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Antirreumáticos , Artritis Reumatoide , Diagnóstico Precoz , Humanos , Inmunomodulación , TenascinaRESUMEN
OBJECTIVES: Controlled immune responses rely on integrated crosstalk between cells and their microenvironment. We investigated whether targeting proinflammatory signals from the extracellular matrix that persist during pathological inflammation provides a viable strategy to treat rheumatoid arthritis (RA). METHODS: Monoclonal antibodies recognising the fibrinogen-like globe (FBG) of tenascin-C were generated by phage display. Clones that neutralised FBG activation of toll-like receptor 4 (TLR4), without impacting pathogenic TLR4 activation, were epitope mapped by crystallography. Antibodies stained synovial biopsies of patients at different stages of RA development. Antibody efficacy in preventing RA synovial cell cytokine release, and in modulating collagen-induced arthritis in rats, was assessed. RESULTS: Tenascin-C is expressed early in the development of RA, even before disease diagnosis, with higher levels in the joints of people with synovitis who eventually developed RA than in people whose synovitis spontaneously resolved. Anti-FBG antibodies inhibited cytokine release by RA synovial cells and prevented disease progression and tissue destruction during collagen-induced arthritis. CONCLUSIONS: Early changes in the synovial microenvironment contribute to RA progression; blocking proinflammatory signals from the matrix can ameliorate experimental arthritis. These data highlight a new drug class that could offer early, disease-specific immune modulation in RA, without engendering global immune suppression.
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Anticuerpos Monoclonales/uso terapéutico , Artritis Reumatoide/inmunología , Microambiente Celular/inmunología , Inmunoterapia/métodos , Membrana Sinovial/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Artritis Experimental , Colágeno , Citocinas/metabolismo , Progresión de la Enfermedad , Fibrinógeno/inmunología , Humanos , Ratas , Tenascina/metabolismo , Receptor Toll-Like 4/inmunologíaRESUMEN
OBJECTIVES: Findings relating to dietary intake of n-3 polyunsaturated fatty acids (PUFA) and risk of rheumatoid arthritis (RA) are mixed. Erythrocyte membrane PUFA is an accurate objective biomarker of PUFA status; however, there are little data on erythrocyte membrane PUFA and risk of RA. The objective was therefore to compare erythrocyte membrane PUFA between pre-RA individuals and matched controls from a population-based sample, and specifically to test the hypothesis that higher levels of longer chain n-3 PUFA are associated with lower risk of RA. METHODS: The European Prospective Investigation into Cancer and Nutrition (EPIC) is a large European prospective cohort study of apparently healthy populations. We undertook a nested case-control study by identifying RA cases with onset after enrolment (pre-RA) in four EPIC cohorts in Italy and Spain. Confirmed pre-RA cases were matched with controls by age, sex, centre, and date, time and fasting status at blood collection. Conditional logistic regression analysis was used to estimate associations of PUFA with the development of RA, adjusting for potential confounders including body mass index, waist circumference, education level, physical activity, smoking status and alcohol intake. RESULTS: The study analysed samples from 96 pre-RA subjects and 258 matched controls. In this analysis, the median time to diagnosis (defined as time between date of blood sample and date of diagnosis) was 6.71 years (range 0.8-15). A significant inverse association was observed with n-6 PUFA linoleic acid (LA) levels and pre-RA in the fully adjusted model (highest tertile: OR 0.29; 95% CI 0.12 to 0.75; P for trend 0.01). No association was observed with any individual n-3 PUFA, total n-3 PUFA or total n-3:n-6 ratio. CONCLUSIONS: Erythrocyte levels of the n-6 PUFA LA were inversely associated with risk of RA, whereas no associations were observed for other n-6 or n-3 PUFA. Further work is warranted to replicate these findings and to investigate if lower LA levels are a bystander or contributor to the process of RA development.
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Artritis Reumatoide/sangre , Artritis Reumatoide/epidemiología , Eritrocitos/metabolismo , Ácidos Grasos Omega-3/sangre , Ácidos Grasos Omega-6/sangre , Adulto , Distribución por Edad , Anciano , Artritis Reumatoide/diagnóstico , Biomarcadores/sangre , Estudios de Casos y Controles , Citocinas/sangre , Eritrocitos/química , Europa (Continente)/epidemiología , Femenino , Humanos , Incidencia , Italia/epidemiología , Masculino , Persona de Mediana Edad , Valores de Referencia , Medición de Riesgo , Índice de Severidad de la Enfermedad , Distribución por Sexo , España/epidemiologíaRESUMEN
BACKGROUND: Antibodies to citrullinated proteins (ACPA) occur years before RA diagnosis. Porphyromonas gingivalis expresses its own peptidylarginine deiminase (PPAD), and is a proposed aetiological factor for the ACPA response. Smoking is a risk factor for both ACPA-positive RA and periodontitis. We aimed to study the relation of these factors to the risk of RA in a prospective cohort. METHODS: We performed a nested case-control study by identifying pre-RA cases in four populations from the European Prospective Investigation into Cancer and nutrition, matched with three controls. Data on smoking and other covariates were obtained from baseline questionnaires. Antibodies to CCP2 and citrullinated peptides from α-enolase, fibrinogen, vimentin and PPAD were measured. Antibodies to arginine gingipain (RgpB) were used as a marker for P.gingivalis infection and validated in a separate cohort of healthy controls and subjects with periodontitis. RESULTS: We studied 103 pre-RA cases. RA development was associated with several ACPA specificities, but not with antibodies to citrullinated PPAD peptides. Antibody levels to RgpB and PPAD peptides were higher in smokers but were not associated with risk of RA or with pre-RA autoimmunity. Former but not current smoking was associated with antibodies to α-enolase (OR 4.06; 95 % CI 1.02, 16.2 versus 0.54; 0.09-3.73) and fibrinogen peptides (OR 4.24; 95 % CI 1.2-14.96 versus 0.58; 0.13-2.70), and later development of RA (OR 2.48; 95 % CI 1.27-4.84 versus 1.57; 0.85-2.93), independent of smoking intensity. CONCLUSIONS: Smoking remains a risk factor for RA well before the clinical onset of disease. In this cohort, P.gingivalis is not associated with pre-RA autoimmunity or risk of RA in an early phase before disease-onset. Antibodies to PPAD peptides are not an early feature of ACPA ontogeny.
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Artritis Reumatoide/inmunología , Hidrolasas/inmunología , Péptidos Cíclicos/inmunología , Porphyromonas gingivalis/enzimología , Fumar/efectos adversos , Adhesinas Bacterianas/inmunología , Adulto , Artritis Reumatoide/epidemiología , Artritis Reumatoide/microbiología , Autoantígenos/inmunología , Estudios de Casos y Controles , Cisteína Endopeptidasas/inmunología , Europa (Continente)/epidemiología , Femenino , Cisteína-Endopeptidasas Gingipaínas , Humanos , Masculino , Persona de Mediana Edad , Periodontitis/complicaciones , Periodontitis/inmunología , Estudios Prospectivos , Desiminasas de la Arginina Proteica , Fumar/inmunologíaRESUMEN
OBJECTIVE: To examine the potential of chronic severe bacterial infection to generate rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs), by studying patients with bronchiectasis (BR) alone and BR patients with rheumatoid arthritis (BR/RA). METHODS: We studied 122 patients with BR alone, 50 patients with BR/RA, and 50 RA patients without lung disease, as well as 87 patients with asthma and 79 healthy subjects as controls. RF levels were measured using an automated analyzer, and cyclic citrullinated peptide 2 (CCP-2) was used to detect ACPAs. The fine specificities of citrullinated α-enolase peptide 1 (CEP-1), Cit-vimentin, and Cit-fibrinogen to their arginine-containing control peptides (arginine-containing α-enolase peptide 1 [REP-1], vimentin, and fibrinogen) were measured by enzyme-linked immunosorbent assay. RESULTS: Among the BR patients and control subjects, 39% and 42%, respectively, were ever-smokers. The frequency of RF positivity in serum was increased in BR patients compared with controls (25% versus 10%), as were the frequencies of antibodies to CCP-2 (5% versus 0%), CEP-1 (7% versus 4%), Cit-vimentin (7% versus 4%), and Cit-fibrinogen (12% versus 4%), although only the differences for RF and Cit-fibrinogen were significant (P < 0.05). We observed a corresponding increase in the frequency of antibodies to the arginine-containing control peptides in BR patients compared with controls (for REP-1, 19% versus 4% [P < 0.01]; for vimentin, 16% versus 4% [P < 0.05]), demonstrating that the ACPA response in patients with BR is not citrulline specific. The lack of citrulline specificity was further confirmed by absorption studies. In BR/RA patients, all ACPA responses were highly citrulline specific. CONCLUSION: Bronchiectasis is an unusual but potent model for the induction of autoimmunity in RA by bacterial infection in the lung. Our study suggests that the ACPA response is not citrulline specific during the early stages of tolerance breakdown but becomes more specific in patients with BR in whom BR/RA develops.
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Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Autoinmunidad/inmunología , Bronquiectasia/inmunología , Fumar/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Asma/inmunología , Infecciones Bacterianas/inmunología , Estudios de Casos y Controles , Enfermedad Crónica , Citrulina/inmunología , Citrulina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibrinógeno/inmunología , Fibrinógeno/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Péptidos Cíclicos/inmunología , Fosfopiruvato Hidratasa/inmunología , Fosfopiruvato Hidratasa/metabolismo , Factor Reumatoide/inmunología , Vimentina/inmunología , Vimentina/metabolismoRESUMEN
BACKGROUND AND PURPOSE: To investigate whether a narrow spectrum kinase inhibitor RV1088, which simultaneously targets specific MAPKs, Src and spleen tyrosine kinase (Syk), is more effective at inhibiting inflammatory signalling in rheumatoid arthritis (RA) than single kinase inhibitors (SKIs). EXPERIMENTAL APPROACH: elisas were used to determine the efficacy of RV1088, clinically relevant SKIs and the pharmaceutical Humira on pro-inflammatory cytokine production by activated RA synovial fibroblasts, primary human monocytes and macrophages, as well as spontaneous cytokine synthesis by synovial membrane cells from RA patients. In human macrophages, RNAi knockdown of individual kinases was used to reveal the effect of inhibition of kinase expression on cytokine synthesis. KEY RESULTS: RV1088 reduced TNF-α, IL-6 and IL-8 production in all individual activated cell types with low, nM, IC50 s. SKIs, and combinations of SKIs, were significantly less effective than RV1088. RNAi of specific kinases in macrophages also caused only modest inhibition of pro-inflammatory cytokine production. RV1088 was also significantly more effective at inhibiting IL-6 and IL-8 production by monocytes and RA synovial fibroblasts compared with Humira. Finally, RV1088 was the only inhibitor that was effective in reducing TNF-α, IL-6 and IL-8 synthesis in RA synovial membrane cells with low nM IC50 s. CONCLUSIONS AND IMPLICATIONS: This study demonstrates potent anti-inflammatory effect of RV1088, highlighting that distinct signalling pathways drive TNF-α, IL-6 and IL-8 production in the different cell types found in RA joints. As such, targeting numerous signalling pathways simultaneously using RV1088 could offer a more powerful method of reducing inflammation in RA than targeting individual kinases.
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Acetamidas/farmacología , Antiinflamatorios/farmacología , Artritis Reumatoide/enzimología , Artritis Reumatoide/patología , Inhibidores de Proteínas Quinasas/farmacología , Membrana Sinovial/patología , Urea/análogos & derivados , Adalimumab/farmacología , Artritis Reumatoide/metabolismo , Dasatinib/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Naftalenos/farmacología , Oxazinas/farmacología , Piperidinas/farmacología , Cultivo Primario de Células , Pirazoles/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Membrana Sinovial/enzimología , Membrana Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Urea/farmacologíaRESUMEN
BACKGROUND: Rheumatoid arthritis (RA) is a debilitating and painful disease leading to increased morbidity and mortality and novel therapeutic approaches are needed. The purpose of this study was to elucidate if mesenchymal stem cells (MSCs) injected in the joints of mice with arthritis are therapeutic, reducing joint swelling and cartilage destruction. METHODS: Murine mesenchymal stem cells (mMSCs) were isolated from bone marrow of C57Bl/6 mice and expanded in culture. Cells were tested for immunophenotype and their ability to form colonies and to differentiate into chondrocytes, osteocytes and adipocytes. Antigen-induced arthritis (AIA) was induced by intra-articular injection of methylated bovine serum albumin into the knee joints of preimmunized C57Bl/6 mice. After one day, when peak swelling occurs, 500,000 mMSCs labelled with red fluorescent cell tracker CM-DiI were injected intra-articularly in the right knee joint. Left knee joints were treated as controls by receiving PBS injections. Differences between groups were calculated by Mann Whitney U test or unpaired t tests using GraphPad Prism software version 5. RESULTS: Knee joint diameter (swelling) was measured as a clinical indication of joint inflammation and this parameter was significantly less in MSC-treated mice compared to control-treated animals 48 hours after arthritis induction. This difference continued for ~7 days. CM-DiI-labelled MSCs were clearly visualised in the lining and sublining layers of synovium, in the region of the patella and femoral and tibial surfaces. By day 3, parameters indicative of disease severity, including cartilage depletion, inflammatory exudate and arthritic index were shown to be significantly reduced in MSC-treated animals. This difference continued for 7 days and was further confirmed by histological analysis. The serum concentration of tumour necrosis factor α was significantly decreased following MSC administration. CONCLUSIONS: Our results reveal that MSCs injected in the joints of mice with AIA are therapeutic, reducing inflammation, joint swelling and cartilage destruction. These cells also integrate into the synovium in AIA.
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Antígenos/administración & dosificación , Artritis Experimental/prevención & control , Cartílago Articular/patología , Inflamación/prevención & control , Articulaciones , Trasplante de Células Madre Mesenquimatosas , Animales , Artritis Experimental/patología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Chemokine receptors on leukocytes mediate the recruitment and accumulation of these cells within affected joints in chronic inflammatory diseases such as rheumatoid arthritis (RA). Identification of involved receptors offers potential for development of therapeutic interventions. The objective of this study was to investigate the expression of orphan receptor GPR15/BOB in the synovium of RA and non-RA patients and in peripheral blood of RA patients and healthy donors. GPR15/BOB protein and messenger RNA expression were examined in RA and non-RA synovium by immunofluorescence and reverse-transcription polymerase chain reaction (RT-PCR) respectively. GPR15/BOB expression on peripheral blood leukocytes was analysed by flow cytometry and GPR15/BOB messenger RNA was examined in peripheral blood monocytes by RT-PCR. GPR15/BOB protein was observed in CD68+ and CD14+ macrophages in synovia, with greater expression in RA synovia. GPR15/BOB protein was expressed in all patient synovia whereas in non-RA synovia expression was low or absent. Similarly GPR15/BOB messenger RNA was detected in all RA and a minority of non-RA synovia. GPR15/BOB protein was expressed on peripheral blood leukocytes from RA and healthy individuals with increased expression by monocytes and neutrophils in RA. GPR15/BOB messenger RNA expression was confirmed in peripheral blood monocytes. In conclusion GPR15/BOB is expressed by macrophages in synovial tissue and on monocytes and neutrophils in peripheral blood, and expression is up-regulated in RA patients compared to non-RA controls. This orphan receptor on monocytes/macrophages and neutrophils may play a role in RA pathophysiology.
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Artritis Reumatoide/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Regulación hacia Arriba , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/sangre , Artritis Reumatoide/genética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Neutrófilos/metabolismo , Receptores de Quimiocina/metabolismo , Receptores Acoplados a Proteínas G/sangre , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/sangre , Receptores de Péptidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Membrana Sinovial/metabolismoRESUMEN
INTRODUCTION: Monocytes/macrophages accumulate in the rheumatoid (RA) synovium where they play a central role in inflammation and joint destruction. Identification of molecules involved in their accumulation and differentiation is important to inform therapeutic strategies. This study investigated the expression and function of chemokine receptor CCR9 in the peripheral blood (PB) and synovium of RA, non-RA patients and healthy volunteers. METHODS: CCR9 expression on PB monocytes/macrophages was analysed by flow cytometry and in synovium by immunofluorescence. Chemokine receptor CCR9 mRNA expression was examined in RA and non-RA synovium, monocytes/macrophages from PB and synovial fluid (SF) of RA patients and PB of healthy donors using the reverse transcription polymerase chain reaction (RT-PCR). Monocyte differentiation and chemotaxis to chemokine ligand 25 (CCL25)/TECK were used to study CCR9 function. RESULTS: CCR9 was expressed by PB monocytes/macrophages in RA and healthy donors, and increased in RA. In RA and non-RA synovia, CCR9 co-localised with cluster of differentiation 14+ (CD14+) and cluster of differentiation 68+ (CD68+) macrophages, and was more abundant in RA synovium. CCR9 mRNA was detected in the synovia of all RA patients and in some non-RA controls, and monocytes/macrophages from PB and SF of RA and healthy controls. CCL25 was detected in RA and non-RA synovia where it co-localised with CD14+ and CD68+ cells. Tumour necrosis factor alpha (TNFα) increased CCR9 expression on human acute monocytic leukemia cell line THP-1 monocytic cells. CCL25 induced a stronger monocyte differentiation in RA compared to healthy donors. CCL25 induced significant chemotaxis of PB monocytes but not consistently among individuals. CONCLUSIONS: CCR9 expression by monocytes is increased in RA. CCL25 may be involved in the differentiation of monocytes to macrophages particularly in RA.
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Artritis Reumatoide/patología , Quimiocinas CC/genética , Macrófagos/patología , Monocitos/patología , Receptores CCR/genética , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Diferenciación Celular/inmunología , Línea Celular , Quimiocinas CC/metabolismo , Femenino , Citometría de Flujo , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Receptores CCR/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
Freshly isolated bovine articular chondrocytes were seeded into a resorbable gelatin sponge and cultured in the absence or presence of extrinsic high molecular weight hyaluronan (HA) for up to 1 month. The gelatin sponge could be uniformly and reproducibly loaded with chondrocytes. Immunostaining demonstrated that accumulation of pericellular HA increased in the presence of extrinsic HA. However, this approach could not differentiate between extrinsic and endogenous HA. More chondrocytes were retained within the loaded sponges in the presence of HA. Both cell number and matrix synthesis were increased in the presence of high molecular weight HA throughout the time course. Proteoglycan synthesis per cell increased by 22-fold in the presence of HA at 500 microg/mL. Our model demonstrates that HA can be used as a tool not only to expand freshly isolated chondrocyte numbers but also to increase matrix synthesis and deposition within a resorbable gelatin sponge. Autologous chondrocytes for tissue engineering are always in short supply, so this could be a useful tool with which to increase the retention of cells seeded into other types of scaffold matrices before implanting them into a cartilage defect.