RESUMEN
Adipokines such as adiponectin and visfatin/pre-B-cell colony-enhancing factor (PBEF) have been recently shown to contribute to synovial inflammation in rheumatoid arthritis (RA). In this study, we evaluated the pathophysiological implication of visfatin/PBEF in the molecular patterns of RA synovial tissue, focusing on RA synovial fibroblasts (RASFs), key players in RA synovium. Expression of visfatin/PBEF in synovial fluid and tissue of RA patients was detected by immunoassays and immunohistochemistry. RASFs were stimulated with different concentrations of visfatin/PBEF over varying time intervals, and changes in gene expression were evaluated at the RNA and protein levels using Affymetrix array, real-time PCR, and immunoassays. The signaling pathways involved were identified. The influence of visfatin/PBEF on fibroblast motility and migration was analyzed. In RA synovium, visfatin/PBEF was predominantly expressed in the lining layer, lymphoid aggregates, and interstitial vessels. In RASFs, visfatin/PBEF induced high amounts of chemokines such as IL-8 and MCP-1, proinflammatory cytokines such as IL-6, and matrix metalloproteinases such as MMP-3. Phosphorylation of p38 MAPK was observed after visfatin/PBEF stimulation, and inhibition of p38 MAPK showed strong reduction of visfatin-induced effects. Directed as well as general fibroblast motility was increased by visfatin/PBEF-induced factors. The results of this study indicate that visfatin/PBEF is involved in synovial fibroblast activation by triggering fibroblast motility and promoting cytokine synthesis at central sites in RA synovium.
Asunto(s)
Artritis Reumatoide/enzimología , Movimiento Celular , Citocinas/biosíntesis , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica , Mediadores de Inflamación/metabolismo , Nicotinamida Fosforribosiltransferasa/biosíntesis , Membrana Sinovial/enzimología , Artritis Reumatoide/patología , Quimiocina CCL2/biosíntesis , Femenino , Fibroblastos/patología , Perfilación de la Expresión Génica , Humanos , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Masculino , Metaloproteinasa 3 de la Matriz/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Membrana Sinovial/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: Fibrin deposits are characteristic of the synovial tissues in rheumatoid arthritis (RA). Once citrullinated, fibrin becomes an autoantigen and is thought to contribute in this way to perpetuate the disease. Our study aimed to analyse the responses of RA synovial fibroblasts (RASF) to native and citrullinated fibrin. METHODS: The transcriptome induced by fibrin in RASF was approached with whole-genome-based gene expression arrays. The upregulation of selected pro-inflammatory genes by fibrin was confirmed in additional primary cell cultures using quantitative PCR and ELISA. Citrullination reactions were carried out with recombinant human peptidylarginine deiminases (PAD) 2 and 4. RESULTS: In the whole-genome array native fibrin was found to modulate the gene expression profile of RASF, particularly upregulating mRNA levels of several pro-inflammatory cytokines. The induction of interleukin (IL)-6 and IL-8 by fibrin was confirmed in additional samples at both the mRNA and the protein level. Blocking and knockdown experiments showed the participation of toll-like receptor (TLR)4 in the induction of both cytokines. As compared with the native macromolecule, PAD2-citrullinated fibrin induced significantly higher expression of the pro-inflammatory cytokines in these cells. CONCLUSIONS: Our results suggest that fibrin mediates inflammatory responses in RASF via a TLR4 pathway. In this way, fibrin and particularly its citrullinated form may contribute to sustain the cytokine burst in RA.
Asunto(s)
Artritis Reumatoide/metabolismo , Fibrina/farmacología , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Artritis Reumatoide/patología , Supervivencia Celular , Células Cultivadas , Citrulina/metabolismo , Citocinas/genética , Citocinas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Hidrolasas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Arginina Deiminasa Proteína-Tipo 2 , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMEN
OBJECTIVES: Several clinical studies have suggested the adipocytokine adiponectin is involved in the progression of rheumatoid arthritis (RA). From this point of view, adiponectin might present a new therapeutic target. However, as adiponectin also exerts beneficial effects in the human organism, a strategy that would allow its detrimental effects to be abolished while maintaining the positive effects would be highly favourable. To elucidate such a strategy, the authors analysed whether the different adiponectin isoforms induce diverging effects, especially with regard to rheumatoid arthritis synovial fibroblasts (RASF), a central cell type in RA pathogenesis capable of invading into and destroying cartilage. METHODS: Affymetrix microarrays were used to screen for changes in gene expression of RASF. Messenger RNA levels were quantified by real-time PCR, protein levels by immunoassay. The migration of RASF and primary human lymphocytes was analysed using a two-chamber migration assay. RESULTS: In RASF, the individual adiponectin isoforms induced numerous genes/proteins relevant in RA pathogenesis to clearly different extents. In general, the most potent isoforms were the high molecular weight/middle molecular weight isoforms and the globular isoform, while the least potent isoform was the adiponectin trimer. The chemokines secreted by RASF upon adiponectin stimulation resulted in an increased migration of RASF and lymphocytes. CONCLUSION: The results clearly suggest a pro-inflammatory and joint-destructive role of all adiponectin isoforms in RA pathophysiology, indicating that in chronic inflammatory joint diseases the detrimental effects outweigh the beneficial effects of adiponectin.
Asunto(s)
Adiponectina/metabolismo , Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Adiponectina/inmunología , Artritis Reumatoide/inmunología , Células Cultivadas , Quimiocinas/biosíntesis , Fibroblastos/inmunología , Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Membrana Sinovial/metabolismoRESUMEN
OBJECTIVE: To analyse the expression of SIRT1 in synovial tissues and cells of patients with rheumatoid arthritis (RA) and to study the function of SIRT1 in inflammation and apoptosis in RA. METHODS: Levels of SIRT1 expression were analysed in synovial tissues and cells from patients with RA by real-time PCR and western blotting before and after stimulation with toll-like receptor ligands, tumour necrosis factor α (TNFα) and interleukin 1ß (IL-1ß). Immunohistochemistry was used to study the localisation of SIRT1. Fluorescence activated cell sorting analysis was performed to investigate the effect of SIRT1 on apoptosis. Peripheral blood monocytes and rheumatoid arthritis synovial fibroblasts (RASFs) were transfected with wild-type or enzymatically inactive SIRT1 expression vectors or with siRNA targeting SIRT1. Cytokine analysis of IL-6, IL-8 and TNFα were performed by ELISA to study the role of SIRT1 on proinflammatory mediators of RA. RESULTS: SIRT1 was found to be constitutively upregulated in synovial tissues and cells from patients with RA compared to osteoarthritis. TNFα stimulation of RASFs and monocytes resulted in further induced expression levels of SIRT1. Silencing of SIRT1 promoted apoptosis in RASFs, whereas SIRT1 overexpression protected cells from apoptosis. Inhibition of SIRT1 enzymatic activity by inhibitors, siRNA and overexpression of an enzymatically inactive form of SIRT1 reduced lipopolysaccharide-induced levels of TNFα in monocytes. Similarly, knockdown of SIRT1 resulted in a reduction of proinflammatory IL-6 and IL-8 in RASFs. CONCLUSION: The TNFα-induced overexpression of SIRT1 in RA synovial cells contributes to chronic inflammation by promoting proinflammatory cytokine production and inhibiting apoptosis.
Asunto(s)
Apoptosis/fisiología , Artritis Reumatoide/metabolismo , Citocinas/biosíntesis , Sirtuina 1/fisiología , Membrana Sinovial/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/patología , Biopsia , Carbazoles/farmacología , Células Cultivadas , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Masculino , Persona de Mediana Edad , Monocitos/efectos de los fármacos , FN-kappa B/fisiología , Osteoartritis/metabolismo , Osteoartritis/patología , ARN Mensajero/genética , Transducción de Señal/fisiología , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/biosíntesis , Sirtuina 1/genética , Membrana Sinovial/patología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVES: To dissect the role of toll-like receptor (TLR) signalling and B cell survival/proliferating factors in the crosstalk between rheumatoid arthritis synovial fibroblasts (RASF) and B cells. METHODS: RASF, rheumatoid arthritis dermal fibroblasts (RADF) and osteoarthritis synovial fibroblasts (OASF) were analysed for the expression of B cell survival/proliferating factors BAFF and APRIL in resting conditions and upon stimulation with TLR2/TLR3/TLR4 ligands. Unswitched IgD+ B cells were co-cultured with RASF/OASF/RADF in the presence/absence of TLR ligands and with/without BAFF/APRIL blocking antibodies. Activation-induced cytidine deaminase (AID) mRNA expression, Iγ-Cµ and Iα-Cµ circular transcripts (CTs; markers of ongoing class-switching to IgG and IgA) and IgM/A/G production were measured to assess functional activation of B cells. RESULTS: TLR3 and to a lesser extent TLR4, but not TLR2 stimulation, induced up to â¼1000-fold BAFF mRNA and increased soluble BAFF release. APRIL was less significantly regulated by TLR3. Resting and TLR3-stimulated RASF released higher levels of BAFF/APRIL compared with RADF. TLR3 stimulation of RASF but not RADF in co-culture with B cells strongly enhanced AID expression, Iγ-Cµ and Iα-Cµ CTs and class-switching to IgG/IgA. Blockade of BAFF/APRIL signalling completely inhibited TLR3-induced, RASF-dependent expression of AID, CTs and the secretion of IgG/IgA. CONCLUSIONS: RASF produce high levels of BAFF and APRIL constitutively and in response to TLR3 stimulation. These factors are critical in directly modulating AID expression, class-switch recombination and IgG/IgA production in IgD+ B cells. Overall, this work highlights a novel and fundamental role for the TLR3/B cell survival factor axis in sustaining B cell activation in the rheumatoid arthritis synovium.
Asunto(s)
Artritis Reumatoide/inmunología , Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Membrana Sinovial/inmunología , Receptor Toll-Like 3/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Anciano , Factor Activador de Células B/biosíntesis , Factor Activador de Células B/genética , Técnicas de Cocultivo , Citidina Desaminasa/biosíntesis , Citidina Desaminasa/genética , Femenino , Fibroblastos/inmunología , Humanos , Inmunoglobulina A/biosíntesis , Cambio de Clase de Inmunoglobulina/inmunología , Inmunoglobulina D/análisis , Inmunoglobulina G/biosíntesis , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Osteoartritis/inmunología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/genética , Transducción de Señal/inmunología , Piel/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/biosíntesis , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genéticaRESUMEN
OBJECTIVE: Rheumatoid arthritis (RA) is associated with increased production of adipokines, which are cytokine-like mediators that are produced mainly in adipose tissue but also in synovial cells. Since RA synovial fibroblasts (RASFs), lymphocytes, endothelial cells, and chondrocytes are key players in the pathophysiology of RA, this study was undertaken to analyze the effects of the key adipokine adiponectin on proinflammatory and prodestructive synovial effector cells. METHODS: Lymphocytes were activated in part prior to stimulation. All cells were stimulated with adiponectin, and changes in gene and protein expression were determined by Affymetrix and protein arrays. Messenger RNA and protein levels were confirmed using semiquantitative reverse transcription-polymerase chain reaction (PCR), real-time PCR, and immunoassays. Intracellular signal transduction was evaluated using chemical signaling inhibitors. RESULTS: Adiponectin stimulation of human RASFs predominantly induced the secretion of chemokines, as well as proinflammatory cytokines, prostaglandin synthases, growth factors, and factors of bone metabolism and matrix remodeling. Lymphocytes, endothelial cells, and chondrocytes responded to adiponectin stimulation with enhanced synthesis of cytokines and various chemokines. Additionally, chondrocytes released increased amounts of matrix metalloproteinases. In RASFs, adiponectin-mediated effects were p38 MAPK and protein kinase C dependent. CONCLUSION: Our previous findings indicated that adiponectin was present in inflamed synovium, at sites of cartilage invasion, in lymphocyte infiltrates, and in perivascular areas. The findings of the present study indicate that adiponectin induces gene expression and protein synthesis in human RASFs, lymphocytes, endothelial cells, and chondrocytes, supporting the concept of adiponectin being involved in the pathophysiologic modulation of RA effector cells. Adiponectin promotes inflammation through cytokine synthesis, attraction of inflammatory cells to the synovium, and recruitment of prodestructive cells via chemokines, thus promoting matrix destruction at sites of cartilage invasion.
Asunto(s)
Adiponectina/fisiología , Artritis Reumatoide/inmunología , Artritis Reumatoide/fisiopatología , Quimiocinas/metabolismo , Fibroblastos/metabolismo , Membrana Sinovial/fisiopatología , Estudios de Casos y Controles , Células Cultivadas , Condrocitos/metabolismo , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Humanos , Inflamación/fisiopatología , Articulación de la Rodilla/fisiopatología , Linfocitos/metabolismo , Osteoartritis de la Rodilla , Análisis por Matrices de ProteínasRESUMEN
OBJECTIVE: Since fibroblasts in the synovium of patients with rheumatoid arthritis (RA) express the serine proteases fibroblast activation protein (FAP) and dipeptidylpeptidase 4 (DPP-4)/CD26, we undertook the current study to determine the functional role of both enzymes in the invasion of RA synovial fibroblasts (RASFs) into articular cartilage. METHODS: Expression of FAP and DPP-4/CD26 by RASFs was analyzed using fluorescence-activated cell sorting and immunocytochemistry. Serine protease activity was measured by cleavage of fluorogenic substrates and inhibited upon treatment with L-glutamyl L-boroproline. The induction and expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in RASFs were detected using real-time polymerase chain reaction. Densitometric measurements of MMPs using immunoblotting confirmed our findings on the messenger RNA level. Stromal cell-derived factor 1 (SDF-1 [CXCL12]), MMP-1, and MMP-3 protein levels were measured using enzyme-linked immunosorbent assay. The impact of FAP and DPP-4/CD26 inhibition on the invasiveness of RASFs was analyzed in the SCID mouse coimplantation model of RA using immunohistochemistry. RESULTS: Inhibition of serine protease activity of FAP and DPP-4/CD26 in vitro led to increased levels of SDF-1 in concert with MMP-1 and MMP-3, which are downstream effectors of SDF-1 signaling. Using the SCID mouse coimplantation model, inhibition of enzymatic activity in vivo significantly promoted invasion of xenotransplanted RASFs into cotransplanted human cartilage. Zones of cartilage resorption were infiltrated by FAP-expressing RASFs and marked by a significantly higher accumulation of MMP-1 and MMP-3, when compared with controls. CONCLUSION: Our results indicate a central role for the serine protease activity of FAP and DPP-4/CD26 in protecting articular cartilage against invasion by synovial fibroblasts in RA.
Asunto(s)
Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Cartílago Articular/patología , Dipeptidil Peptidasa 4/metabolismo , Gelatinasas/metabolismo , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Cartílago Articular/metabolismo , Células Cultivadas , Quimiocina CXCL12/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV , Modelos Animales de Enfermedad , Endopeptidasas , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/patología , Gelatinasas/antagonistas & inhibidores , Humanos , Metaloproteinasas de la Matriz/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones SCID , Membrana Sinovial/enzimología , Membrana Sinovial/patologíaRESUMEN
Rheumatoid arthritis (RA) is a chronic inflammatory disease that ultimately leads to the progressive destruction of cartilage and bone in numerous joints. There is mounting evidence for an important function of innate immunity in the pathogenesis of RA. Activation of cells by microbial components and also by endogenous molecules via Toll-like receptor (TLR) results in the production of a variety of proinflammatory cytokines, chemokines, and destructive enzymes, some of which can characteristically be found in RA.By immunohistochemistry we found elevated TLR2, 3, and 4 expressions in the rheumatoid synovium. In the synovial lining layer and at sites of invasion into cartilage, RA synovial fibroblasts (RASF) are the major cells expressing TLR2, 3, and 4. Stimulation of cultured RASF in vitro with the TLR2 ligand bacterial lipoprotein (bLP), the TLR3 ligand poly(I-C), and the TLR4 ligand LPS was shown to upregulate IL-6 as well as matrix metalloproteinases (MMPs) 1 and 3. These results suggest an important role for TLR2, 3, and 4 in the activation of synovial fibroblasts in RA leading to chronic inflammation and joint destruction.
Asunto(s)
Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Receptores Toll-Like/análisis , Receptores Toll-Like/metabolismo , Artritis Reumatoide/genética , Separación Celular , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Secciones por Congelación , Humanos , Inmunohistoquímica , Interleucina-6/inmunología , Interleucina-6/metabolismo , Ligandos , Reacción en Cadena de la Polimerasa , ARN/aislamiento & purificación , Receptores Toll-Like/genéticaRESUMEN
OBJECTIVE: Since pattern-recognition receptors (PRRs), in particular Toll-like receptors (TLRs), were found to be overexpressed in the synovium of rheumatoid arthritis (RA) patients and to play a role in the production of disease-relevant molecules, we sought to determine the expression, regulation, and function of the PRR nucleotide-binding oligomerization domain 2 (NOD-2) in RA. METHODS: Expression of NOD-2 in synovial tissues was analyzed by immunohistochemistry. Expression and induction of NOD-2 in RA synovial fibroblasts (RASFs) were measured by conventional and real-time polymerase chain reaction (PCR) analyses. Levels of interleukin-6 (IL-6) and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA) and expression of matrix metalloproteinases (MMPs) by ELISA and/or real-time PCR. NOD-2 expression was silenced with small interfering RNA. Western blotting with antibodies against phosphorylated and total p38, JNK, and ERK, as well as inhibitors of p38, JNK, and ERK was performed. Activation of NF-kappaB was measured by electrophoretic mobility shift assay. RESULTS: NOD-2 was expressed by fibroblasts and macrophages in the synovium of RA patients, predominantly at sites of invasion into articular cartilage. In cultured RASFs, no basal expression of messenger RNA for NOD-2 was detectable, but was induced by poly(I-C), lipopolysaccharide, and tumor necrosis factor alpha. After up-regulation of NOD-2 by TLR ligands, its ligand muramyl dipeptide (MDP) increased the expression of IL-6 and IL-8 via p38 and NF-kappaB. Stimulation with MDP further induced the expression of MMP-1, MMP-3, and MMP-13. CONCLUSION: Not only TLRs, but also the PRR NOD-2 is expressed in the synovium of RA patients, and activation of NOD-2 acts synergistically with TLRs in the production of proinflammatory and destructive mediators. Therefore, NOD-2 might contribute to the initiation and perpetuation of chronic, destructive inflammation in RA.
Asunto(s)
Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Membrana Sinovial/metabolismo , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacología , Artritis Reumatoide/patología , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Expresión Génica/efectos de los fármacos , Silenciador del Gen , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolisacáridos/farmacología , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , FN-kappa B/biosíntesis , Proteína Adaptadora de Señalización NOD2/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/patología , Receptores Toll-Like/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia ArribaRESUMEN
OBJECTIVE: We have recently shown a significant reduction in cytokine-induced transcription of type I collagen and fibronectin in systemic sclerosis (SSc) skin fibroblasts upon treatment with trichostatin A (TSA). Moreover, in a mouse model of fibrosis, TSA prevented the dermal accumulation of extracellular matrix. The purpose of this study was to analyze the silencing of histone deacetylase 7 (HDAC-7) as a possible mechanism by which TSA exerts its antifibrotic function. METHODS: Skin fibroblasts from patients with SSc were treated with TSA and/or transforming growth factor beta. Expression of HDACs 1-11, extracellular matrix proteins, connective tissue growth factor (CTGF), and intercellular adhesion molecule 1 (ICAM-1) was analyzed by real-time polymerase chain reaction, Western blotting, and the Sircol collagen assay. HDAC-7 was silenced using small interfering RNA. RESULTS: SSc fibroblasts did not show a specific pattern of expression of HDACs. TSA significantly inhibited the expression of HDAC-7, whereas HDAC-3 was up-regulated. Silencing of HDAC-7 decreased the constitutive and cytokine-induced production of type I and type III collagen, but not fibronectin, as TSA had done. Most interestingly, TSA induced the expression of CTGF and ICAM-1, while silencing of HDAC-7 had no effect on their expression. CONCLUSION: Silencing of HDAC-7 appears to be not only as effective as TSA, but also a more specific target for the treatment of SSc, because it does not up-regulate the expression of profibrotic molecules such as ICAM-1 and CTGF. This observation may lead to the development of more specific and less toxic targeted therapies for SSc.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/uso terapéutico , Esclerodermia Sistémica/tratamiento farmacológico , Western Blotting , Células Cultivadas , Colágeno Tipo I/biosíntesis , Colágeno Tipo III/biosíntesis , Factor de Crecimiento del Tejido Conjuntivo/análisis , Proteínas de la Matriz Extracelular/análisis , Fibroblastos/efectos de los fármacos , Histona Desacetilasas/análisis , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Reacción en Cadena de la Polimerasa , Piel/efectos de los fármacos , Factor de Crecimiento Transformador beta/uso terapéuticoRESUMEN
OBJECTIVE: MicroRNAs (miRNA) have recently emerged as a new class of modulators of gene expression. In this study we investigated the expression, regulation, and function of miR-155 and miR-146a in rheumatoid arthritis (RA) synovial fibroblasts (RASFs) and RA synovial tissue. METHODS: Locked nucleic acid microarray was used to screen for differentially expressed miRNA in RASFs treated with tumor necrosis factor alpha (TNFalpha). TaqMan-based real-time polymerase chain reaction was applied to measure the levels of miR-155 and miR-146a. Enforced overexpression of miR-155 was used to investigate the function of miR-155 in RASFs. RESULTS: Microarray analysis of miRNA expressed in RASFs treated with TNFalpha revealed a prominent up-regulation of miR-155. Constitutive expression of both miR-155 and miR-146a was higher in RASFs than in those from patients with osteoarthritis (OA), and expression of miR-155 could be further induced by TNFalpha, interleukin-1beta, lipopolysaccharide, poly(I-C), and bacterial lipoprotein. The expression of miR-155 in RA synovial tissue was higher than in OA synovial tissue. Enforced expression of miR-155 in RASFs was found to repress the levels of matrix metalloproteinase 3 (MMP-3) and reduce the induction of MMPs 3 and 1 by Toll-like receptor ligands and cytokines. Moreover, compared with monocytes from RA peripheral blood, RA synovial fluid monocytes displayed higher levels of miR-155. CONCLUSION: This study provides the first description of increased expression of miRNA miR-155 and miR-146a in RA. Based on these findings, we postulate that the inflammatory milieu may alter miRNA expression profiles in resident cells of the rheumatoid joints. Considering the repressive effect of miR-155 on the expression of MMPs 3 and 1 in RASFs, we hypothesize that miR-155 may be involved in modulation of the destructive properties of RASFs.
Asunto(s)
Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , MicroARNs/metabolismo , Membrana Sinovial/fisiopatología , Artritis Reumatoide/fisiopatología , Estudios de Cohortes , Humanos , Metaloproteinasa 3 de la Matriz/inmunología , Metaloproteinasa 3 de la Matriz/metabolismo , MicroARNs/inmunología , Monocitos/inmunología , Monocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Líquido Sinovial/citología , Líquido Sinovial/inmunología , Membrana Sinovial/metabolismoRESUMEN
OBJECTIVE: To analyze the expression, regulation, and biologic relevance of Toll-like receptors (TLRs) 1-10 in synovial and skin fibroblasts and to determine the expression levels of TLRs 2, 3, and 4 in synovial tissues from patients with early rheumatoid arthritis (RA), longstanding RA, and osteoarthritis (OA). METHODS: Expression of TLRs 1-10 in RA synovial fibroblasts (RASFs), OASFs, and skin fibroblasts was analyzed by real-time polymerase chain reaction (PCR). Fibroblasts were stimulated with tumor necrosis factor alpha, interleukin-1beta (IL-1beta), bacterial lipopeptide, poly(I-C), lipopolysaccharide, and flagellin. Production of IL-6 was determined by enzyme-linked immunosorbent assay and induction of TLRs 2-5, matrix metalloproteinases (MMPs) 3 and 13 messenger RNA by real-time PCR. Expression of TLRs 2-4 in synovial tissues was analyzed by immunohistochemistry. RESULTS: Synovial fibroblasts expressed TLRs 1-6, but not TLRs 7-10. Among the expressed TLRs, TLR-3 and TLR-4 were the most abundant in synovial fibroblasts, and stimulation of synovial fibroblasts with the TLR-3 ligand poly(I-C) led to the most pronounced increase in IL-6, MMP-3, and MMP-13. In contrast, skin fibroblasts did not up-regulate MMP-3 or MMP-13 after stimulation with any of the tested stimuli. In synovial tissues from patients with early RA, TLR-3 and TLR-4 were highly expressed and were comparable to the levels of patients with longstanding RA. These expression levels were elevated as compared with those in OA. CONCLUSION: Our findings of high expression of TLRs, particularly TLRs 3 and 4, at an early stage of RA and the reactivity of synovial fibroblasts in vitro to TLR ligands suggest that TLR signaling pathways resulting in persistent inflammation and joint destruction are activated early in the disease process.
Asunto(s)
Artritis Reumatoide/fisiopatología , Receptor Toll-Like 3/genética , Receptor Toll-Like 4/genética , Adulto , Anciano , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Células Cultivadas , Citocinas/metabolismo , Progresión de la Enfermedad , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Expresión Génica/fisiología , Humanos , Masculino , Persona de Mediana Edad , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoartritis/fisiopatología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citología , Membrana Sinovial/citología , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
OBJECTIVE: To study possible mechanisms that mediate induction of the recently described adipocytokine pre-B cell colony-enhancing factor (PBEF) in joints of patients with rheumatoid arthritis (RA), and to analyze whether levels of PBEF correlate with disease severity and whether PBEF itself has the potential to act as a proinflammatory and destructive mediator in RA. METHODS: RA synovial fibroblasts (RASFs) and monocytes were stimulated with Toll-like receptor (TLR) ligands, cytokines, and recombinant human PBEF or were transfected with PBEF expression constructs or with PBEF-specific small interfering RNA. Production of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNFalpha) was measured by enzyme-linked immunosorbent assay, and expression of matrix metalloproteinases (MMPs) was assessed by real-time polymerase chain reaction. PBEF expression in synovial tissue, synovial fluid, serum, and SFs was assessed by immunohistochemistry, in situ hybridization, Western blotting, and enzyme immunoassays. RESULTS: In RASFs, PBEF was up-regulated by TLR ligands and cytokines that are characteristically present in the joints of patients with RA. In synovial tissue, RASFs were the major PBEF-expressing cells. A predominance of PBEF was found in the synovial lining layer and at sites of invasion into cartilage. Levels of PBEF in serum and synovial fluid correlated with the degree of inflammation and clinical disease activity. Moreover, PBEF itself activated the transcription factors NF-kB and activator protein 1 and induced IL-6, IL-8, MMP-1, and MMP-3 in RASFs as well as IL-6 and TNFalpha in monocytes. PBEF knockdown in RASFs significantly inhibited basal and TLR ligand-induced production of IL-6, IL-8, MMP-1, and MMP-3. CONCLUSION: Our findings establish PBEF as a proinflammatory and destructive mediator of joint inflammation in RA and identify PBEF as a potential therapeutic target.
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Artritis Reumatoide/inmunología , Fibroblastos/inmunología , Inflamación/inmunología , Artritis Reumatoide/sangre , Artritis Reumatoide/patología , Citocinas/análisis , Humanos , Inflamación/sangre , Metaloproteinasas de la Matriz/biosíntesis , Nicotinamida Fosforribosiltransferasa/análisis , Líquido Sinovial/química , Membrana Sinovial/inmunologíaRESUMEN
Recent evidence highlighted the role of Toll-like receptors (TLRs) as key recognition structures of the innate immune system. The activation of TLRs initiates the production of inflammatory cytokines, chemokines, tissue destructive enzymes, and type I interferons. In addition, TLR signalling plays an important role in the activation and direction of the adaptive immune system by the upregulation of costimulatory molecules of antigen presenting cells. Considering the important role of TLR signalling as a critical link between innate and adaptive immunity it has been proposed that a dysregulation in TLR signalling might be associated with autoimmunity. In this review, recent studies on TLR signal transduction pathways activated by corresponding ligands are summarized and evidence for a possible role of TLR signalling in the pathogenesis of rheumatoid arthritis is discussed.
Asunto(s)
Artritis Reumatoide/etiología , Artritis Reumatoide/inmunología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Animales , Artritis Experimental/etiología , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Humanos , Receptores Toll-LikeRESUMEN
OBJECTIVE: To assess the expression of Toll-like receptor 3 (TLR-3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR-3 ligands. METHODS: TLR-3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence-activated cell sorting and real-time polymerase chain reaction techniques. TLR-3 signaling was assessed by incubating RASFs with poly(I-C), lipopolysaccharide, palmitoyl-3-cysteine-serine-lysine-4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon-beta (IFNbeta), CXCL10, CCL5, and interleukin-6 (IL-6) protein production in the culture supernatants was performed by enzyme-linked immunosorbent assays. RESULTS: TLR-3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR-3 expression was localized predominantly in the synovial lining, with a majority of the TLR-3-expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR-3 ligand poly(I-C) resulted in the production of high levels of IFNbeta, CXCL10, CCL5, and IL-6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up-regulation of these cytokines and chemokines in a TLR-3-dependent manner. CONCLUSION: Our findings demonstrate the expression of TLR-3 in RA synovial tissue and the activation of RASFs in vitro by the TLR-3 ligand poly(I-C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR-3 ligand for the stimulation of proinflammatory gene expression in RASFs.
Asunto(s)
Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Glicoproteínas de Membrana/metabolismo , ARN/metabolismo , Receptores de Superficie Celular/metabolismo , Líquido Sinovial/metabolismo , Artritis Reumatoide/patología , Células Cultivadas , Quimiocinas/metabolismo , Combinación de Medicamentos , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Técnicas para Inmunoenzimas , Inductores de Interferón/farmacología , Interferón beta/genética , Interferón beta/metabolismo , Ligandos , Necrosis , Osteoartritis/metabolismo , Osteoartritis/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Líquido Sinovial/citología , Receptor Toll-Like 3 , Receptores Toll-Like , Regulación hacia ArribaRESUMEN
To analyze the role of Toll-like receptors (TLR) in the pathogenesis of rheumatoid arthritis, we have assessed the effects of stimulation of cultured synovial fibroblasts by the TLR-2 ligand bacterial peptidoglycan. By using high density oligonucleotide microarray analysis we identified 74 genes that were up-regulated >2.5-fold. Fourteen CC and CXC chemokine genes were among the genes with the highest up-regulation. Quantitative real-time PCR analysis confirmed up-regulation of granulocyte chemotactic protein (GCP)-2, RANTES, monocyte chemoattractant protein (MCP)-2, IL-8, growth-related oncogene-2, and to a lesser extent, macrophage-inflammatory protein 1alpha, MCP-1, EXODUS, and CXCL-16. GCP-2, RANTES, and MCP-2 were detected in culture supernatants of synovial fibroblasts stimulated with peptidoglycan. Chemokine secretion induced by stimulation of rheumatoid arthritis synovial fibroblasts via TLR-2 was functionally relevant as demonstrated by chemotaxis assays. GCP-2 and MCP-2 expression, which have not been reported previously in rheumatoid arthritis, was demonstrated in synovial tissue sections of patients diagnosed with rheumatoid arthritis but not in those with osteoarthritis. Correspondingly, synovial fluid levels were significantly higher in patients diagnosed with rheumatoid arthritis as compared with osteoarthritis. Thus, we present evidence for an induction of chemokine secretion by activation of synovial fibroblasts via TLR-2, possibly contributing to the formation of inflammatory infiltrates characteristically found in rheumatoid arthritis joints.