/ml). The NO and PGE2 levels were determined in the culture medium using the Griess reaction and a specific ELISA, respectively. Production of COX-2 and synthesis and expression of iNOS were quantitated by Western blot and Northern blot, respectively. RESULTS: The IL- 1beta induced NO production was inhibited by both diacerhein and rhein in a time and dose dependent fashion, with statistical significance reached at the therapeutic concentration of 20 microg/ml. A decrease over 80% was found at 24, 48, and 72 h incubation. This was consistent for both chondrocytes and cartilage explants even in the presence of high IL-1beta concentration (100 units/ml). Moreover, this effect appeared to result from iNOS transcriptional and/or post-transcriptional events as indicated by a decrease in this enzyme level for both the mRNA and protein. Naproxen, however, showed only a slight inhibition of IL-1beta induced NO production at the highest dose used, 90 microg/ml. A maximum decrease of 23% in IL-1beta induced NO production was recorded after a 72 h incubation. In contrast to naproxen, which abrogated PGE2 and had no effect on COX-2 synthesis, rhein and diacerhein at 5 and 10 microg/ml produced an enhancement in their levels. CONCLUSION: Diacerhein and rhein, in contrast to an NSAID, are potent inhibitors of IL-1beta induced NO production by chondrocytes and cartilage, without reducing PGE2 production.
Asunto(s)
Antraquinonas/farmacología , Condrocitos/efectos de los fármacos , Interleucina-1/farmacología , Isoenzimas/efectos de los fármacos , Óxido Nítrico Sintasa/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Anciano , Antiinflamatorios no Esteroideos/farmacología , Condrocitos/citología , Condrocitos/enzimología , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Isoenzimas/biosíntesis , Masculino , Proteínas de la Membrana , Persona de Mediana Edad , Naproxeno/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Osteoartritis/tratamiento farmacológico , Osteoartritis/enzimología , Osteoartritis/patología , Prostaglandina-Endoperóxido Sintasas/biosíntesisRESUMEN
OBJECTIVE: In this study we investigated the effect of interleukin-13 (IL-13), an anti-inflammatory cytokine, for potential therapeutic use in osteoarthritis (OA). DESIGN: We examined the effect of IL-13 on the synthesis and expression of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), IL-1 receptor antagonist (IL-1Ra) and stromelysin-1 on human OA synovial membrane in ex vivo cultures. In addition, we explored the effect of IL-13 on both the IL-1 receptor (IL-1R) and TNF-receptor (TNF-R) systems on OA synovial fibroblasts. This included determination of the levels of IL-1 beta and TNF-alpha receptor binding, IL-1Ra and TNF-soluble receptors 55 and 75 (TNF-sR55 and TNF-sR75). RESULTS: In OA synovial membrane treated with LPS, IL-13 inhibited the synthesis of IL-1 beta, TNF-alpha and stromelysin-1, but increased IL-1Ra production. In addition, IL-13 reduced the level of IL-1 beta mRNA and stimulated the level of IL-1Ra mRNA. In synovial fibroblasts, IL-13 decreased the level of IL-1 binding, an effect related to the increased production of IL-1Ra. Although IL-13 had no effect on the TNF-R level, this cytokine markedly decreased the shedding of TNF-R75. CONCLUSION: These experiments suggest that IL-13 is potentially useful in the therapeutic treatment of OA, as it could regulate the major pathological process of this disease by reducing the production of proinflammatory cytokines and metalloproteases, and favoring the production of IL-1Ra.
Asunto(s)
Citocinas/efectos de los fármacos , Interleucina-13/farmacología , Osteoartritis/metabolismo , Receptores de Citocinas/efectos de los fármacos , Membrana Sinovial/efectos de los fármacos , Anciano , Northern Blotting , Técnicas de Cultivo de Célula , Citocinas/biosíntesis , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/metabolismo , Masculino , Metaloproteinasa 3 de la Matriz/biosíntesis , Metaloproteinasa 3 de la Matriz/efectos de los fármacos , Persona de Mediana Edad , ARN Mensajero/genética , Receptores de Citocinas/biosíntesis , Sialoglicoproteínas/biosíntesis , Membrana Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
IL-17 is a newly described, T cell-derived cytokine with ill-defined physiologic properties. As such, we examined the release of proinflammatory mediators by human macrophages in response to recombinant human (rh) IL-17. IL-1beta and TNF-alpha expression and synthesis were up-regulated by rhIL-17 in a dose (ED50 was 50 +/- 9 ng/ml)- and time-dependent fashion, with cytokine accumulation reaching a zenith after 9 h. Release of IL-6, PGE2, IL-10, IL-12, IL-1R antagonist, and stromelysin was also stimulated by rhIL-17. IL-1beta and TNF-alpha mRNA expression levels were controlled by rhIL-17 in a complex manner with an initial 30-min inhibitory phase, and then up-regulation beginning at 1 h and reaching a plateau at about 3 h. The latter expression pattern closely mirrored the nuclear accumulation of the transcription factor nuclear factor-kappaB. cAMP mimetics isobutyl-1-methylxanthine (IBMX), forskolin, PGE2, and cholera toxin reversed rhIL-17-induced release of TNF-alpha, but had no consistent effect on induced IL-1beta synthesis. Induced release of TNF-alpha was also inhibited by serine/threonine protein kinase inhibitors KT-5720 (protein kinase A) and Calphostin C (protein kinase C), mitogen-activated protein kinase kinase inhibitor PD098059, and a nonspecific tyrosine kinase inhibitor, genistein. Calphostin C alone abrogated the rhIL-17-induced release of IL-1beta. The antiinflammatory cytokines IL-4 (p < 0.01) and IL-10 (p < 0.02) completely reversed rhIL-17-stimulated IL-1beta release, while IL-13 and TGF-beta2 were partially effective (59 and 43% diminution, respectively). IL-10 exerted a significant suppressive effect on IL-17-induced TNF-alpha release (99%, p < 0.02), while the inhibitory effects of IL-4, IL-13, and TGF-beta2 on TNF-alpha secretion were partial (48, 10, and 23%, respectively). The data suggest a pivotal role for IL-17 in initiating and/or sustaining an inflammatory response.
Asunto(s)
Interleucina-1/biosíntesis , Interleucinas/farmacología , Macrófagos/inmunología , Macrófagos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Adulto , Antiinflamatorios no Esteroideos/farmacología , Calcio/metabolismo , Células Cultivadas , AMP Cíclico/agonistas , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Relación Dosis-Respuesta Inmunológica , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Interleucina-1/metabolismo , Interleucina-10/farmacología , Interleucina-17 , Interleucina-4/farmacología , Activación de Macrófagos , Macrófagos/enzimología , Persona de Mediana Edad , FN-kappa B/biosíntesis , Inhibidores de Proteínas Quinasas , Factores de Tiempo , Factor de Transcripción AP-1/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To evaluate the in vitro effects of diacerhein, a new drug for the treatment of osteoarthritis (OA), and its active metabolite, rhein, on interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) synthesis and expression in human OA synovial membrane, and on the IL-1beta and TNF-alpha receptors on human OA chondrocytes. METHODS: Levels of IL-1beta and TNF-alpha were determined using specific ELISA in culture medium of human synovial membrane explants incubated in the presence of 1 microg/ml of lipopolysaccharide with or without therapeutic concentrations of diacerhein (1.4, 2.7, 5.4 x 10(-5) M) and rhein (1.7, 3.5, 7.0 x 10(-5) M). IL-1beta mRNA level was quantitated by Northern blotting. Using radioligand binding experiments, we determined the effects of these agents on the density and affinity of chondrocyte IL-1 and TNF receptors. RESULTS: IL-1beta synthesis was significantly inhibited by diacerhein and rhein, with maximum inhibition at 5.4 x 10(-5) M for diacerhein (p < 0.02) and 3.5 x 10(-5) M for rhein (p < 0.05). The effect of both agents on IL-1beta was found to be translational and/or post-translational, judging by the absence of effect on gene expression level. Both agents produced dose and time dependent decreases in the number of IL-1 receptors (IL-1R) on OA chondrocytes. This effect was mediated through a reduction in the level of the type I IL-1R as shown by experiments using a blocking monoclonal antibody against this receptor type. Both agents also markedly reduced the IL-1 induced synthesis and expression of stromelysin 1. Neither diacerhein nor rhein significantly affected the level of synthesis of TNF-alpha or the level of TNF-R. CONCLUSION: Diacerhein and rhein can effectively inhibit the synthesis of IL-1beta on human OA synovium, as well as the action of this cytokine at the cartilage level, by reducing the number of chondrocyte IL-1R. The effects of these agents seemed "selective" to the IL-1 system.
Asunto(s)
Antraquinonas/farmacología , Condrocitos/metabolismo , Interleucina-1/metabolismo , Osteoartritis/metabolismo , Membrana Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anciano , Northern Blotting , Células Cultivadas , Condrocitos/efectos de los fármacos , Técnicas de Cultivo , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Lipopolisacáridos/farmacología , Masculino , Metaloproteinasa 3 de la Matriz/metabolismo , Persona de Mediana Edad , Receptores de Interleucina-1/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Membrana Sinovial/efectos de los fármacosRESUMEN
OBJECTIVES: To evaluate the effect of therapeutic and pharmacologic concentrations of two non-steroidal anti-inflammatory drugs (NSAIDs), nimesulide and naproxen, on the synthesis of urokinase (uPA), plasminogen activator inhibitor (PAI-1) and interleukin-6 (IL-6) in human synovial fibroblasts isolated from osteoarthritis (OA) patients. METHODS: Urokinase, PAI-1, and IL-6 production were measured by specific ELISA. RESULTS: Nimesulide and naproxen induced a dose-dependent decrease in uPA synthesis. The two drugs, at therapeutic concentrations, exerted a stimulatory effect on the synthesis of PAI-1 whereas the synthesis of IL-6 was significantly reduced by both NSAIDs. CONCLUSION: The results of this study indicate some of the mechanisms by which nimesulide and naproxen could exert their effects on the arthritis process. The suppressive action of the two drugs on the synthesis of uPA, while stimulating PAI-1 production, may have a positive impact on the balance of plasminogen activator/inhibitor, which could help reduce cartilage catabolism.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Fibroblastos/metabolismo , Interleucina-6/metabolismo , Osteoartritis/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Anciano , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibroblastos/efectos de los fármacos , Humanos , Masculino , Naproxeno/farmacología , Sulfonamidas/farmacología , Líquido Sinovial/citologíaRESUMEN
OBJECTIVE: To study, in vivo, the therapeutic effectiveness of tenidap, an antirheumatic drug, on the progression of lesions in an experimental osteoarthritis (OA) dog model. The action of tenidap on the activity and expression of metalloproteases in cartilage, as well as on the bioactivity of interleukin-1 (IL-1) in synovial fluid, was determined. METHODS: The anterior cruciate ligament of the right stifle joint of 20 mongrel dogs was sectioned through a stab wound. Dogs were divided into 3 groups: group I (n = 7) received no treatment, group II (n = 6) was treated with oral omeprazole (20 mg/day), and group III (n = 7) received oral omeprazole (20 mg/day) and a therapeutic dosage of oral tenidap (3 mg/kg twice daily). Four weeks following surgery, the untreated dogs (group I) were killed, and drug treatments were begun for the other dogs (groups II and III). These dogs received medications for 8 weeks (weeks 4-12) and then were killed. Evaluations were made of the incidence and size of osteophytes as well as of the size and grade of cartilage erosions on both the condyles and plateaus. Histologic examination of the severity of the cartilage lesions and synovial inflammation was also performed. Activity levels of collagenase, stromelysin, and gelatinase as well as collagenase-1, collagenase-3, and stromelysin-1 messenger RNA were determined in the cartilage. The level of IL-1 activity in the synovial fluid was also measured. RESULTS: Among the dogs with OA, lesions were more severe at 12 weeks than at 4 weeks. Group III (tenidap-treated) dogs had a slightly reduced incidence of osteophytes compared with the group II (12-week OA) dogs (71% versus 100%), and the size of the osteophytes was significantly diminished (mean +/- SEM 1.75 +/- 0.69 mm versus 4.38 +/- 0.64 mm). Macroscopically, tenidap decreased the size (condyles 6.00 +/- 2.18 mm2 versus 21.08 +/- 6.70 mm2, plateaus 15.50 +/- 4.77 mm2 versus 35.0 +/- 3.64 mm2) and the grade (condyles 0.57 +/- 0.20 versus 1.17 +/- 0.21, plateaus 1.07 +/- 0.22 versus 2.00 +/- 0.25) of the cartilage lesions compared with the 12-week OA dogs. At the histologic level, the severity of cartilage lesions was also decreased in the tenidap-treated dogs versus the 12-week OA dogs, both on the condyles (3.43 +/- 0.54 versus 5.55 +/- 0.38) and on the plateaus (3.39 +/- 0.35 versus 5.54 +/- 0.60). All 3 OA groups showed a significant and similar level of synovial inflammation. Tenidap markedly decreased collagenase, stromelysin, and gelatinase activity, as well as the level of expression of collagenase-3 in the cartilage. Interestingly, the activity level of IL-1 in synovial fluid was also significantly reduced in the tenidap-treated dogs. CONCLUSION: Tenidap markedly reduced the severity of OA lesions, indicating the effect of this drug in decreasing the progression of disease. It appears that the drug acts by reducing the activity and/or expression of metalloproteases in cartilage, a process known to play a major role in the pathophysiology of OA lesions. This effect could be mediated by the suppressive effect of tenidap on IL-1 activity.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Indoles/farmacología , Osteoartritis/fisiopatología , Animales , Cartílago Articular/enzimología , Colagenasas/metabolismo , Modelos Animales de Enfermedad , Perros , Gelatinasas/metabolismo , Expresión Génica , Interleucina-1/antagonistas & inhibidores , Interleucina-1/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/genética , Osteoartritis/tratamiento farmacológico , Oxindoles , Líquido Sinovial/química , Membrana Sinovial/anatomía & histologíaRESUMEN
OBJECTIVE: To investigate the in vivo effect of recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) on the development of lesions and the expression of metalloproteases in the canine experimental osteoarthritis (OA) model. METHODS: The right anterior cruciate ligament was sectioned percutaneously in 3 groups of dogs. The control group (n = 5) received an intraarticular injection of sterile physiologic saline (1 ml) twice weekly for 4 weeks starting on the day of surgery. The remaining 2 groups received intraarticular injections of either 2 mg (n = 6) or 4 mg (n = 5) rHuIL-1Ra in 1 ml of physiologic saline according to the same schedule as the first group. All dogs were killed 4 weeks after surgery. The macroscopic appearance of femoral condyle osteophytes and the size and severity of cartilage lesions on femoral condyles and tibial plateaus were evaluated, as were the histologic features of cartilage and synovial membrane. Levels of collagenase-1 and stromelysin-1 messenger RNA expression in cartilage and synovium were determined by Northern blotting. RESULTS: Recombinant human IL-1Ra exerted a dose-dependent protective effect on the development of osteophytes and cartilage lesions in vivo. Treatment with rHuIL-1Ra reduced the incidence (saline-treated group 70%, 2 mg rHuIL-1Ra-treated group 42%, 4 mg rHuIL-1Ra-treated group 20%) and size (saline-treated group 2.3 +/- 0.7 mm [mean +/- SEM], 2 mg rHuIL-1Ra-treated group 0.7 +/- 0.3 mm, 4 mg rHuIL-1Ra-treated group 0.5 +/- 0.3 mm) of femoral condyle osteophytes. In addition, a dose-dependent decrease in the size (saline-treated group 24.40 +/- 8.17 mm2, 2 mg rHuIL-1Ra-treated group 20.90 +/- 8.01 mm2, 4 mg rHuIL-1Ra-treated group 7.70 +/- 5.16 mm2) and the grade (0-4 scale; saline-treated group 1.20 +/- 0.29, 2 mg rHuIL-1Ra-treated group 1.00 +/- 0.26, 4 mg rHuIL-1Ra-treated group 0.30 +/- 0.21) of the tibial plateau cartilage lesions was found, with a significant difference (P < 0.04) reached only with 4 mg rHuIL-1Ra. Similarly, the histologic lesions in dogs treated with 4 mg rHuIL-1Ra (Mankin scale; mean +/- SEM 2.95 +/- 0.53) were significantly less severe (P < 0.002) compared with those in the saline-treated group (4.95 +/- 0.54). Importantly, rHuIL-1Ra treatment led to a significant reduction (P < 0.005) of collagenase-1 expression in OA cartilage. CONCLUSION: This study demonstrated that intraarticular injections of rHuIL-1Ra can protect against the development of experimentally induced OA lesions. This effect could result, at least in part, from a reduction of collagenase-1 expression. However, other catabolic processes involved in the degradation of OA cartilage may also be affected.
Asunto(s)
Colagenasas/metabolismo , Cabeza Femoral/patología , Osteoartritis/prevención & control , Sialoglicoproteínas/farmacología , Animales , Secuencia de Bases , Modelos Animales de Enfermedad , Perros , Femenino , Humanos , Inyecciones Intraarticulares , Proteína Antagonista del Receptor de Interleucina 1 , Masculino , Metaloproteinasa 3 de la Matriz/metabolismo , Osteoartritis/enzimología , Osteoartritis/patología , ARN Mensajero/metabolismo , Sialoglicoproteínas/administración & dosificación , Sialoglicoproteínas/análisis , Líquido Sinovial/química , Membrana Sinovial/patología , Tibia/patologíaRESUMEN
The degradation of osteoarthritic (OA) cartilage is likely related to the synthesis and the release of catabolic factors by chondrocytes. Nitric oxide (NO) has recently been suggested as playing a role in cartilage degradation. Since NO production is largely dependent on stimulation by IL-1, its effects on factors regulating the IL-1 biological activity, such as IL-1ra, are of the utmost importance. This study examined and compared the level of NO production by normal and OA cartilage and chondrocytes, as well as studied the effect of IL-1-induced NO production on the synthesis and steady-state mRNA of interleukin-1 receptor antagonist (IL-1ra). The NO baseline production by normal cartilage explants was undetectable but inducible by rhIL-1 beta. OA cartilage spontaneously produced NO. About a two-fold increase in NO production was found in OA rhIL-1 beta-stimulated (0.5-100 units/ml) cartilage as compared with the similarly stimulated normal cartilage. on chondrocytes rhIL-1 beta-stimulation (0.5-100 units/ml) produced a dose-dependent enhancement of both NO production and IL-1ra synthesis. Treatment with 200 microM N(g)-monomethyl-L-arginine (L-NMA), a well known NO synthase inhibitor, induced over 70% inhibition of the NO production and a marked increased IL-1ra synthesis (average of 84%) and expression (mRNA level). Inhibition of prostaglandin synthesis by indomethacin had no effect on both the NO production or the IL-1ra level. In the present study, we demonstrated the capacity of OA cartilage to produce a larger amount of NO than the normal controls, both in spontaneous and IL-1-stimulated conditions. These data support the notion that, in vivo, OA chondrocytes are stimulated by factors, possibly IL-1, which in turn may induce the expression of NO synthase, thus the synthesis of NO itself. Importantly, our results showed that the elevation of of NO production may be an important factor in the pathophysiology of OA since it can reduce IL-1ra synthesis by chondrocytes. As such, an increased level of IL-1, associated with a decreased IL-1ra level, may be responsible for the stimulation of OA chondrocytes by this cytokine, leading to an enhancement of cartilage matrix degradation.
Asunto(s)
Cartílago Articular/metabolismo , Óxido Nítrico/biosíntesis , Osteoartritis/metabolismo , ARN Mensajero/metabolismo , Sialoglicoproteínas/biosíntesis , Anciano , Secuencia de Bases , Northern Blotting , Cartílago Articular/efectos de los fármacos , Cartílago Articular/patología , Células Cultivadas , Densitometría , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/farmacología , Persona de Mediana Edad , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/química , Osteoartritis/patología , Sialoglicoproteínas/genéticaRESUMEN
OBJECTIVE: To compare the effects of tenidap, a new antirheumatic drug, with a nonsteroidal anti-inflammatory drug, naproxen, on the synthesis and expression of interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF-alpha), and interleukin-6 (IL-6) in rheumatoid synovium. METHODS: Human synovial membrane explants from patients with rheumatoid arthritis (RA) were incubated in the absence or presence of 20 micrograms/ml lipopolysaccharides (LPS) and tenidap at 50, 20 (therapeutic concentration), and 5 micrograms/ml or naproxen at 90 (therapeutic concentration) and 30 micrograms/ml. The levels of IL-1 beta, TNF-alpha, and IL-6 in the culture medium were measured by specific enzyme linked immunosorbent assays. The cytokine mRNA levels were quantitated by Northern blotting. RESULTS: In the absence of LPS, tenidap at 20 micrograms/ml produced a significant (p < 0.04) decrease in the IL-1 synthesis level. Under LPS stimulation, IL-1 beta synthesis was inhibited by tenidap at all concentrations tested (p < 0.01) and by naproxen at only 90 micrograms/ml (p < 0.01). Very small amounts of TNF-alpha could be detected only when the synovial membranes were stimulated with LPS. Tenidap significantly reduced LPS stimulated TNF-alpha synthesis; the maximum inhibition was noted at 20 micrograms/ml (69%, p < 0.002). Naproxen, at 90 micrograms/ml, reduced TNF-alpha synthesis by about 40% (p < 0.03) and values were similar to those with subtherapeutic concentrations (5 micrograms/ml) of tenidap. The spontaneous and LPS induced synthesis of IL-6 was significantly inhibited by tenidap at all concentrations tested, whereas neither concentration of naproxen demonstrated a significant effect. Tenidap induced a somewhat similar reduction pattern of IL-1 beta and IL-6 mRNA to that observed for cytokine synthesis. Naproxen only slightly reduced the LPS induced expression of IL-6, while enhancing the IL-1 beta expression. CONCLUSION: Tenidap and naproxen showed differences in their effects on cytokine synthesis and mRNA expression. Tenidap, at the therapeutic concentration, was most clearly differentiated from naproxen by its inhibition of IL-6, but was also a more potent modulator of IL-1 beta and TNF-alpha in RA synovial explants. The significance of these findings lies in the possible therapeutic benefit of proinflammatory cytokine suppression in joint disease.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Antirreumáticos/farmacología , Artritis Reumatoide/tratamiento farmacológico , Citocinas/efectos de los fármacos , Indoles/farmacología , Mediadores de Inflamación/farmacología , Naproxeno/farmacología , Artritis Reumatoide/patología , Northern Blotting , Citocinas/fisiología , Humanos , Técnicas In Vitro , Interleucina-1/fisiología , Interleucina-6/fisiología , Lipopolisacáridos/farmacología , Oxindoles , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/fisiopatología , Factor de Necrosis Tumoral alfa/efectos de los fármacosRESUMEN
OBJECTIVE: To investigate the effects of tenidap, a new antirheumatic drug, sodium diclofenac, a non-steroidal antiinflammatory drug, and a disease modifying antirheumatic drug, hydroxychloroquine, on the level and expression of interleukin 1 receptors (IL-1R) on synovial fibroblasts from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). In addition, the effect of tenidap on IL-1 stimulated collagenase gene expression was studied. METHODS: Binding assays were performed using [125I]-IL-1 beta as radioligand. Flow cytometry was done using a specific antibody against type I IL-1R. Protein synthesis was determined by [3H]-leucine incorporation. Levels of expression were determined by Northern Blot for collagenase and by reverse transcription polymerase chain reaction (RT-PCR) for type I IL-1R. RESULTS: Tenidap produced for both OA and RA synovial fibroblasts a dose dependent decrease in the number of IL-1 binding sites/cell. A reduction of 41% (2.5 micrograms/ml) to 81% (at therapeutic concentration, 20 micrograms/ml) was noted for OA, while 29% (2.5 micrograms/ml) to 89% (20 micrograms/ml) was found for RA cells. Diclofenac produced no effect on OA cells, and minimal inhibition of RA synovial fibroblasts was observed only at pharmacological concentration (12%, 300 mg/ml). Hydroxychloroquine had effects similar to diclofenac. The decreased number of IL-1 binding sites/cell by tenidap was time dependent and reached 93% inhibition after 48 h. The effect of tenidap appears to be posttranscriptional, judged by the marked reduction of the type I IL-1R protein/cell and the absence of effect on its mRNA level. Tenidap also markedly reduced the IL-1 induced collagenase expression in synovial fibroblasts. CONCLUSION: At therapeutic concentrations tenidap is a potent inhibitor of type I IL-1R in OA and RA synovial fibroblasts. The effect of tenidap was considerably more marked than diclofenac or hydroxychloroquine.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Artritis Reumatoide/tratamiento farmacológico , Colagenasas/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Indoles/farmacología , Osteoartritis/tratamiento farmacológico , Receptores de Interleucina-1/efectos de los fármacos , Membrana Sinovial/efectos de los fármacos , Artritis Reumatoide/metabolismo , Northern Blotting , Células Cultivadas , Colagenasas/biosíntesis , Diclofenaco/farmacología , Fibroblastos/citología , Fibroblastos/enzimología , Citometría de Flujo , Humanos , Hidroxicloroquina/farmacología , Osteoartritis/metabolismo , Oxindoles , Reacción en Cadena de la Polimerasa , Membrana Sinovial/citología , Membrana Sinovial/enzimología , Factores de TiempoRESUMEN
OBJECTIVE: To examine the effects of tenidap and diclofenac on osteoarthritic lesions and metalloprotease activity in experimental osteoarthritis (OA). METHODS: The anterior cruciate ligament of the right stifle joint of 25 mongrel dogs was sectioned by a stab wound. Seven dogs received no treatment, 6 were treated with oral omeprazole (20 mg/day), another 6 were treated with diclofenac (0.25 mg/kg/twice daily) plus omeprazole (20 mg/day), and 6 received oral tenidap (3 mg/kg/twice daily) plus omeprazole (20 mg/day). The dogs received medication for 8 weeks; all dogs were killed at the end of this period. Eight normal dogs were used as controls. Lesions were evaluated macroscopically for the incidence and size of osteophytes and the area and grade of cartilage erosions on the condyles and plateaus, along with histologic evaluation of the severity of the cartilage lesions and synovial inflammation. Stromelysin and collagenase activities and the collagenase messenger RNA (mRNA) level were measured in cartilage and synovial membrane. RESULTS: Compared with the untreated or omeprazole-treated OA groups, the dogs treated with tenidap exhibited significant reduction in the incidence (P < or = 0.001) and size (P < or = 0.0001) of osteophytes. Tenidap also significantly decreased the size and grade of cartilage macroscopic lesions, as well as the histologic severity of cartilage lesions on both condyles and plateaus. The histologic severity of synovial inflammatory reaction was also significantly reduced (P < or = 0.003) in the tenidap group. Tenidap markedly decreased stromelysin and collagenase activity in both cartilage (stromelysin P < or = 0.003; collagenase P < or = 0.01) and synovial membrane (stromelysin P < or = 0.003; collagenase P < or = 0.005). Moreover, tenidap also decreased the collagenase mRNA level in cartilage (P < or = 0.005) and synovial membrane (P < or = 0.002). Diclofenac slightly reduced the incidence and size of osteophytes and cartilage lesions, but these changes were not statistically significant. Diclofenac had no effect on the severity of synovial inflammation, metalloprotease activity, or collagenase expression. CONCLUSION: This study showed that tenidap had a more potent anti-osteoarthritic effect than diclofenac in this model. The effect of the drug in suppressing metalloprotease synthesis, a process known to play a major role in the pathophysiology of osteoarthritic lesions, may explain its mechanism of action.