RESUMO
Objectives: To investigate the expression of IL-18 in symptomatic and asymptomatic muscle tissues of patients with PM and DM and the effects of conventional immunosuppressive treatment on such expression. Methods: Two cohorts of patients were included in this study. The first cohort consisted of 10 new-onset myositis patients. IL-18 expression was compared between symptomatic and asymptomatic muscle biopsies that were taken prior to treatment. The second cohort consisted of another 10 patients with repeated muscle biopsies before and after 8 months with conventional immunosuppressive treatment. Using immunohistochemistry, IL-18 expression in muscle tissues was compared before and after treatment. Biopsies from seven healthy individuals were included as controls. Results: IL-18 expression was predominantly localized to inflammatory cells and capillaries in patients and mostly to capillaries in healthy controls. Total IL-18 expression in muscle tissues from the new-onset patients, at both symptomatic and asymptomatic sites, was significantly higher compared with healthy controls (P = 0.007 and P = 0.002) with no statistical difference in appearances between symptomatic and asymptomatic sites. The number of IL-18 positive capillaries was not different among symptomatic, asymptomatic and healthy muscles. Total IL-18 expression appeared lower in biopsies from patients receiving and improving with immunosuppressive treatment, particularly the number of IL-18 positive inflammatory cells but not the number of IL-18 positive capillaries, which was consistent with significantly decreased expression of CD68+ macrophages (P = 0.04). Conclusion: IL-18 is highly expressed in muscle tissue in the context of inflammatory myopathies and based on its plausible effector functions could provide a novel therapeutic target in future.
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Dermatomiosite/metabolismo , Imunossupressores/uso terapêutico , Interleucina-18/metabolismo , Músculo Esquelético/metabolismo , Polimiosite/metabolismo , Adulto , Idoso , Biópsia , Estudos de Coortes , Dermatomiosite/tratamento farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimiosite/tratamento farmacológico , Resultado do TratamentoRESUMO
PURPOSE: Many attempts with contradictory results have been made to correlate different features of OA with pain. One reason may be that pain at rest and pain with movement are seldom considered separately although the mechanisms may be quite different. Furthermore, pain ratings are subject to individual interpretation making an inter-individual comparison questionable. By instead calculating the absolute and relative changes in pain on an intra-individual level after total knee arthroplasty (TKA), we aimed at exploring a relationship between pain and radiological and histological changes. METHODS: In 69 patients undergoing TKA, preoperative radiographs and perioperative histological samples of the synovial membrane were graded for severity of osteoarthritic and inflammatory changes. The findings were related to the intensity of pain at rest and with movement both preoperatively and 18 months postoperatively according to the visual analogue scale (VAS). RESULTS: The radiographic and histological findings showed no significant correlation with the mean pre- or postoperative pain scores. Instead, change in pain with movement from pre- to postoperative was significantly related to the grade of radiographic osteoarthritis. Best pain relief by TKA was achieved in patients with severe radiographic changes. This, however, only applied to pain with movement. CONCLUSIONS: Pain at rest and pain with movement may have different mechanisms. We believe that assessing the intensity of pain at rest and pain with movement separately and considering changes in pain on an individual level will be helpful strategies in future follow-up studies and efforts aimed at explaining the mechanisms of pain in OA.
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Artralgia/diagnóstico por imagem , Artralgia/patologia , Artroplastia do Joelho , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/patologia , Membrana Sinovial/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Artralgia/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/cirurgia , Cuidados Pré-Operatórios , RadiografiaRESUMO
INTRODUCTION: Arthroscopy is a minimally invasive technique for retrieving synovial biopsies in rheumatology during the past 20 years. Vital for its use is continual evaluation of its safety and efficacy. Important for sampling is the fact of intraarticular variation for synovial markers. For microscopic measurements scoring systems have been developed and validated, but for macroscopic evaluations there is a need for further comprehensive description and validation of equivalent scoring systems. METHODS: We studied the complication rate and yield of arthroscopies performed at our clinic between 1998 and 2005. We also created and evaluated a macroscopic score set of instructions for synovitis. RESULTS: Of 408 procedures, we had two major and one minor complication; two haemarthrosis and one wound infection, respectively. Pain was most often not a problem, but 12 procedures had to be prematurely ended due to pain. Yield of biopsies adequate for histology were 83% over all, 94% for knee joints and 34% for smaller joints. Video printer photographs of synovium taken during arthroscopy were jointly and individually reviewed by seven raters in several settings, and intra and inter rater variation was calculated. A macroscopic synovial scoring system for arthroscopy was created (Macro-score), based upon hypertrophy, vascularity and global synovitis. These written instructions were evaluated by five control-raters, and when evaluated individual parameters were without greater intra or inter rater variability, indicating that the score is reliable and easy to use. CONCLUSIONS: In our hands rheumatologic arthroscopy is a safe method with very few complications. For knee joints it is a reliable method to retrieve representative tissue in clinical longitudinal studies. We also created an easy to use macroscopic score, that needs to be validated against other methodologies. We hope it will be of value in further developing international standards in this area.
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Artroscopia/métodos , Artroscopia/normas , Projetos de Pesquisa/normas , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Articulação do Joelho/patologia , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/diagnóstico , Osteoartrite do Joelho/cirurgia , Membrana Sinovial/patologia , Adulto JovemRESUMO
OBJECTIVE: A CD30-CD153 mast cell axis has been described in skin inflammations and Hodgkin's lymphoma. We investigated if a soluble form of CD153 is present in the serum and synovial fluid (SF) of patients with rheumatoid arthritis (RA), and determined whether mast cells express CD153 in the synovium of these patients. METHODS: Soluble forms of CD30 and CD153 were quantified in serum and SF of patients with RA by ELISA. Consecutive sections of synovial biopsies from 12 patients were stained against tryptase (mast-cell marker), CD30, and CD153. RESULTS: Elevated concentrations of the soluble form of CD153 were found in serum from 14/15 RA patients. In the SF, 11/20 patients had detectable levels of soluble CD153. CD30 and CD153 were expressed in all biopsies that were studied. Mast cells were present in all the synovial biopsies, and expressed CD153 in one-third of the cases. CONCLUSION: We observed that CD153 was expressed in the synovium of patients with RA and we were able to correlate the serum levels of soluble CD153 with SF levels in the same patients. Because CD30 can activate mast cells to release chemokines without degranulation, our finding that mast cells express CD153 in RA synovium raises the possibility that a CD30-CD153 axis may contribute to the activation of synovial mast cells in the absence of degranulation.
Assuntos
Artrite Reumatoide/sangue , Ligante CD30/metabolismo , Mastócitos/metabolismo , Líquido Sinovial/metabolismo , Artrite Reumatoide/metabolismo , Ligante CD30/sangue , Estudos de Casos e Controles , Humanos , Antígeno Ki-1/sangue , Antígeno Ki-1/metabolismoRESUMO
OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) in muscle biopsy specimens and serum from patients with polymyositis and patients with dermatomyositis compared with that in healthy control subjects. METHODS: Muscle biopsy specimens from 33 patients with polymyositis or dermatomyositis and 15 healthy control subjects and serum samples from 56 patients and 56 healthy control subjects were analyzed. Patients were categorized into 3 groups, depending on disease duration and the presence or absence of inflammatory infiltrates. The expression of VEGF and the vessel marker CD31 in muscle was analyzed by immunohistochemistry, the expression of VEGF messenger RNA (mRNA) was analyzed by in situ hybridization, and serum levels of VEGF were determined by enzyme-linked immunosorbent assay. RESULTS: Patients with polymyositis or dermatomyositis in the early or chronic phase without inflammatory infiltrates had a decreased total number of capillaries compared with healthy individuals. In patients with early disease without inflammatory infiltrates, the number of VEGF-expressing muscle fibers was increased compared with that in control subjects, whereas VEGF expression was unchanged in the chronic phase of disease. In patients with established disease with inflammatory infiltrates, total VEGF expression was high compared with that in healthy control subjects. In healthy control subjects, VEGF was expressed in endothelial cells and in occasional muscle fibers. VEGF mRNA was expressed in muscle fibers in both healthy individuals and patients. The level of serum VEGF was significantly increased in patients compared with control subjects. CONCLUSION: Our observations support a role of VEGF in the early phases of polymyositis and dermatomyositis. A reduced number of capillaries could lead to induction of VEGF expression in muscle fibers. Furthermore, differences in molecular expression during certain phases of disease may help in the development of specific therapeutic algorithms in the treatment of myositis.
Assuntos
Dermatomiosite/metabolismo , Músculo Esquelético/metabolismo , Polimiosite/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Capilares , Estudos de Casos e Controles , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
BACKGROUND: Impaired immunological tolerance to commensal enteric flora is considered one possible pathogenic mechanism of inflammatory bowel disease (IBD). Given that regulatory T cells and Toll-like receptor (TLR)-positive cells are key actors in mucosal immune regulation, we aimed to identify the dynamics of these actors in the intestinal mucosa in relation to clinical improvement following selective leukapheresis treatment. METHODS: Ten patients with active IBD despite treatment with corticosteroids, immunomodulators, or anti-tumor necrosis factor therapy were assessed by immunohistochemical staining of colorectal mucosal biopsies obtained before and after five sessions (week 7) of granulocyte and monocyte adsorption apheresis (GCAP). The presence of FoxP3-positive regulatory T cells, macrophages, dendritic cells, and TLR-2 and-4 positive cells was determined in relation to short-(week 7) and long-term (week 52) clinical outcome data. RESULTS: Following GCAP, the number of FoxP3-(P = 0.012) and TLR-2 (P = 0.008)-positive cells significantly decreased in biopsies after 7 weeks, in parallel with both clinical improvement at week 7 and a longstanding response after 12 months. CONCLUSIONS: Downregulation of FoxP3 and TLR-2 cells in the colorectal mucosa mirrors both short-and long-term improvement in patients with active IBD responding to GCAP. This observation suggests a potential role of these cells in the pathogenesis of IBD and the induction of immunological tolerance in the mucosa.
Assuntos
Fatores de Transcrição Forkhead/biossíntese , Imunidade Celular/fisiologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Leucaférese/métodos , Receptor 2 Toll-Like/biossíntese , Adulto , Biópsia , Colo/imunologia , Colo/patologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/terapia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Prognóstico , Reto/imunologia , Reto/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
INTRODUCTION: High-mobility group box chromosomal protein 1 (HMGB1) has recently been identified as an endogenous mediator of arthritis. TNF and IL-1beta, pivotal cytokines in arthritis pathogenesis, both have the ability to induce the release of HMGB1 from myeloid and dendritic cells. It was, therefore, decided to investigate whether treatment based on TNF blockade in rheumatoid arthritis (RA) affects the expression of synovial HMGB1. METHODS: Repeated arthroscopy-guided sampling of synovial tissue was performed in nine patients with RA before and nine weeks after initiation of anti-TNF mAb (infliximab) therapy. Synovial biopsy specimens were analysed for HMGB1 protein by immunohistochemical staining and for HMGB1 mRNA expression by real-time reverse transcriptase PCR (RT-PCR). Statistical evaluations were based on Wilcoxon's signed rank tests or Spearman rank sum tests. RESULTS: Aberrant, extranuclear HMGB1 and constitutive nuclear HMGB1 expression, with histological signs of inflammation, were evident in all biopsies obtained before infliximab therapy. Signs of inflammation were still evident in the second biopsies obtained nine weeks after initiation of infliximab therapy. The cytoplasmic and extracellular expression of HMGB1 decreased in five patients, remained unchanged in one patient and increased in three patients, making the overall change in HMGB1 protein expression not significant. No correlation between the clinical response, as measured by disease activity score calculated for 28 joints (DAS28) or the American College of Rheumatology response criteria (ACR 20, 50, and 70), and the direction of change of HMGB1 expression in individual patients could be discerned. In addition, infliximab therapy did not alter HMGB1 mRNA synthesis. CONCLUSION: Pro-inflammatory HMGB1 expression during rheumatoid synovitis was not consistently influenced by TNF-blocking therapy with infliximab. This suggests that TNF is not the main inducer of extranuclear HMGB1 during synovitis and that HMGB1 may represent a TNF-independent molecule that could be considered as a possible target for future therapeutic intervention in RA.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/metabolismo , Proteína HMGB1/biossíntese , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Idoso , Artrite Reumatoide/tratamento farmacológico , Artroscopia , Feminino , Expressão Gênica , Proteína HMGB1/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Infliximab , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/metabolismo , Masculino , Pessoa de Meia-Idade , Projetos Piloto , RNA Mensageiro/análise , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
Erythema Toxicum, a rash frequently present in the healthy newborn infant is an innate, immune response to the first commensal micro flora. Flushing and urtication are seen in this manifestation suggesting mast cell (MC) activation and MC derived mediator release. It has recently become evident that MCs participate in the protective, innate immune response against microbes also by secreting products toxic to pathogens such as cathelicidin peptide antibiotics. We hypothesized that MCs contribute to the process of inflammation in Erythema Toxicum and that skin MCs of human newborns express the cathelicidin peptide antibiotic LL-37. Skin sections were immunostained for MC tryptase. Double immunofluorescence was performed by staining LL-37 in combination with tryptase. We studied ultra structure of skin MCs with transmission (TEM) and immunoelectron microscopy (IEM). Seven infants with and six infants without the rash, as well as three adults were included. We found numerous tryptase-expressing MCs recruited around the hair follicles in the lesions of Erythema Toxicum. TEM analysis of MCs exhibited signs of degranulation in the lesion. Neither skin MCs from newborns nor adults did express LL-37 as judged by confocal and IEM. MCs participate in the inflammatory responses of Erythema Toxicum by taking an active part in the immune system of the hair follicle. However, their immunological activity is not linked to the expression of the cathelicidin antimicrobial peptide LL-37. A pivotal role of MCs in the innate, inflammatory response at the site of pathogen invasion during the critical time of perinatal colonization is suggested.
Assuntos
Eritema/imunologia , Folículo Piloso/imunologia , Mastócitos/imunologia , Pele/imunologia , Triptases/metabolismo , Urticária/imunologia , Adulto , Peptídeos Catiônicos Antimicrobianos/análise , Catelicidinas , Eritema/metabolismo , Feminino , Humanos , Recém-Nascido , Masculino , Mastócitos/enzimologia , Mastócitos/ultraestrutura , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Pele/metabolismo , Pele/ultraestrutura , Urticária/metabolismoRESUMO
At birth, commensal microbes penetrate into the skin of the human newborn, eliciting an acute rash, erythema toxicumn neonatorum. Histologically, the rash is characterized by an upregulation of proinflammatory activity and a local recruitment of immunocytes, including macrophages. High mobility group box chromosomal protein 1, a nuclear and cytosolic protein, is also a pro-inflammatory cytokine released by macrophages in response to microbial stimulation. Here, we reasoned that macrophages but also keratinocytes might upregulate this protein in response to the first colonization and that high mobility group box chromosomal protein 1 might play a role as a proinflammatory mediator in the development and progression of erythema toxicum. Punch biopsy specimens from 1-day-old healthy infants, seven with and four without erythema toxicum were analyzed with indirect immunohistochemistry and two different antihigh mobility group box chromosomal protein 1 antibodies, immunofluorescence, nuclear counterstaining, confocal and immunoelectron imaging. We found relocation of nuclear high mobility group box chromosomal protein 1 into the cytoplasm in keratinocytes and macrophages in erythema toxicum. Cytoplasmatic high mobility group box chromosomal protein 1 was also found in melanocytes and did neither co-locate with lysosomal-associated membrane proteins nor with melanosomes. We speculate that terrestrial adaptation triggers the induction of the endogenous "danger signal" high mobility group box chromosomal protein 1 in the skin of the newborn infant, perhaps in response to the first commensal colonization and that this signal may contribute to alert the immune system and promote a protective immune response.
Assuntos
Eritema/patologia , Proteína HMGB1/metabolismo , Doenças do Recém-Nascido/patologia , Queratinócitos/metabolismo , Pele/patologia , Bactérias/imunologia , Biópsia , Eritema/imunologia , Eritema/microbiologia , Imunofluorescência , Humanos , Recém-Nascido , Doenças do Recém-Nascido/imunologia , Doenças do Recém-Nascido/microbiologia , Queratinócitos/patologia , Queratinócitos/ultraestrutura , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas de Membrana Lisossomal/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/ultraestrutura , Proteínas de Membrana/metabolismo , Microscopia Confocal , Microscopia Imunoeletrônica , Pele/imunologia , Pele/microbiologia , Proteínas de Transporte Vesicular/metabolismoRESUMO
Expanded populations of CD4+ T cells lacking the co-stimulatory molecule CD28 (CD4+CD28null T cells) have been reported in several inflammatory disorders. In rheumatoid arthritis, increased frequencies of CD4+CD28null T cells in peripheral blood have previously been associated with extra-articular manifestations and human cytomegalovirus (HCMV) infection, but their presence in and contribution to joint manifestations is not clear. In the present article we investigated the distribution of CD4+CD28null T cells in the synovial membrane, synovial fluid and peripheral blood of RA patients, and analysed the association with erosive disease and anti-citrullinated protein antibodies. CD4+CD28null T cells were infrequent in the synovial membrane and synovial fluid, despite significant frequencies in the circulation. Strikingly, the dominant TCR-Vbeta subsets of CD4+CD28null T cells in peripheral blood were often absent in synovial fluid. CD4+CD28null T cells in blood and synovial fluid showed specificity for HCMV antigens, and their presence was clearly associated with HCMV seropositivity but not with anti-citrullinated protein antibodies in the serum or synovial fluid, nor with erosive disease. Together these data imply a primary role for CD4+CD28null T cells in manifestations elsewhere than in the joints of patients with HCMV-seropositive rheumatoid arthritis.
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Artrite Reumatoide/sangue , Antígenos CD28/sangue , Antígenos CD4/sangue , Linfócitos Nulos/metabolismo , Linfócitos T/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/patologia , Antígenos CD28/análise , Antígenos CD4/análise , Estudos de Coortes , Feminino , Humanos , Linfócitos Nulos/química , Linfócitos Nulos/citologia , Masculino , Pessoa de Meia-Idade , Líquido Sinovial/química , Líquido Sinovial/citologia , Líquido Sinovial/metabolismo , Membrana Sinovial/química , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo , Linfócitos T/química , Linfócitos T/citologiaRESUMO
OBJECTIVE: To investigate type I interferon (IFN) system activation and its correlation with autoantibodies and organ manifestations in polymyositis (PM), dermatomyositis (DM), and inclusion body myositis. METHODS: Sera from 30 patients and 16 healthy controls, or purified IgG, were combined with material released from necrotized cells to stimulate IFNalpha production by peripheral blood mononuclear cells (PBMCs) from healthy blood donors. Muscle biopsy specimens from 25 patients and 7 healthy controls were investigated for blood dendritic cell antigen 2 (BDCA-2)-positive plasmacytoid dendritic cells (PDCs) and IFNalpha/beta-inducible myxovirus resistance 1 (MX-1) protein. RESULTS: Sera from 13 patients who were positive for anti-Jo-1 or anti-Ro 52/anti-Ro 60 autoantibodies induced IFNalpha production in PBMCs when combined with necrotic cell material. In addition, IgG prepared from anti-Jo-1-positive PM sera induced IFNalpha with necrotic material, but not when the latter was treated with RNase. BDCA-2 expression in PDCs in muscle tissue was increased in PM patients with anti-Jo-1 autoantibodies, while MX-1 staining in capillaries was increased in DM patients, compared with healthy individuals. IFNalpha-inducing capacity correlated with interstitial lung disease, while MX-1 expression in the capillaries correlated with DM. CONCLUSION: Immune complexes containing anti-Jo-1 or anti-Ro 52/anti-Ro 60 autoantibodies and RNA may act as endogenous IFNalpha inducers that activate IFNalpha production in PDCs. These PDCs could be of importance for inducing myositis, whereas in DM patients without autoantibodies the presence of MX-1 protein in capillaries suggests another cellular IFNalpha source and induction mechanism. Consequently, the type I IFN system may be of importance in both PM and DM, but via different pathways.
Assuntos
Autoanticorpos/sangue , Autoantígenos/imunologia , Histidina-tRNA Ligase/imunologia , Interferon Tipo I/fisiologia , Miosite/sangue , Miosite/imunologia , RNA Citoplasmático Pequeno/imunologia , Ribonucleoproteínas/imunologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVES: To evaluate inter-observer agreement for microscopic measurement of inflammation in synovial tissue using manual quantitative, semiquantitative and computerised digital image analysis. METHODS: Paired serial sections of synovial tissue, obtained at arthroscopic biopsy of the knee from patients with rheumatoid arthritis (RA), were stained immunohistochemically for T lymphocyte (CD3) and macrophage (CD68) markers. Manual quantitative and semiquantitative scores for sub-lining layer CD3+ and CD68+ cell infiltration were independently derived in 6 international centres. Three centres derived scores using computerised digital image analysis. Inter-observer agreement was evaluated using Spearman's Rho and intraclass correlation coefficients (ICCs). RESULTS: Paired tissue sections from 12 patients were selected for evaluation. Satisfactory inter-observer agreement was demonstrated for all 3 methods of analysis. Using manual methods, ICCs for measurement of CD3+ and CD68+ cell infiltration were 0.73 and 0.73 for quantitative analysis and 0.83 and 0.78 for semiquantitative analysis, respectively. Corresponding ICCs of 0.79 and 0.58 were observed for the use of digital image analysis. All ICCs were significant at levels of p<0.0001. At each participating centre, use of computerised image analysis produced results that correlated strongly and significantly with those obtained using manual measurement. CONCLUSION: Strong inter-observer agreement was demonstrated for microscopic measurement of synovial inflammation in RA using manual quantitative, semiquantitative and computerised digital methods of analysis. This further supports the development of these methods as outcome measures in RA.
Assuntos
Processamento de Imagem Assistida por Computador , Competência Profissional , Membrana Sinovial/imunologia , Sinovite/imunologia , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Artrite Reumatoide/imunologia , Biomarcadores/análise , Complexo CD3/análise , Humanos , Inflamação , Microscopia , Variações Dependentes do Observador , Coloração e Rotulagem/métodosRESUMO
Activating Fc gamma receptors (FcgammaRs) have been identified as having important roles in the inflammatory joint reaction in rheumatoid arthritis (RA) and murine models of arthritis. However, the role of the inhibitory FcgammaRIIb in the regulation of the synovial inflammation in RA is less known. Here we have investigated synovial tissue from RA patients using a novel monoclonal antibody (GB3) specific for the FcgammaRIIb isoform. FcgammaRIIb was abundantly expressed in synovia of RA patients, in sharp contrast to the absence or weak staining of FcgammaRIIb in synovial biopsies from healthy volunteers. In addition, the expression of FcgammaRI, FcgammaRII and FcgammaRIII was analyzed in synovia obtained from early and late stages of RA. Compared with healthy synovia, which expressed FcgammaRII, FcgammaRIII but not FcgammaRI, all activating FcgammaRs were expressed and significantly up-regulated in RA, regardless of disease duration. Macrophages were one of the major cell types in the RA synovium expressing FcgammaRIIb and the activating FcgammaRs. Anti-inflammatory treatment with glucocorticoids reduced FcgammaR expression in arthritic joints, particularly that of FcgammaRI. This study demonstrates for the first time that RA patients do not fail to up-regulate FcgammaRIIb upon synovial inflammation, but suggests that the balance between expression of the inhibitory FcgammaRIIb and activating FcgammaRs may be in favour of the latter throughout the disease course. Anti-inflammatory drugs that target activating FcgammaRs may represent valuable therapeutics in this disease.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Receptores de IgG/biossíntese , Membrana Sinovial/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Especificidade de Anticorpos , Artrite Reumatoide/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Glucocorticoides/uso terapêutico , Humanos , Imuno-Histoquímica , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de IgG/efeitos dos fármacosRESUMO
Successive studies from one academic center (Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands) have consistently suggested that synovial tissue expression of sublining macrophages may be a biomarker of clinical response to therapeutic intervention in rheumatoid arthritis (RA) clinical trials. A proof-of-concept, randomized clinical trial was completed at a second academic center (St. Vincent's University Hospital, Dublin, Ireland), and the relationship between the change in disease activity and the change in sublining macrophages in distinct treatment cohorts was determined. The preliminary findings were not conclusive, but appeared to support a role for sublining CD68+ macrophages as a biomarker of clinical response to therapeutic intervention in cohorts of patients with RA.
Assuntos
Artrite Reumatoide/terapia , Macrófagos/citologia , Membrana Sinovial/imunologia , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Biomarcadores , Humanos , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Membrana Sinovial/citologiaRESUMO
OBJECTIVE: In rheumatoid arthritis (RA), formation of new blood vessels is necessary to meet the nutritional and oxygen requirements of actively proliferating synovial tissue. The aim of this study was to analyze the specific synovial vascular expression profiles of several angiogenesis-related genes as well as CD82 in RA compared with osteoarthritis (OA), using laser-mediated microdissection (LMM). METHODS: LMM and subsequent real-time polymerase chain reaction were used in combination with immunohistochemical analysis for area-specific analysis of messenger RNA (mRNA) and protein expression of vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR-1), VEGFR-2, hypoxia-inducible factor 1alpha (HIF-1alpha), HIF-2alpha, platelet-derived growth factor receptor alpha (PDGFRalpha), PDGFRbeta, inhibitor of DNA binding/differentiation 2 (Id2), and CD82 in RA and OA synovial microvasculature and synovial lining. RESULTS: Expression of Id2 mRNA was significantly lower in RA synovial vessels compared with OA synovial vessels (P=0.0011), whereas expression of VEGFR-1 was significantly higher in RA (P=0.0433). No differences were observed for the other parameters. At the protein level, no statistically significant differences were observed for any parameter, although Id2 levels were 2.5-fold lower in RA (P=0.0952). However, the number of synovial blood vessels and the number of VEGFR-2-expressing blood vessels were significantly higher in RA compared with OA. CONCLUSION: Our results underscore the importance of area-specific gene expression analysis in studying the pathogenesis of RA and support LMM as a robust tool for this purpose. Of note, our results indicate that previously described differences between RA and OA in the expression of angiogenic molecules are attributable to higher total numbers of synovial and vascular cells expressing these molecules in RA rather than higher expression levels in the individual cells.
Assuntos
Expressão Gênica , Neovascularização Patológica/genética , Membrana Sinovial/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Proteína Kangai-1/genética , Proteína Kangai-1/metabolismo , Masculino , Microdissecção , Pessoa de Meia-Idade , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Osteoartrite , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: The pro-inflammatory calcium-binding protein S100A12 has been recently ascribed to the novel group of damage associated molecular pattern (DAMP) molecules. Serum levels of S100A12 reflect neutrophil activation during synovial inflammation. The aim of this project was to analyse the effect of intra-articular corticosteroids or systemic anti-TNF treatment on synovial expression and serum levels of S100A12 in rheumatoid arthritis (RA). METHODS: Serum and synovial tissue was obtained from 19 RA patients prior to and 2 weeks after intra-articular corticosteroid therapy. Serum was collected for 34 other patients, and in 14 of these patients synovial tissue was additionally obtained prior to and after 8 weeks of infliximab treatment. The expression of S100A12 was analysed by immunohistochemistry on frozen sections. Levels of S100A12 in serum were determined by ELISA. RESULTS: S100A12 serum levels were elevated in patients with active RA prior to therapy and decreased significantly in patients who responded to treatment in both patient groups, but not in non-responders. The synovial expression of S100A12 was reduced 2 weeks after successful intra-articular corticosteroid treatment. A similar decrease in local expression was found after 8 weeks of successful infliximab treatment. CONCLUSIONS: Successful treatment of RA leads to downregulation of the DAMP protein S100A12. Expression and secretion of S100A12 is rapidly diminished after therapy with intra-articular corticosteroids or infliximab. Taking these findings together, decreasing serum concentrations of S100A12 could reflect alleviated synovial neutrophil activation during successful anti-inflammatory therapy in RA.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Glucocorticoides/administração & dosagem , Ativação de Neutrófilo , Triancinolona Acetonida/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Biomarcadores/análise , Feminino , Glucocorticoides/uso terapêutico , Humanos , Infliximab , Injeções Intra-Articulares , Masculino , Pessoa de Meia-Idade , Proteínas S100/análise , Proteína S100A12 , Líquido Sinovial/química , Fatores de Tempo , Triancinolona Acetonida/administração & dosagem , Triancinolona Acetonida/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
We examined the gene expression profiles in arthroscopic biopsies retrieved from 10 rheumatoid arthritis patients before and after anti-TNF treatment with infliximab to investigate whether such profiles can be used to predict responses to the therapy, and to study effects of the therapy on the profiles. Responses to treatment were assessed using European League Against Rheumatism response criteria. Three patients were found to be good responders, five patients to be moderate responders and two patients to be nonresponders. The TNF-alpha status of the biopsies from each of the patients before treatment was also investigated immunohistochemically, and it was detected in biopsies from four of the patients, including all three of the good responders. The gene expression data demonstrate that all patients had unique gene expression signatures, with low intrapatient variability between biopsies. The data also revealed significant differences between the good responding and nonresponding patients (279 differentially expressed genes were detected, with a false discovery rate < 0.025). Among the identified genes we found that MMP-3 was significantly upregulated in good responders (log2 fold change, 2.95) compared with nonresponders, providing further support for the potential of MMP-3 as a marker for good responses to therapy. An even more extensive list of 685 significantly differentially expressed genes was found between patients in whom TNF-alpha was found and nonresponders, indicating that TNF-alpha could be an important biomarker for successful infliximab treatment. Significant differences were also observed between biopsies taken before and after anti-TNF treatment, including 115 differentially expressed genes in the good responding group. Interestingly, the effect was even stronger in the group in which TNF-alpha was immunohistochemically detected before therapy. Here, 1,058 genes were differentially expressed, including many that were novel in this context (for example, CXCL3 and CXCL14). Subsequent Gene Ontology analysis revealed that several 'themes' were significantly over-represented that are known to be affected by anti-TNF treatment in inflammatory tissue; for example, immune response (GO:0006955), cell communication (GO:0007154), signal transduction (GO:0007165) and chemotaxis (GO:0006935). No genes reached statistical significance in the moderately responding or nonresponding groups. In conclusion, this pilot study suggests that further investigation is warranted on the usefulness of gene expression profiling of synovial tissue to predict and monitor the outcome of rheumatoid arthritis therapies.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Expressão Gênica/efeitos dos fármacos , Membrana Sinovial/efeitos dos fármacos , Adulto , Idoso , Artrite Reumatoide/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização In Situ , Infliximab , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Projetos Piloto , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/metabolismo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To investigate the occurrence of selected markers for inflammatory cells and cytokines in patients with chronic polyarthritis (CPA) and temporomandibular joint (TMJ) involvement. MATERIAL AND METHODS: Eleven patients (11 joints) with CPA and TMJ disorder were included in the study. Synovial specimens were obtained during TMJ open surgery and these were subjected to immunohistochemistry on frozen sections post-fixed with paraformaldehyde and with the cell membranes permeabilized by saponin. In all patients, the cytokines IL-1alpha, IL-1beta, IL-1ra, TNFalpha, IFNgamma, IL2, and TGFbeta were investigated using specific antibodies. The occurrence of macrophages and T-lymphocytes was investigated using immunohistochemistry with monoclonal antibodies against antigens CD68 and CD45RO, respectively. In addition, PCNA was used as a marker for cell proliferation. RESULTS: Staining of IL-1alpha, IL-1, and TGF was seen in all 11 specimens, IFN? in 1, TNFalpha in 4, and IL-2 in none. CD45RO-positive T cells were detected in 7 specimens, CD68-positive macrophages in 6, and cell proliferation seen with PCNA was noted in 8. CONCLUSIONS: The predominant cytokines of TMJ CPA were IL-1alpha, IL-1beta, and TGFbeta, and there appeared to be no differences between the subgroups (rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis) involved. Moreover, the cytokine pattern of TMJ CPA patients seemed to differ from patients with osteoarthritis, as shown in our previous study. The main difference was the absence of IFNgamma and TNFalpha in TMJ CPA patients and a stronger TGFbeta and IL-1alpha expression.
Assuntos
Artrite/imunologia , Citocinas/análise , Transtornos da Articulação Temporomandibular/imunologia , Adulto , Idoso , Artrite/patologia , Biomarcadores/análise , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição da Dor , Inquéritos e Questionários , Transtornos da Articulação Temporomandibular/patologiaRESUMO
OBJECTIVE: Intraarticular corticosteroids are frequently used as successful adjuvant therapy for inflammatory arthritides, but little is known about their effects on molecules that regulate bone biology. We undertook this study to investigate the effect of intraarticular corticosteroids on the synovial expression of RANKL and osteoprotegerin (OPG). METHODS: We evaluated RANKL, OPG, and surface marker expression by immunohistochemical methods in synovial knee biopsy samples obtained from 13 patients with inflammatory arthritis before and 2 weeks following intraarticular injection of triamcinolone hexacetonide. We further investigated the effect of dexamethasone (DEX) on RANKL expression by lymphocytes from rheumatoid arthritis synovial fluids (RA SF), using flow cytometric analysis. Finally, we evaluated the in vitro effect of DEX on RANKL and OPG expression in osteoblast-like cells, by Western blotting. RESULTS: Intraarticular corticosteroids induced a decrease in the number of synovial T cells without influencing the number of macrophages, evaluated as both CD68+ and CD163+ cells. This change was paralleled by a decrease of synovial RANKL expression with a concomitant reduction of the RANKL:OPG ratio. DEX down-regulated RANKL expression on lymphocytes derived from RA SF. Moreover, in vitro pretreatment of osteoblast-like cells with tumor necrosis factor favored an antiresorptive effect of DEX treatment through a similar down-regulation of RANKL expression. CONCLUSION: The decrease in inflammation attributed to intraarticular corticosteroids is accompanied by down-modulation of bone destruction markers. These findings offer a rationale for the beneficial effect of corticosteroids on joint erosion in arthritis.
Assuntos
Corticosteroides/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Artrite/tratamento farmacológico , Artrite/metabolismo , Proteínas de Transporte/biossíntese , Glicoproteínas/biossíntese , Glicoproteínas de Membrana/biossíntese , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores do Fator de Necrose Tumoral/biossíntese , Membrana Sinovial/metabolismo , Triancinolona Acetonida/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Feminino , Humanos , Injeções Intra-Articulares , Masculino , Pessoa de Meia-Idade , Osteoprotegerina , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Membrana Sinovial/patologia , Triancinolona Acetonida/administração & dosagemRESUMO
Blockade of tumour necrosis factor (TNF) is an effective treatment in rheumatoid arthritis (RA), but both non-responders and partial responders are quite frequent. This suggests that other pro-inflammatory cytokines may be of importance in the pathogenesis of RA and as possible targets for therapy. In this study we investigated the effect of TNF blockade (infliximab) on the synovial expression of IL-15 in RA in relation to different cell types and expression of other cytokines, to elucidate whether or not IL-15 is a possible target for therapy, independently of TNF blockade. Two arthroscopies with multiple biopsies were performed on nine patients with RA and knee-joint synovitis before and after three infusions of infliximab (3 mg/kg). Synovial biopsies were analysed with immunohistochemistry for expression of IL-15, TNF, IL-1alpha, IL-1ss and IFN-gamma, and for the cell surface markers CD3, CD68 and CD163. Stained synovial biopsy sections were evaluated by computerized image analysis. IL-15 expression was detected in all synovial biopsies taken at baseline. After infliximab therapy, the expression of IL-15 was increased in four patients and reduced in five. Synovial expression of IL-15 was not correlated with any CD marker or with the presence of any other cytokine. Synovial cellularity was decreased after 8 to 10 weeks of treatment with a significant reduction of the CD68-positive synovial cells, whereas no significant change was seen in the number of CD3-positive T cells and CD163-expressing macrophages. The number of TNF-producing cells in the synovial tissue at baseline was correlated with a good response to therapy. Thus, in this study the synovial expression of IL-15 in RA was not consistently influenced by TNF blockade, being apparently independent of TNF expression in the synovium. Consequently, we propose that IL-15 should remain as a therapeutic target in RA, regardless of the response to TNF blockade.