RESUMEN
Our previous study showed that Leukotriene B4 can directly stimulate osteoclast differentiation independent of RANKL. In order to determine whether Leukotriene B4 could indirectly stimulate human osteoclast differentiation through increasing RANKL expression of rheumatoid arthritis fibroblast-like synoviocytes, we utilize the coculture model of rheumatoid arthritis fibroblast-like synoviocytes and monocyte, which were stimulated in the presence of 2.5 ng/ml M-CSF in the control group, 2.5 ng/ml M-CSF+10(-8)M LTB4 in the experimental group a, and 2.5 ng/ml M-CSF+10(-8)M LTB4+100 ng/ml OPG in the experimental group b. After culture for 3 weeks, the number of multinucleated TRAP staining positive osteoclast-like cells stained with TRAP was counted to evaluate the differentiation effect in each group. There was almost no osteoclast-like cell in the control group and the experimental group b. There were many osteoclast-like cells in the experimental group a. These results indicated that Leukotriene B4 is capable of inducing osteoclast differentiation by a RANKL-dependent mechanism.
Asunto(s)
Artritis Reumatoide/patología , Diferenciación Celular/efectos de los fármacos , Fibroblastos/metabolismo , Leucotrieno B4/farmacología , Osteoclastos/metabolismo , Osteoclastos/patología , Ligando RANK/biosíntesis , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/fisiopatología , Resorción Ósea , Células Cultivadas , Técnicas de Cocultivo , Fibroblastos/patología , Humanos , Factor Estimulante de Colonias de Macrófagos/farmacología , Monocitos/metabolismo , Monocitos/patología , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteogénesis/inmunología , Ligando RANK/genética , Ligando RANK/inmunología , Proteínas Recombinantes/farmacología , Membrana Sinovial/patologíaRESUMEN
OBJECTIVE: To explore the effect of programmed cell death 5(PDCD5) on apoptosis of rheumatoid arthritis fibroblast-like synoviocytes(RA FLS) induced by triptolide. METHODS: Cultured synovial cells in vitro from RA patients were transfected with Ad-PDCD5.At protein level, expression of PDCD5 in RA FLS infected with Ad-PDCD5 was detected by Western blot.RA FLS infected with Ad-PDCD5 were cultured in presence or absence of triptolide and apoptosis of RA FLS was determined by flow cytometry. RESULTS: Infection of RA FLS with increasing concentrations of Ad-PDCD5(50-300 MOI) resulted in a does-dependent increase in the production of PDCD5. Apoptotic cells percentage for noinfection group, Ad-null group and Ad-PDCD5 group were(22.41 +/- 3.87)%, (28.77 +/- 12.97)% and (48.87 +/- 12.69)%, respectively. Alternatively, infection without addition of triptolide stimuli had no effect. The data showed that gene transfection of PDCD5 alone without addition of triptolide was not sufficient to activate RA FLS apoptosis, PDCD5 acted as an enhancer rather than inductor of apoptosis. CONCLUSION: Overexpression of PDCD5 could enhance apoptosis of RA FLS induced by triptolide, PDCD5 may be a potential therapeutic target to RA.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Artritis Reumatoide/patología , Diterpenos/farmacología , Proteínas de Neoplasias/metabolismo , Fenantrenos/farmacología , Membrana Sinovial/patología , Adenoviridae/genética , Proteínas Reguladoras de la Apoptosis/genética , Artritis Reumatoide/metabolismo , Células Cultivadas , Compuestos Epoxi/farmacología , Fibroblastos/patología , Humanos , Proteínas de Neoplasias/genética , Membrana Sinovial/metabolismo , TransfecciónRESUMEN
Apoptosis is reduced in the synovial tissue of patients with rheumatoid arthritis (RA), possibly due to decreased expression of pro-apoptotic genes. Programmed Cell Death 5 (PDCD5) has been recently identified as a protein that mediates apoptosis. Although PDCD5 is down-regulated in many human tumors, the role of PDCD5 in RA has not been investigated. Here we report that reduced levels of PDCD5 mRNA and protein are detected in RA synovial tissue (ST) and fibroblast-like synoviocytes (FLS) than in tissue and cells from patients with osteoarthritis (OA). We also report differences in the PDCD5 expression pattern in tissues from patients with these two types of arthritis. PDCD5 showed a scattered pattern in rheumatoid synovium compared with OA, in which the protein labeling was stronger in the synovial lining layer than in the sublining. We also observed increased expression and nuclear translocation of PDCD5 in RA patient-derived FLS undergoing apoptosis. Finally, overexpression of PDCD5 led to enhanced apoptosis and activation of caspase-3 in triptolide-treated FLS. We propose that PDCD5 may be involved in the pathogenesis of RA. These data also suggest that PDCD5 may serve as a therapeutic target to enhance sensitivity to antirheumatic drug-induced apoptosis in RA.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis , Artritis Reumatoide/genética , Fibroblastos/patología , Proteínas de Neoplasias/fisiología , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Antiinflamatorios no Esteroideos/farmacología , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Caspasa 3/metabolismo , Células Cultivadas , Diterpenos/farmacología , Compuestos Epoxi/farmacología , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fenantrenos/farmacología , Distribución Tisular , TransfecciónRESUMEN
OBJECTIVE: To investigate quantification of expression of LTB4 inducing IL-1beta and TNF-alpha at mRNA level in synovial membrane cells of rheumatoid arthritis. METHODS: Primary cultured synovial cells from RA patients were treated with exogenous LTB4, MK-886 (inhibitor of 5-lipoxygenase activating protein) and Bestatin(inhibitor of leukotriene A4 hydrolase) in the presence of LIT respectively, expressions of TNF-alpha and IL-1beta were detected at mRNA level by Real-time Quantitative PCR. RESULTS: Expressions of basic TNF-alpha (TNF-alpha/GAPDH) and IL-beta (IL-beta/GAPDH) at mRNA level in primary cultured synovial cells were 0.02 +/- 0.00 and 0.16 +/- 0.01 respectively. LTB4 (10(-9) mol/L-10(-8) mol/L) was shown to induce dose-dependent increase of mRNA expression of TNF-alpha. (7-15 times) and IL-1beta (1 time) , endogenous product of LTB4 by LIT significantly increased mRNA expressions of TNF-alpha (145 times) and IL-1beta (12 times) respectively. LIT-treated synoviocytes with addition of MK-886 (5-LOX exciting protein FLAP inhibitor) (1-10 micromol/L) were inhibited to secrete LTB4 dose-dependently, following the markedly down-regulated expressions of TNF-alpha (15%-66%) and IL-1beta (41%-71%) at mRNA level . Bestatin(100 mg/L) could also remarkably diminish LTB4-induced mRNA expressions of TNF-alpha(86%) and IL-1beta (79%). CONCLUSION: LTB4 of synovial membrance cells in rheumatoid arthritis could induce expressions of TNF-alpha and IL-1beta at mRNA level, and their expression at mRNA level had been quantified successfully. It is a beneficial help to quantify all kinds of cytokines in methodology.
Asunto(s)
Interleucina-1beta/genética , Leucotrieno B4/farmacología , Membrana Sinovial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Células Cultivadas , Expresión Génica/efectos de los fármacos , Humanos , Indoles/farmacología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Membrana Sinovial/citología , Membrana Sinovial/metabolismoRESUMEN
Leukotriene B4, as a kind of 5-lipoxygenase metabolite of arachidonic acid, is known to influence osteoclast formation and bone resorption. In order to determine whether Leukotriene B4 could directly stimulate human osteoclast differentiation and activation independent of RANKL (ODF), three different concentrations of Leukotriene B4 (10(-9)M, 10(-8)M, 10(-7)M) were added to the culture of CD14+ monocyte fraction of peripheral blood mononuclear cell (PBMC) in the presence of macrophage colony-stimulating factor (M-CSF). Under these conditions, Leukotriene B4 could induce multinucleated cells, which were positive for Tartrate-resistant acidic phosphatase (TRAP) staining and capable of bone resorption. Addition of osteoprotegerin (OPG) to PBMC cultures does not abrogate osteoclast formation induced by LTB4. Osteoclastogenesis induced by Leukotriene B4 were dose-dependently increased and weaker than that of RANKL. These results indicated that Leukotriene B4, elevated in many inflammatory diseases, is directly capable of inducing osteoclast formation by a RANKL-independent mechanism.
Asunto(s)
Leucotrieno B4/farmacología , Monocitos/efectos de los fármacos , Osteoclastos/citología , Resorción Ósea , Proteínas Portadoras/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Glicoproteínas/farmacología , Humanos , Factor Estimulante de Colonias de Macrófagos , Glicoproteínas de Membrana/farmacología , Monocitos/citología , Osteoprotegerina , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares , Receptores del Factor de Necrosis TumoralRESUMEN
OBJECTIVE: To investigate the expression of extracellular matrix metalloproteinase induced (EMMPRIN) in the interface tissue, and explore the role of EMMPRIN in the aseptic loosening of prostheses. METHODS: Immunohistochemistry was performed to characterize the EMMPRIN-expressing cells at sites of interface tissue around aseptic loosened hip prostheses in 16 cases. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to study the existence of EMMPRIN mRNA in interface tissue samples. And it was followed up by computer assisted image analysis in order to detect the A values of their expression. Synovium of hip joint of 8 femoral neck fracture were in control group. RESULTS: Strong immunostaining of EMMPRIN was found in the macrophages and fibroblasts of lining-like layers and vascular endothelium of synovial membrane-like interface tissue around loosened prostheses. Expression of EMMPRIN was significantly higher in interface tissue than the control synovium (z=-3.252, P=0.001). RT-PCR of interface tissue samples disclosed the presence of EMMPRIN mRNA of 14 cases. In interface tissue, the A value of EMMPRIN increased significantly compared to control synovium (P<0.01). CONCLUSION: Over-expression of EMMPRIN up-regulates the production of matrix metalloproteinase (MMPs) in the interface tissue. And it can promote the bone destruction around prostheses. Thereby it may be one of methods to prevent and treat aseptic loosening of prostheses by repression the biology activity of EMMPRIN.
Asunto(s)
Artroplastia de Reemplazo de Cadera , Basigina/fisiología , Prótesis de Cadera , Osteólisis/fisiopatología , Falla de Prótesis , Membrana Sinovial/metabolismo , Adulto , Anciano , Basigina/genética , Femenino , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasas de la Matriz/metabolismo , Persona de Mediana Edad , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: ADAM8 is a protein of a disintegrin and a metalloproteinase family that can induce osteoclast fusion and activity, perhaps via interactions involving integrin receptors and their cysteine-rich/disintegrin domains. Because loosening of hip replacement implants is characterized by foreign body giant cells and peri-implant osteoclasts, it was speculated that this molecule might be (over)expressed in the synovial membrane-like interface tissues. METHODS: In situ hybridization; immunohistochemical staining with or without tartrate-resistant acid phosphatase (TRAP) staining; image analysis/morphometry; isolation, amplification, and cloning of ADAM8; nucleotide sequencing; quantitative reverse transcriptase-polymerase chain reaction (RT-PCR); and Western blot. RESULTS: In situ hybridization disclosed ADAM8 mRNA in mono- and multinuclear cells in both interface and control synovial samples. Quantitative RT-PCR revealed high ADAM8 mRNA copy numbers in interface tissue (p < 0.01). Accordingly, extensive ADAM8 immunoreactivity was observed in the lining-like layers and sublining areas of interface tissue (p < 0.001). A 65 kDa ADAM8 band in Western blot of tissue extracts confirmed these findings. ADAM8/TRAP double staining showed close spatial relationships of ADAM8 positive precursor cells with other precursors and/or TRAP-positive multinuclear cells. CONCLUSION: ADAM8 is (over)expressed in tissues around aseptically loosened total hip implants, which are characterized by chronic foreign body inflammation and peri-implant bone loss. This is compatible with a role for ADAM8 in the formation of foreign body giant cells and osteoclasts.