RESUMEN
OBJECTIVE: The mechanistic link between Janus kinase (JAK) signaling and structural damage to arthritic joints in rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate how selective inhibition of JAK with tofacitinib (CP-690,550) affects osteoclast-mediated bone resorption in a rat adjuvant-induced arthritis (AIA) model, as well as human T lymphocyte RANKL production and human osteoclast differentiation and function. METHODS: Hind paw edema, inflammatory cell infiltration, and osteoclast-mediated bone resorption in rat AIA were assessed using plethysmography, histopathologic analysis, and immunohistochemistry; plasma and hind paw tissue levels of cytokines and chemokines (including RANKL) were also assessed. In vitro RANKL production by activated human T lymphocytes was evaluated by immunoassay, while human osteoclast differentiation and function were assessed via quantitative tartrate-resistant acid phosphatase staining and degradation of human bone collagen, respectively. RESULTS: Edema, inflammation, and osteoclast-mediated bone resorption in rats with AIA were dramatically reduced after 7 days of treatment with the JAK inhibitor, which correlated with reduced numbers of CD68/ED-1+, CD3+, and RANKL+ cells in the paws; interleukin-6 (transcript and protein) levels were rapidly reduced in paw tissue within 4 hours of the first dose, whereas it took 4-7 days of therapy for RANKL levels to decrease. Tofacitinib did not impact human osteoclast differentiation or function, but did decrease human T lymphocyte RANKL production in a concentration-dependent manner. CONCLUSION: These results suggest that the JAK inhibitor tofacitinib suppresses osteoclast-mediated structural damage to arthritic joints, and this effect is secondary to decreased RANKL production.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Artritis Experimental/metabolismo , Quinasas Janus/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Ligando RANK/metabolismo , Animales , Artritis Experimental/inmunología , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/inmunología , Resorción Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Quinasas Janus/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Monocitos/citología , Monocitos/efectos de los fármacos , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Piperidinas , Ratas , Ratas Endogámicas Lew , Transducción de Señal/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimologíaRESUMEN
In this study, a chronic yet synchronized version of the K/BxN mouse, the KRN-cell transfer model (KRN-CTM), was developed and extensively characterized. The transfer of purified splenic KRN T cells into T cell-deficient B6.TCR.Calpha(-/-)H-2(b/g7) mice induced anti-glucose 6-phosphate isomerase antibody-dependent chronic arthritis in 100% of the mice with uniform onset of disease 7 days after T cell transfer. Cellular infiltrations were assessed by whole-ankle transcript microarray, cytokine and chemokine levels, and microscopic and immunohistochemical analyses 7 through 42 days after T cell transfer. Transcripts identified an influx of monocytes/macrophages and neutrophils into the ankles and identified temporal progression of cartilage damage and bone resorption. In both serum and ankle tissue there was a significant elevation in interleukin-6, whereas macrophage inflammatory protein-1 alpha and monocyte chemotactic protein-1 were only elevated in tissue. Microscopic and immunohistochemical analyses revealed a time course for edema, synovial hypertrophy and hyperplasia, infiltration of F4/80-positive monocytes/macrophages and myeloperoxidase-positive neutrophils, destruction of articular cartilage, pannus invasion, bone resorption, extra-articular fibroplasia, and joint ankylosis. The KRN cell transfer model replicates many features of chronic rheumatoid arthritis in humans in a synchronized manner and lends itself to manipulation of adoptively transferred T cells and characterizing specific genes and T cell subsets responsible for rheumatoid arthritis pathogenesis and progression.
Asunto(s)
Artritis Reumatoide/patología , Modelos Animales de Enfermedad , Articulaciones/patología , Linfocitos T/patología , Linfocitos T/trasplante , Animales , Artritis Reumatoide/etiología , Artritis Reumatoide/metabolismo , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunohistoquímica , Inflamación , Articulaciones/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Transgénicos , Monocitos/metabolismo , Monocitos/patología , Linfocitos T/metabolismoRESUMEN
In this report, we show that apoptosis signal-regulating kinase 1(-/-) (ASK1 KO) mice were resistant to inflammatory arthritis induced in the K/BxN serum transfer model of rheumatoid arthritis (RA). The p38 inhibitor, SD-0006 was administered to wild type (WT) mice as a comparator. Both ASK1 KO and p38 inhibition resulted in marked attenuation of edema, cartilage damage, bone resorption, and general inflammatory responses. Transcriptional profiling of mRNA prepared from paw tissue demonstrated that the production of many proinflammatory genes including cytokines, chemokines, and extracellular matrix degradative enzymes were maintained at basal levels by either ASK1 KO or prophylactic p38 MAPK inhibition. In the mouse whole blood (MWB) assay, tumor necrosis factor-α (TNF-α)-induced KC and CCL2 levels and also LPS-induced interleukin-6 (IL-6), CCL2, and KC levels in MWB from ASK1 KO were significantly lower than those from WT. Furthermore, both p38 and JNK were activated by TNF-α in human synovial fibroblasts isolated from RA patients (RASF). SD-0006 or SP600125, a JNK inhibitor, partially blocked the elevation of IL-6 production in RASF following stimulation with TNF-α. In contrast, dual inhibition with both p38/JNK inhibitors almost completely abolished TNF-α-induced IL-6 production from these cells. Ablation of ASK1 expression in RASF using siRNA for ASK1 resulted in inhibition of TNF-α-induced IL-6 and PGE(2) production. This study is the first to suggest that ASK1 is critical for the development of RA and that ASK1 may be involved in the production of proinflammatory mediators in response to TNF-α stimulation in the RA joint.
Asunto(s)
Apoptosis/fisiología , Artritis/inducido químicamente , MAP Quinasa Quinasa Quinasa 5/metabolismo , Animales , Artritis/patología , Artritis Reumatoide/metabolismo , Células Cultivadas , Dinoprostona/genética , Dinoprostona/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Interleucina-6 , MAP Quinasa Quinasa Quinasa 5/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pirazoles/toxicidad , Pirimidinas/toxicidad , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.
Asunto(s)
Enfermedades Gastrointestinales/inducido químicamente , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Enfermedades Linfáticas/inducido químicamente , Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Linfocitos B/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colon/efectos de los fármacos , Colon/patología , Perros , Femenino , Enfermedades Gastrointestinales/patología , Hemorragia Gastrointestinal/inducido químicamente , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Lineales , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Enfermedades Linfáticas/patología , Macaca fascicularis , Masculino , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Bazo/citología , Bazo/metabolismo , Linfocitos T/metabolismo , Pruebas de Toxicidad Aguda , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Zileuton, a redox and iron chelator 5-lipoxygenase (5-LOX) inhibitor and, leukotriene receptor antagonists are presently used clinically in the long term treatment of asthma. Recent data implicate 5-LOX pathway in pain signaling. We report 5-LOX expression in the central nervous system (CNS) and analyze the pain efficacy of a new class of non redox, non iron chelating 5-LOX inhibitor. CJ-13610, 4-(3-(4-(2-methyl-1H-imidazol-1-yl) phenylthio) phenyl)-tetrahydro-2H-pyran-4-carboxamide, demonstrated antihyperalgesic activity in inflammatory pain models including the acute carrageenan model and the chronic inflammatory model using complete Freund's adjuvant. Following complete Freund's adjuvant stimulus leukotrieneB(4) concentration in the brain was elevated (9+/-1 ng/g, mean+/-S.E.M.) by about 3 times that of the control group (3+/-0.11, mean+/-S.E.M.). Hyperalgesia and leukotrieneB(4) concentration were both reversed following CJ-13610 treatment. Furthermore, we demonstrate CJ-13610 efficacy against osteoarthritis like pain using the rat medial meniscal transection model. CJ-13610 at oral doses of 0.6, 2 and 6 mg/kg/day reversed two modalities of pain in this model; tactile allodynia and weight bearing differential. Taken together, these data suggest that 5-LOX pathway and the leukotriene products are important mediators of pain.