RESUMO
All organisms are exposed constantly to a variety of infectious and injurious stimuli. These induce inflammatory responses tailored to the threat posed. While the innate immune system is the front line of response to each stimulant, it has been considered traditionally to lack memory, acting in a generic fashion until the adaptive immune arm can take over. This outmoded simplification of the roles of innate and acquired arms of the immune system has been challenged by evidence of myeloid cells altering their response to subsequent encounters based on earlier exposure. This concept of 'innate immune memory' has been known for nearly a century, and is accepted among myeloid biologists. In recent years other innate immune cells, such as natural killer cells, have been shown to display memory, suggesting that innate immune memory is a trait common to several cell types. During the last 30 years, evidence has slowly accumulated in favour of not only haematopoietic cells, but also stromal cells, being imbued with memory following inflammatory episodes. A recent publication showing this also to be true in epithelial cells suggests innate immune memory to be widespread, if under-appreciated, in non-haematopoietic cells. In this review, we will examine the evidence supporting the existence of innate immune memory in stromal cells. We will also discuss the ramifications of memory in long-lived tissue-resident cells. Finally, we will pose questions we feel to be important in the understanding of these forgotten cells in the field of innate memory.
Assuntos
Células Endoteliais/imunologia , Fibroblastos/imunologia , Imunidade Inata/imunologia , Memória Imunológica/imunologia , Células Estromais/imunologia , Humanos , Inflamação/imunologia , Inflamação/patologiaRESUMO
OBJECTIVE: In rheumatoid arthritis, the enzyme 11 beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is highly expressed at sites of inflammation, where it converts inactive glucocorticoids (GC) to their active counterparts. In conditions of GC excess it has been shown to be a critical regulator of muscle wasting and bone loss. Here we examine the contribution of 11ß-HSD1 to the pathology of persistent chronic inflammatory disease. METHODS: To determine the contribution of 11ß-HSD1 to joint inflammation, destruction and systemic bone loss associated with persistent inflammatory arthritis, we generated mice with global and mesenchymal specific 11ß-HSD1 deletions in the TNF-transgenic (TNF-tg) model of chronic polyarthritis. Disease severity was determined by clinical scoring. Histology was assessed in formalin fixed sections and fluorescence-activated cell sorting (FACS) analysis of synovial tissue was performed. Local and systemic bone loss were measured by micro computed tomography (micro-CT). Measures of inflammation and bone metabolism were assessed in serum and in tibia mRNA. RESULTS: Global deletion of 11ß-HSD1 drove an enhanced inflammatory phenotype, characterised by florid synovitis, joint destruction and systemic bone loss. This was associated with increased pannus invasion into subchondral bone, a marked polarisation towards pro-inflammatory M1 macrophages at sites of inflammation and increased osteoclast numbers. Targeted mesenchymal deletion of 11ß-HSD1 failed to recapitulate this phenotype suggesting that 11ß-HSD1 within leukocytes mediate its protective actions in vivo. CONCLUSIONS: We demonstrate a fundamental role for 11ß-HSD1 in the suppression of synovitis, joint destruction, and systemic bone loss. Whilst a role for 11ß-HSD1 inhibitors has been proposed for metabolic complications in inflammatory diseases, our study suggests that this approach would greatly exacerbate disease severity.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Artrite Reumatoide/imunologia , Artrite/imunologia , Reabsorção Óssea/imunologia , Inflamação/imunologia , Articulações/patologia , Macrófagos/imunologia , Sinovite/imunologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Animais , Doença Crônica , Modelos Animais de Doenças , Glucocorticoides/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoclastos/patologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation, local and systemic bone loss and a lack of compensatory bone repair. Fibroblast-like synoviocytes (FLS) are the most abundant cells of the stroma and a key population in autoimmune diseases such as RA. An increasing body of evidence suggests that these cells play not only an important role in chronic inflammation and synovial hyperplasia, but also impact bone remodelling. Under inflammatory conditions FLS release inflammatory cytokines, regulate bone destruction and formation and communicate with immune cells to control bone homeostasis. Other stromal cells, such as osteoblasts and terminally differentiated osteoblasts, termed osteocytes, are also involved in the regulation of bone homeostasis and are dysregulated during inflammation. This review highlights our current understanding of how stromal cells influence the balance between bone formation and bone destruction. Increasing our understanding of these processes is critical to enable the development of novel therapeutic strategies with which to treat bone loss in RA.
Assuntos
Artrite Reumatoide/complicações , Reabsorção Óssea/imunologia , Osso e Ossos/patologia , Osteócitos/imunologia , Células Estromais/citologia , Sinoviócitos/citologia , Artrite Reumatoide/imunologia , Remodelação Óssea/imunologia , Reabsorção Óssea/terapia , Osso e Ossos/citologia , Citocinas/imunologia , Citocinas/farmacologia , Humanos , Hiperplasia , Inflamação/patologia , Células Estromais/imunologia , Sinoviócitos/imunologia , Via de Sinalização Wnt/imunologiaRESUMO
OBJECTIVES: Tristetraprolin (TTP), a negative regulator of many pro-inflammatory genes, is strongly expressed in rheumatoid synovial cells. The mitogen-activated protein kinase (MAPK) p38 pathway mediates the inactivation of TTP via phosphorylation of two serine residues. We wished to test the hypothesis that these phosphorylations contribute to the development of inflammatory arthritis, and that, conversely, joint inflammation may be inhibited by promoting the dephosphorylation and activation of TTP. METHODS: The expression of TTP and its relationship with MAPK p38 activity were examined in non-inflamed and rheumatoid arthritis (RA) synovial tissue. Experimental arthritis was induced in a genetically modified mouse strain, in which endogenous TTP cannot be phosphorylated and inactivated. In vitro and in vivo experiments were performed to test anti-inflammatory effects of compounds that activate the protein phosphatase 2A (PP2A) and promote dephosphorylation of TTP. RESULTS: TTP expression was significantly higher in RA than non-inflamed synovium, detected in macrophages, vascular endothelial cells and some fibroblasts and co-localised with MAPK p38 activation. Substitution of TTP phosphorylation sites conferred dramatic protection against inflammatory arthritis in mice. Two distinct PP2A agonists also reduced inflammation and prevented bone erosion. In vitro anti-inflammatory effects of PP2A agonism were mediated by TTP activation. CONCLUSIONS: The phosphorylation state of TTP is a critical determinant of inflammatory responses, and a tractable target for novel anti-inflammatory treatments.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/enzimologia , Proteína Fosfatase 2/metabolismo , Tristetraprolina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Amino Álcoois/uso terapêutico , Animais , Apolipoproteínas E/uso terapêutico , Artrite Reumatoide/imunologia , Artrite Reumatoide/prevenção & controle , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Fosforilação , Proteína Fosfatase 2/efeitos dos fármacos , RNA Mensageiro/metabolismo , Serina/metabolismo , Membrana Sinovial/metabolismo , Tristetraprolina/genéticaRESUMO
BACKGROUND AND OBJECTIVES: For our understanding of the pathogenesis of rheumatoid arthritis (RA), it is important to elucidate the mechanisms underlying early stages of synovitis. Here, synovial cytokine production was investigated in patients with very early arthritis. METHODS: Synovial biopsies were obtained from patients with at least one clinically swollen joint within 12â weeks of symptom onset. At an 18-month follow-up visit, patients who went on to develop RA, or whose arthritis spontaneously resolved, were identified. Biopsies were also obtained from patients with RA with longer symptom duration (>12â weeks) and individuals with no clinically apparent inflammation. Synovial mRNA expression of 117 cytokines was quantified using PCR techniques and analysed using standard and novel methods of data analysis. Synovial tissue sections were stained for CXCL4, CXCL7, CD41, CD68 and von Willebrand factor. RESULTS: A machine learning approach identified expression of mRNA for CXCL4 and CXCL7 as potentially important in the classification of early RA versus resolving arthritis. mRNA levels for these chemokines were significantly elevated in patients with early RA compared with uninflamed controls. Significantly increased CXCL4 and CXCL7 protein expression was observed in patients with early RA compared with those with resolving arthritis or longer established disease. CXCL4 and CXCL7 co-localised with blood vessels, platelets and CD68(+) macrophages. Extravascular CXCL7 expression was significantly higher in patients with very early RA compared with longer duration RA or resolving arthritis CONCLUSIONS: Taken together, these observations suggest a transient increase in synovial CXCL4 and CXCL7 levels in early RA.
Assuntos
Artrite Reumatoide/genética , Citocinas/genética , Macrófagos/metabolismo , Fator Plaquetário 4/genética , RNA Mensageiro/metabolismo , Membrana Sinovial/metabolismo , beta-Tromboglobulina/genética , Adulto , Idoso , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Artrite Reumatoide/metabolismo , Citocinas/metabolismo , Progressão da Doença , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Fator Plaquetário 4/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/citologia , beta-Tromboglobulina/metabolismo , Fator de von Willebrand/metabolismoRESUMO
OBJECTIVE: A population of synovial inflammatory dendritic cells (infDCs) has recently been identified in rheumatoid arthritis (RA) and is thought to be monocyte-derived. Here, we investigated the role and source of granulocyte macrophage-colony-stimulating factor (GM-CSF) in the differentiation of synovial infDC in RA. METHODS: Production of GM-CSF by peripheral blood (PB) and synovial fluid (SF) CD4+ T cells was assessed by ELISA and flow cytometry. In vitro CD4+ T-cell polarisation experiments were performed with T-cell activating CD2/CD3/CD28-coated beads in the absence or presence of pro-Th1 or pro-Th17 cytokines. CD1c+ DC and CD16+ macrophage subsets were flow-sorted and analysed morphologically and functionally (T-cell stimulatory/polarising capacity). RESULTS: RA-SF CD4+ T cells produced abundant GM-CSF upon stimulation and significantly more than RA-SF mononuclear cells depleted of CD4+ T cells. GM-CSF-producing T cells were significantly increased in RA-SF compared with non-RA inflammatory arthritis SF, active RA PB and healthy donor PB. GM-CSF-producing CD4+ T cells were expanded by Th1-promoting but not Th17-promoting conditions. Following coculture with RA-SF CD4+ T cells, but not healthy donor PB CD4+ T cells, a subpopulation of monocytes differentiated into CD1c+ infDC; a process dependent on GM-CSF. These infDC displayed potent alloproliferative capacity and enhanced GM-CSF, interleukin-17 and interferon-γ production by CD4+ T cells. InfDC with an identical phenotype to in vitro generated cells were significantly enriched in RA-SF compared with non-RA-SF/tissue/PB. CONCLUSIONS: We demonstrate a therapeutically tractable feedback loop of GM-CSF secreted by RA synovial CD4+ T cells promoting the differentiation of infDC with potent capacity to induce GM-CSF-producing CD4+ T cells.
Assuntos
Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Antígenos CD1/análise , Técnicas de Cocultura , Citocinas/biossíntese , Glicoproteínas/análise , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Humanos , Imunofenotipagem , Receptores de Lipopolissacarídeos/análise , Macrófagos/imunologia , Monócitos/imunologia , Osteoartrite/imunologia , Líquido Sinovial/imunologia , Células Th1/imunologiaRESUMO
OBJECTIVES: The success of B cell targeting therapies has highlighted the importance of B cells in rheumatoid arthritis pathogenesis. We have previously shown that B cells in the RA synovium are capable of producing pro-inflammatory and bone-destructive cytokines including RANKL. Here we sought to characterise the nature and functional relevance of the RANKL-producing B cell subset in the RA synovium. METHODS: Synovial fluid and peripheral blood B cells from patients with RA were analysed by flow cytometry for markers of B cell differentiation and activation and for chemokine receptors. FcRL4(+) and FcRL4(-) B cells sorted from synovial fluid were analysed for cytokine expression using Taqman low-density arrays. Synovial tissue biopsies obtained from patients with RA were analysed by immunofluorescence for CD20, RANKL and FcRL4. FCRL4 mRNA expression was determined in synovial tissue of RA patients and non-inflammatory control subjects by real-time PCR. RESULTS: RANKL-producing B cells in RA synovial tissue and fluid were identified as belonging to a distinct subset of B cells defined by expression of the transmembrane protein FcRL4. FcRL4+ B cells express a distinct combination of cytokines and surface proteins indicating a function distinct from that of FcRL4- B cells. Notably, FcRL4+ B cells expressed high levels of TNF-α and RANKL mRNA. CONCLUSIONS: We have identified a novel pro-inflammatory B cell population in the RA synovium which is defined by expression of FcRL4 and responsible for RANKL production. This B cell population expresses high levels of CD20, and its removal by rituximab may contribute to the anti-inflammatory effect of this drug.
Assuntos
Artrite Reumatoide/imunologia , Subpopulações de Linfócitos B/imunologia , Ligante RANK/genética , RNA Mensageiro/metabolismo , Receptores Fc/genética , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/genética , Adulto , Idoso , Antígenos CD20/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Subpopulações de Linfócitos B/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Feminino , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Ligante RANK/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Fc/metabolismo , Líquido Sinovial , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To determine the tolerability, safety and yield of synovial tissue in an early arthritis cohort using a minimally invasive, ultrasound (US)-guided, synovial biopsy technique in small, medium and large joints. METHODS: 93 sequential biopsy procedures were assessed from a total of 57 patients (baseline and 36 repeat biopsies at 6 months) recruited as part of the 'Pathobiology of Early Arthritis Cohort' study. Patients completed a tolerability questionnaire prior to and following the synovial biopsy procedure. The synovial biopsy was performed under US guidance with US images of the joint recorded prior to each procedure. Synovial tissue was harvested for immunohistochemistry and RNA extraction. RESULTS: Five different joint sites were biopsied (knee, elbow, wrist, metacarpal phalangeal and proximal interphalangeal). No significant complications were reported following the procedure. No difference in pain, swelling and stiffness of the biopsied joint from before and after the procedure was demonstrated. A median of 14 biopsy samples was retrieved from each procedure with 93% of biopsy procedures yielding good quality tissue. RNA yield was good in all joints and in repeat biopsies. Multivariant analysis demonstrated a significantly greater yield of RNA and graded tissue in relation to a high prebiopsy, grey-scale synovitis score (0-3, semiquantitative). CONCLUSIONS: A minimally invasive approach to synovial tissue harvesting, using US guidance, is both safe and well-tolerated by patients. Tissue quality/RNA yield is preserved in subsequent biopsies following therapeutic intervention. A high US grey-scale synovitis score is a predictor of good quality/quantity of tissue and RNA.
Assuntos
Artrite Reumatoide/patologia , Articulação do Cotovelo/patologia , Articulação da Mão/patologia , Biópsia Guiada por Imagem , Articulação do Joelho/patologia , RNA/análise , Membrana Sinovial/patologia , Sinovite/patologia , Adulto , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/metabolismo , Estudos de Coortes , Articulação do Cotovelo/diagnóstico por imagem , Feminino , Articulação da Mão/diagnóstico por imagem , Humanos , Articulação do Joelho/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Membrana Sinovial/diagnóstico por imagem , Membrana Sinovial/metabolismo , Sinovite/diagnóstico por imagem , Sinovite/metabolismo , UltrassonografiaRESUMO
Inflammation is an unstable state; it either resolves or persists. Inflammatory reactions often have a propensity for specific anatomical sites. Why inflammation persists with specific tissue tropism remains obscure. Increasing evidence suggests that stromal cells which define tissue architecture are the key cells involved, and therefore make attractive therapeutic targets. Research on stromal cells in general and fibroblasts in particular has so far been hampered by a lack of fibroblast-specific cell markers. This review highlights our increasing understanding of the role of fibroblasts in inflammation, and suggests that these cells provide the cellular basis for site specific chronic inflammation.
Assuntos
Inflamação/imunologia , Células Estromais/imunologia , Animais , Doença Crônica , Fibroblastos/imunologia , Humanos , Leucócitos/imunologia , CamundongosRESUMO
Liberation of elastin peptides from damaged lung may be a mechanism of autoimmune lung disease. Citrullination, and anti-citrullinated protein antibody formation occurs in smokers, but the role of smoking in autoantibody generation relevant to pulmonary disease is unclear. Anti-elastin, anti-cyclic citrullinated peptide (anti-CCP) and anti-mutated citrullinated vimentin (anti-MCV) antibodies were measured in 257 subjects with α1-antitrypsin deficiency (AATD), 113 subjects with usual chronic obstructive pulmonary disease (COPD) and 22 healthy nonsmokers. Levels were compared between groups, against phenotypic features and against smoke exposure. Anti-elastin antibodies were higher in controls relative to AATD (p = 0.008) and usual COPD (p < 0.00001), and in AATD relative to usual COPD (p < 0.00001). Anti-elastin levels showed a threshold at 10 pack-yrs, being higher in those who had smoked less (p = 0.004). No relationships between antibody levels and clinical phenotype were seen after adjustment for smoke exposure. Anti-CCP antibodies were higher in usual COPD than AATD (p = 0.002) but the relationship to smoke exposure was less clear. Smoke exposure is the main determinant of anti-elastin antibody levels, which fall after 10 pack-yrs. Local antibody complexes may be a better measure of elastin directed autoimmunity than circulating levels.
Assuntos
Elastina/imunologia , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Fumar/efeitos adversos , Deficiência de alfa 1-Antitripsina/imunologia , Adulto , Idoso , Doenças Autoimunes/imunologia , Elastina/metabolismo , Feminino , Volume Expiratório Forçado , Humanos , Pneumopatias/imunologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Risco , Fatores de TempoRESUMO
OBJECTIVES: Synovial fibroblasts and osteoblasts generate active glucocorticoids by means of the 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) enzyme. This activity increases in response to proinflammatory cytokines or glucocorticoids. During inflammatory arthritis synovium and bone are exposed to both these factors. This study hypothesised that glucocorticoids magnify the effects of inflammatory cytokines on local glucocorticoid production in both synovium and bone. METHODS: The effects of inflammatory cytokines (IL-1beta/tumour necrosis factor alpha; TNFalpha) and glucocorticoids, alone or combined, were assessed on the expression and activity of 11beta-HSD1 in primary synovial fibroblasts, primary human osteoblasts and MG-63 osteosarcoma cells. A range of other target genes and cell types were used to examine the specificity of effects. Functional consequences were assessed using IL-6 ELISA. RESULTS: In synovial fibroblasts and osteoblasts, treatment with cytokines or glucocorticoids in isolation induced 11beta-HSD1 expression and activity. However, in combination, 11beta-HSD1 expression, activity and functional consequences were induced synergistically to a level not seen with isolated treatments. This effect was seen in normal skin fibroblasts but not foreskin fibroblasts or adipocytes and was only seen for the 11beta-HSD1 gene. Synergistic induction had functional consequences on IL-6 production. CONCLUSIONS: Combined treatment with inflammatory cytokines and glucocorticoids synergistically induces 11beta-HSD1 expression and activity in synovial fibroblasts and osteoblasts, providing a mechanism by which synovium and bone can interact to enhance anti-inflammatory responses by increasing localised glucocorticoid levels. However, the synergistic induction of 11beta-HSD1 might also cause detrimental glucocorticoid accumulation in bone or surrounding tissues.
Assuntos
Citocinas/farmacologia , Glucocorticoides/biossíntese , Osteoblastos/efeitos dos fármacos , Membrana Sinovial/efeitos dos fármacos , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/biossíntese , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Mediadores da Inflamação/farmacologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Células Tumorais CultivadasRESUMO
Integrin-mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli, including chemokines, antigen receptors, and adhesion receptors. How cellular signals from CD31 and other adhesion amplifiers are integrated with those from classical mitogenic stimuli to regulate leukocyte function remains poorly understood. Here, we show that the cytoplasmic tail of CD31, an important integrin adhesion amplifier, propagates signals that induce T cell adhesion via beta1 (VLA-4) and beta2 (LFA-1) integrins. We identify the small GTPase, Rap1, as a critical mediator of this effect. Importantly, CD31 selectively activated the small Ras-related GTPase, Rap1, but not Ras, R-Ras, or Rap2. An activated Rap1 mutant stimulated T lymphocyte adhesion to intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), as did the Rap1 guanine nucleotide exchange factor C3G and a catalytically inactive mutant of RapGAP. Conversely, negative regulators of Rap1 signaling blocked CD31-dependent adhesion. These findings identify a novel important role for Rap1 in regulating ligand-induced cell adhesion and suggest that Rap1 may play a more general role in coordinating adhesion-dependent signals during leukocyte migration and extravasation. Our findings also suggest an alternative mechanism, distinct from interference with Ras-proximal signaling, by which Rap1 might mediate transformation reversion.
Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular/fisiologia , Integrinas/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Receptores de Retorno de Linfócitos/fisiologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Antígenos CD/fisiologia , Humanos , Integrina alfa4beta1 , Células Jurkat , Molécula-1 de Adesão Celular Endotelial a Plaquetas/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Linfócitos T/fisiologia , Transfecção , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
BACKGROUND: Despite their efficacy in the treatment of chronic inflammation, the prolonged application of therapeutic glucocorticoids (GCs) is limited by significant systemic side effects including glucocorticoid-induced osteoporosis (GIOP). 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is a bi-directional enzyme that primarily activates GCs in vivo, regulating tissue-specific exposure to active GC. We aimed to determine the contribution of 11ß-HSD1 to GIOP. METHODS: Wild type (WT) and 11ß-HSD1 knockout (KO) mice were treated with corticosterone (100 µg/ml, 0.66% ethanol) or vehicle (0.66% ethanol) in drinking water over 4 weeks (six animals per group). Bone parameters were assessed by micro-CT, sub-micron absorption tomography and serum markers of bone metabolism. Osteoblast and osteoclast gene expression was assessed by quantitative RT-PCR. RESULTS: Wild type mice receiving corticosterone developed marked trabecular bone loss with reduced bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N). Histomorphometric analysis revealed a dramatic reduction in osteoblast numbers. This was matched by a significant reduction in the serum marker of osteoblast bone formation P1NP and gene expression of the osteoblast markers Alp and Bglap. In contrast, 11ß-HSD1 KO mice receiving corticosterone demonstrated almost complete protection from trabecular bone loss, with partial protection from the decrease in osteoblast numbers and markers of bone formation relative to WT counterparts receiving corticosterone. CONCLUSIONS: This study demonstrates that 11ß-HSD1 plays a critical role in GIOP, mediating GC suppression of anabolic bone formation and reduced bone volume secondary to a decrease in osteoblast numbers. This raises the intriguing possibility that therapeutic inhibitors of 11ß-HSD1 may be effective in preventing GIOP in patients receiving therapeutic steroids.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Osso Esponjoso/patologia , Corticosterona/efeitos adversos , Osteoporose/induzido quimicamente , Animais , Osso Esponjoso/efeitos dos fármacos , Osso Esponjoso/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Glucocorticoides/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteoporose/metabolismo , Osteoporose/patologia , Microtomografia por Raio-XRESUMO
BACKGROUND: Isolated, primary synovial fibroblasts generate active glucocorticoids through expression of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). This enzyme produces cortisol from inactive cortisone (and prednisolone from prednisone). OBJECTIVE: To determine how intact synovial tissue metabolises glucocorticoids and to identify the local and systemic consequences of this activity by examination of glucocorticoid metabolism in patients with rheumatoid arthritis (RA). METHODS: Synovial tissue was taken from patients with RA during joint replacement surgery. Glucocorticoid metabolism in explants was assessed by thin-layer chromatography and specific enzyme inhibitors. RT-PCR and immunohistochemistry were used to determine expression and distribution of 11beta-HSD enzymes. Systemic glucocorticoid metabolism was examined in patients with RA using gas chromatography/mass spectrometry. RESULTS: Synovial tissue synthesised cortisol from cortisone, confirming functional 11beta-HSD1 expression. In patients with RA, enzyme activity correlated with donor erythrocyte sedimentation rate (ESR). Synovial tissues could also convert cortisol back to cortisone. Inhibitor studies and immunohistochemistry suggested this was owing to 11beta-HSD2 expression in synovial macrophages, whereas 11beta-HSD1 expression occurred primarily in fibroblasts. Synovial fluids exhibited lower cortisone levels than matched serum samples, indicating net local steroid activation. Urinary analyses indicated high 11beta-HSD1 activity in untreated patients with RA compared with controls and a significant correlation between total body 11beta-HSD1 activity and ESR. CONCLUSIONS: Synovial tissue metabolises glucocorticoids, the predominant effect being glucocorticoid activation, and this increases with inflammation. Endogenous glucocorticoid production in the joint is likely to have an impact on local inflammation and bone integrity.
Assuntos
Artrite Reumatoide/metabolismo , Glucocorticoides/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/fisiologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/fisiologia , Idoso , Artrite Reumatoide/enzimologia , Cortisona/antagonistas & inibidores , Cortisona/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Hidrocortisona/farmacologia , Interleucina-6/biossíntese , Masculino , Pessoa de Meia-Idade , Osteoartrite/enzimologia , Osteoartrite/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/enzimologia , Membrana Sinovial/metabolismo , Técnicas de Cultura de TecidosRESUMO
A characteristic feature of many chronic inflammatory diseases is their persistence and predilection for certain sites. The molecular basis for such tissue tropism and failure of the inflammatory response to resolve has until relative recently remained obscure. Recent studies have strongly implicated fibroblasts as cells which contribute to disease persistence and which help define anatomical location. Therefore fibroblasts make an attractive therapeutic target as they help orchestrate the inflammatory infiltrate. Current anti-inflammatory therapies target immune cells in an attempt to inhibit the production of pro-inflammatory mediators. However an equally important target is the active induction of pro-resolution programmes responsible for the resolution of inflammation. Fibroblasts are likely to be an important source of these anti-inflammatory mediators. Therapeutic manipulation of fibroblasts and their biologically active products is an emerging concept in treating cancer and is likely to provide a novel method to achieve improved control of chronic inflammatory disease.
Assuntos
Anti-Inflamatórios/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Inflamação/tratamento farmacológico , Inflamação/fisiopatologia , Animais , Artrite/tratamento farmacológico , Artrite/fisiopatologia , Doença Crônica , Fibroblastos/imunologia , Humanos , Inflamação/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/fisiopatologiaRESUMO
OBJECTIVE: Effective treatment can only be given during the early stages of RA if patients are seen early. However, many patients delay for prolonged periods before seeking medical advice. This study explores factors influencing the decision to seek medical advice in RA patients. METHODS: In-depth, semi-structured interviews were carried out with 24 patients. Purposive sampling ensured a cross-section in terms of time to presentation, gender, age and ethnic background. Interview transcripts were analysed and themes identified using established methods. RESULTS: Four main themes influenced the decision to seek medical advice: (i) symptom experience: the severity of symptoms and their impact on functional ability; (ii) symptom evaluation: the patient's explanation for their symptoms and recognition of their significance; (iii) knowledge of RA and available therapies; and (iv) experience of and attitudes towards health care providers. A significant and rapid impact of the disease on functional ability characterized those presenting early. Many developed an explanation for their symptoms that related to preceding activities. Recognition that this explanation was inadequate to explain symptom progression frequently prompted a consultation. Only one patient sought advice because she thought that she might have RA. CONCLUSIONS: Symptom evaluation is a key factor influencing how quickly medical advice is sought in other diseases. In contrast to the situation with many cancers where there is widespread association of symptoms and signs with the eventual diagnosis, this was not the case in RA. Our findings should inform strategies to reduce delays in help-seeking in people with early RA.
Assuntos
Artrite Reumatoide/diagnóstico , Tomada de Decisões , Aceitação pelo Paciente de Cuidados de Saúde/psicologia , Adulto , Idoso , Artrite Reumatoide/complicações , Artrite Reumatoide/fisiopatologia , Artrite Reumatoide/psicologia , Atitude Frente a Saúde , Diagnóstico Precoce , Inglaterra , Medicina de Família e Comunidade , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Dor/etiologia , Seleção de PacientesRESUMO
OBJECTIVE: To assess whether patients with RA and SLE who are of South Asian origin have different beliefs about medicines in general, and about DMARDs in particular, compared with patients of White British/Irish origin. METHODS: One hundred patients of South Asian origin (50 RA; 50 SLE) and 100 patients of White British/Irish origin (50 RA; 50 SLE) were recruited. Demographic and disease-related details and responses to the Beliefs about Medicines Questionnaire (BMQ), the SF-36 and the HAQ were collected. RESULTS: Patients of South Asian origin had significantly higher General Overuse (GO), General Harm (GH) and Specific Concern (SC) scores compared with patients of White British/Irish origin. Forward stepwise multivariable regression analysis showed that ethnic origin was an independent predictor of the GO, GH and SC scores with patients of South Asian origin having higher scores in these three scales of the BMQ. CONCLUSION: RA and SLE patients of South Asian origin have very high levels of concern about DMARDs and are generally worried about prescribed medicines. This may have an impact on adherence in this group of patients and further work is needed to understand the reasons underlying these beliefs.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/psicologia , Cultura , Lúpus Eritematoso Sistêmico/psicologia , Adulto , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/etnologia , Sudeste Asiático/etnologia , Povo Asiático , Feminino , Nível de Saúde , Humanos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/etnologia , Masculino , Pessoa de Meia-Idade , Qualidade de Vida , Análise de Regressão , Inquéritos e Questionários , Reino Unido , População BrancaRESUMO
OBJECTIVE: Annexin-1 (Anx-A1) has been recently shown to play a key role in T-cell activation and to be highly expressed in T cells from RA patients. Here, we investigated the effects of glucocorticoids (GCs) on Anx-A1 expression in T cells in vitro and in vivo. METHODS: To evaluate the effects of dexamethasone (Dex) on Anx-A1 expression, human peripheral blood T cells were incubated with Dex and then analysed by real-time PCR and western blotting. Similar experiments were carried out in vivo by measuring Anx-A1 levels in T cells from patients with RA before and after administration of steroids. RESULTS: Incubation of T cells with Dex decreased Anx-A1 levels in a time-dependent fashion and almost abolished its expression after 12 h. Stimulation of T cells pre-incubated with Dex for 12 h with anti-CD3/CD28 led to significant reduction of IL-2 production. Addition of human recombinant Anx-A1 to Dex-treated cells reversed the inhibitory effects of the steroids on anti-CD3/CD28-induced IL-2 production. Treatment of RA patients with steroid decreased Anx-A1 expression in T cells. CONCLUSIONS: GCs suppress Anx-A1 expression in T cells in vitro and in vivo. These results provide evidence for a novel pathway by which steroids regulate the adaptive immune response and suggest that Anx-A1 may represent a target for the treatment of autoimmune diseases.
Assuntos
Anexina A1/análise , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/química , Dexametasona/uso terapêutico , Glucocorticoides/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Anexina A1/metabolismo , Anticorpos Monoclonais/farmacologia , Western Blotting , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Depressão Química , Relação Dose-Resposta a Droga , Feminino , Humanos , Interleucina-2/imunologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: CD4 and CD8 T-cell subsets accumulate in distinct microdomains within the inflamed rheumatoid synovium. The molecular basis for their differential distribution remains unclear. Since chemokines and adhesion molecules play an important role in the positioning of leucocytes at sites of inflammation, we tested the hypothesis that the differential expression and function of chemokine and/or adhesion molecules explains why CD4(+) T cells accumulate within perivascular cuffs, whereas CD8(+) T cells distribute diffusely within the tissue. METHODS: Expression of an extensive panel of chemokine receptors and adhesion molecules on matched CD4(+) and CD8(+) T cells from peripheral blood (PB) and synovial fluid (SF) was analysed by multicolour flow cytometry. Migration assays and flow-based adhesion assays were used to assess the functional consequences of any differences in the expression of chemokine and adhesion receptors. RESULTS: CD4(+) and CD8(+) T cells from PB and SF expressed unique yet consistent patterns of chemokine and adhesion receptors. SF CD8(+) T cells were much less promiscuous in their expression of chemokine receptors than SF CD4(+) T cells. The alpha(6)beta(1) integrin was highly expressed on PB CD4(+) T cells, but not on PB CD8(+) T cells. Laminin, the ligand for alpha(6)beta(1), retained CD4(+) T cells, but less so CD8(+) T cells, within inflamed synovial tissue. CONCLUSION: Infiltrating PB CD4(+) T cells, but not CD8(+) T cells, express functional levels of the alpha(6)beta(1) integrin. We propose that this leads to their retention within the rheumatoid synovium in perivascular cuffs, which are defined and delineated by the expression of laminin.
Assuntos
Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Integrina alfa6beta1/imunologia , Membrana Sinovial/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Integrina alfa6/metabolismo , Laminina/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de Quimiocinas/metabolismo , Líquido Sinovial/imunologiaRESUMO
The C-type lectin domain containing group 14 family members CLEC14A and CD93 are proteins expressed by endothelium and are implicated in tumour angiogenesis. CD248 (alternatively known as endosialin or tumour endothelial marker-1) is also a member of this family and is expressed by tumour-associated fibroblasts and pericytes. Multimerin-2 (MMRN2) is a unique endothelial specific extracellular matrix protein that has been implicated in angiogenesis and tumour progression. We show that the group 14 C-type lectins CLEC14A, CD93 and CD248 directly bind to MMRN2 and only thrombomodulin of the family does not. Binding to MMRN2 is dependent on a predicted long-loop region in the C-type lectin domain and is abrogated by mutation within the domain. CLEC14A and CD93 bind to the same non-glycosylated coiled-coil region of MMRN2, but the binding of CD248 occurs on a distinct non-competing region. CLEC14A and CD248 can bind MMRN2 simultaneously and this occurs at the interface between endothelium and pericytes in human pancreatic cancer. A recombinant peptide of MMRN2 spanning the CLEC14A and CD93 binding region blocks CLEC14A extracellular domain binding to the endothelial cell surface as well as increasing adherence of human umbilical vein endothelial cells to the active peptide. This MMRN2 peptide is anti-angiogenic in vitro and reduces tumour growth in mouse models. These findings identify novel protein interactions involving CLEC14A, CD93 and CD248 with MMRN2 as targetable components of vessel formation.