RESUMO
OBJECTIVE: Activated synovial fibroblasts are key effector cells in RA. Selectively depleting these based upon their expression of fibroblast activation protein (FAP) is an attractive therapeutic approach. Here we introduce FAP imaging of inflamed joints using 68Ga-FAPI-04 in a RA patient, and aim to assess feasibility of anti-FAP targeted photodynamic therapy (FAP-tPDT) ex vivo using 28H1-IRDye700DX on RA synovial explants. METHODS: Remnant synovial tissue from RA patients was processed into 6 mm biopsies and, from several patients, into primary fibroblast cell cultures. Both were treated using FAP-tPDT. Cell viability was measured in fibroblast cultures and biopsies were evaluated for histological markers of cell damage. Selectivity of the effect of FAP-tPDT was assessed using flow cytometry on primary fibroblasts and co-cultured macrophages. Additionally, one RA patient intravenously received 68Ga-FAPI-04 and was scanned using PET/CT imaging. RESULTS: In the RA patient, FAPI-04 PET imaging showed high accumulation of the tracer in arthritic joints with very low background signal. In vitro, FAP-tPDT induced cell death in primary RA synovial fibroblasts in a light dose-dependent manner. An upregulation of cell damage markers was observed in the synovial biopsies after FAP-tPDT. No significant effects of FAP-tPDT were noted on macrophages after FAP-tPDT of neighbouring fibroblasts. CONCLUSION: In this study the feasibility of selective FAP-tPDT in synovium of rheumatoid arthritis patients ex vivo is demonstrated. Furthermore, this study provides the first indication that FAP-targeted PET/CT can be used to image arthritic joints, an important step towards application of FAP-tPDT as a targeted locoregional therapy for RA.
Assuntos
Artrite Reumatoide , Fotoquimioterapia , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Humanos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Membrana Sinovial/metabolismoRESUMO
Systemic sclerosis (SSc) is a rare, severe, auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of this fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells. In this study, we explored the use of FAP-tPDT in primary skin fibroblasts from SSc patients, both in 2D and 3D cultures. Method: The FAP targeting antibody 28H1 was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous outgrowth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer construct, washed after 4 h and exposed to λ = 689 nm light. Cell viability was measured using CellTiter Glo®®. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Results: FAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with an irrelevant antibody-photosensitizer construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. Conclusions: Here we have shown, for the first time, the potential of FAP-tPDT for the treatment of fibrosis in SSc skin.
Assuntos
Endopeptidases/administração & dosagem , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibrose/prevenção & controle , Proteínas de Membrana/administração & dosagem , Miofibroblastos/efeitos dos fármacos , Fotoquimioterapia/métodos , Escleroderma Sistêmico/tratamento farmacológico , Colágeno Tipo I/metabolismo , Fibroblastos/patologia , Fibrose/metabolismo , Fibrose/patologia , Humanos , Miofibroblastos/patologia , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologiaRESUMO
OBJECTIVE: In RA, synovial fibroblasts become activated. These cells express fibroblast activation protein (FAP) and contribute to the pathogenesis by producing cytokines, chemokines and proteases. Selective depletion in inflamed joints could therefore constitute a viable treatment option. To this end, we developed and tested a new therapeutic strategy based on the selective destruction of FAP-positive cells by targeted photodynamic therapy (tPDT) using the anti-FAP antibody 28H1 coupled to the photosensitizer IRDye700DX. METHODS: After conjugation of IRDye700DX to 28H1, the immunoreactive binding and specificity of the conjugate were determined. Subsequently, tPDT efficiency was established in vitro using a 3T3 cell line stably transfected with FAP. The biodistribution of [111In]In-DTPA-28H1 with and without IRDye700DX was assessed in healthy C57BL/6N mice and in C57BL/6N mice with antigen-induced arthritis. The potential of FAP-tPDT to induce targeted damage was determined ex vivo by treating knee joints from C57BL/6N mice with antigen-induced arthritis 24 h after injection of the conjugate. Finally, the effect of FAP-tPDT on arthritis development was determined in mice with collagen-induced arthritis. RESULTS: 28H1-700DX was able to efficiently induce FAP-specific cell death in vitro. Accumulation of the anti-FAP antibody in arthritic knee joints was not affected by conjugation with the photosensitizer. Arthritis development was moderately delayed in mice with collagen-induced arthritis after FAP-tPDT. CONCLUSION: Here we demonstrate the feasibility of tPDT to selectively target and kill FAP-positive fibroblasts in vitro and modulate arthritis in vivo using a mouse model of RA. This approach may have therapeutic potential in (refractory) arthritis.
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Artrite Experimental/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Fotoquimioterapia/métodos , Células 3T3/efeitos dos fármacos , Animais , Feminino , Indóis/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Compostos de Organossilício/uso terapêuticoRESUMO
OBJECTIVE: One of the disease hallmarks of RA is progressive cartilage and bone destruction in the joints. The exact mechanism underlying this disease process is largely unknown. Nod1, an intracellular pattern recognition receptor expressed by the innate immune system, has been previously shown to display anti-inflammatory effects in experimental arthritis. Furthermore, an insertion/deletion polymorphism in NOD1 has been demonstrated to modulate cytokine responses of immune cells. In this study, the effect of the insertion/deletion polymorphism in NOD1 on RA susceptibility and severity was assessed. METHODS: Ex vivo stimulation of primary immune cells and osteoclasts with microbial triggers was performed to measure cytokine responses and osteoclast-specific gene expression in relation to the NOD1 genotype. In total, 1047 RA patients from two centres were genotyped for the NOD1 polymorphism and compared with 431 healthy controls. Clinical scores of joint inflammation and destruction were correlated with the NOD1 genotype. RESULTS: Functional analysis revealed increased production of pro-inflammatory cytokines in cells from individuals bearing the NOD1 +32656 insertion allele. Furthermore, osteoclast bone resorption activity was elevated, as reflected by increased expression of the lysosomal protease cathepsin K. However, the insertion allele of the NOD1 +32656 polymorphism was not associated with either susceptibility to, or clinical parameters of, inflammation or bone destruction in RA patients. CONCLUSION: These findings demonstrate that the NOD1 polymorphism modulates pro-inflammatory cytokine responses induced through Toll-like receptor or Nod-like receptor ligands. Nevertheless, these effects of genetic variation in NOD1 appear to be redundant in RA susceptibility and severity.
Assuntos
Artrite Reumatoide/genética , Citocinas/metabolismo , Predisposição Genética para Doença , Proteína Adaptadora de Sinalização NOD1/genética , Polimorfismo Genético , Adulto , Artrite Reumatoide/fisiopatologia , Reabsorção Óssea/genética , Reabsorção Óssea/fisiopatologia , Estudos de Casos e Controles , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos , Valores de Referência , Medição de RiscoRESUMO
Macrophages play a crucial role in the initiation and progression of rheumatoid arthritis (RA). Liposomes can be used to deliver therapeutics to macrophages by exploiting their phagocytic ability. However, since macrophages serve as the immune system's first responders, it is inadvisable to systemically deplete these cells. By loading the liposomes with the photosensitizer IRDye700DX, we have developed and tested a novel way to perform photodynamic therapy (PDT) on macrophages in inflamed joints. PEGylated liposomes were created using the film method and post-inserted with micelles containing IRDye700DX. For radiolabeling, a chelator was also incorporated. RAW 264.7 cells were incubated with liposomes with or without IRDye700DX and exposed to 689 nm light. Viability was determined using CellTiterGlo. Subsequently, biodistribution and PDT studies were performed on mice with collagen-induced arthritis (CIA). PDT using IRDye700DX-loaded liposomes efficiently induced cell death in vitro, whilst no cell death was observed using the control liposomes. Biodistribution of the two compounds in CIA mice was comparable with excellent correlation of the uptake with macroscopic and microscopic arthritis scores. Treatment with 700DX-loaded liposomes significantly delayed arthritis development. Here we have shown the proof-of-principle of performing PDT in arthritic joints using IRDye700DX-loaded liposomes, allowing locoregional treatment of arthritis.
RESUMO
Osteoarthritis (OA) is a destructive disease of the joint with age and obesity being its most important risk factors. Around 50% of OA patients suffer from inflammation of the synovial joint capsule, which is characterized by increased abundance and activation of synovial macrophages that produce reactive oxygen species (ROS) via NADPH-oxidase 2 (NOX2). Both ROS and high blood levels of low-density lipoprotein (LDL) are implicated in OA pathophysiology, which may interact to form oxidized LDL (oxLDL) and thereby promote disease. Therefore, targeting NOX2 could be a viable treatment strategy for OA. Collagenase-induced OA (CiOA) was used to compare pathology between wild-type (WT) and Nox2 knockout (Nox2-/-) C57Bl/6 mice. Mice were either fed a standard diet or Western diet (WD) to study a possible interaction between NOX2-derived ROS and LDL. Synovial inflammation, cartilage damage and ectopic bone size were assessed on histology. Extracellular ROS production by macrophages was measured in vitro using the Amplex Red assay. Nox2-/- macrophages produced basal levels of ROS but were unable to increase ROS production in response to the alarmin S100A8 or the phorbol ester PMA. Interestingly, Nox2 deficiency reduced cartilage damage, synovial lining thickness and ectopic bone size, whereas these disease parameters were not affected by WD-feeding. These results suggest that NOX2-derived ROS are involved in CiOA development.
RESUMO
Background: Injection of adipose-derived mesenchymal stromal cells (ASCs) into murine knee joints after induction of inflammatory collagenase-induced osteoarthritis (CiOA) reduces development of joint pathology. This protection is only achieved when ASCs are applied in early CiOA, which is characterized by synovitis and high S100A8/A9 and IL-1ß levels, suggesting that inflammation is a prerequisite for the protective effect of ASCs. Our objective was to gain more insight into the interplay between synovitis and ASC-mediated amelioration of CiOA pathology. Methods: CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with hematoxylin/eosin and immunolocalization of polymorphonuclear cells (PMNs) and ASCs was performed using antibodies for NIMP-R14 and CD271, respectively. Chemokine expression induced by IL-1ß or S100A8/A9 was assessed with qPCR and Luminex. ASC-PMN co-cultures were analyzed microscopically and with Luminex for inflammatory mediators. Migration of PMNs through transwell membranes toward conditioned medium of non-stimulated ASCs (ASCNS-CM) or IL-1ß-stimulated ASCs (ASCIL-1ß-CM) was examined using flow cytometry. Phagocytic capacity of PMNs was measured with labeled zymosan particles. Results: Intra-articular saline injection on day 7 of CiOA increased synovitis after 6 h, characterized by PMNs scattered throughout the joint cavity and the synovium. ASC injection resulted in comparable numbers of PMNs which clustered around ASCs in close interaction with the synovial lining. IL-1ß-stimulation of ASCs in vitro strongly increased expression of PMN-attracting chemokines CXCL5, CXCL7, and KC, whereas S100A8/A9-stimulation did not. In agreement, the number of clustered PMNs per ASC was significantly increased after 6 h of co-culturing with IL-1ß-stimulated ASCs. Also migration of PMNs toward ASCIL-1ß-CM was significantly enhanced (287%) when compared to ASCNS-CM. Interestingly, association of PMNs with ASCs significantly diminished KC protein release by ASCs (69% lower after 24 h), accompanied by reduced release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with ASCIL-1ß-CM. Conclusions: Local application of ASCs in inflamed CiOA knee joints results in clustering of attracted PMNs with ASCs in the synovium, which is likely mediated by IL-1ß-induced up-regulation of chemokine release by ASCs. This results in enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis.
Assuntos
Interleucina-1beta/fisiologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Neutrófilos/fisiologia , Osteoartrite do Joelho/terapia , Fagocitose , Animais , Artrite Experimental/terapia , Células Cultivadas , Quimiocinas/fisiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologiaRESUMO
OBJECTIVE: Caspase 1, a known cysteine protease, is a critical component of the inflammasome. Both caspase 1 and neutrophil serine proteases such as proteinase 3 (PR3) can process pro-interleukin-1beta (proIL-1beta), a crucial cytokine linked to the pathogenesis of rheumatoid arthritis. This study was undertaken to establish the relative importance of caspase 1 and serine proteases in mouse models of acute and chronic inflammatory arthritis. METHODS: Acute and chronic arthritis were induced in caspase 1-/- mice, and the lack of caspase 1 was investigated for its effects on joint swelling, cartilage metabolism, and histopathologic features. In addition, caspase 1 activity was inhibited in mice lacking active cysteine proteases, and the effects of dual blockade of caspase 1 and serine proteases on arthritis severity and histopathologic features were evaluated. RESULTS: Surprisingly, caspase 1-/- mice, in a model of acute (neutrophil-dominated) arthritis, developed joint swelling to an extent similar to that in wild-type control mice. Joint fluid concentrations of bioactive IL-1beta were comparable in caspase 1-/- mice and controls. In contrast, induction of chronic arthritis (characterized by minimal numbers of neutrophils) in caspase 1-/- mice led to reduced joint inflammation and less cartilage damage, implying a caspase 1-dependent role in this process. In mice lacking neutrophil serine PR3, inhibition of caspase 1 activity resulted in decreased bioactive IL-1beta concentrations in the synovial tissue and less suppression of chondrocyte anabolic function. In addition, dual blockade of both PR3 and caspase 1 led to protection against cartilage and bone destruction. CONCLUSION: Caspase 1 deficiency does not affect neutrophil-dominated joint inflammation, whereas in chronic arthritis, the lack of caspase 1 results in reduced joint inflammation and cartilage destruction. These findings suggest that inhibitors of caspase 1 are not able to interfere with the whole spectrum of IL-1beta production, and therefore such inhibitors may be of therapeutic value only in inflammatory conditions in which limited numbers of neutrophils are present.
Assuntos
Artrite Reumatoide/enzimologia , Caspase 1/deficiência , Interleucina-1/metabolismo , Mieloblastina/metabolismo , Precursores de Proteínas/metabolismo , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Caspase 1/genética , Caspase 1/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Articulações/metabolismo , Articulações/patologia , Elastase de Leucócito/metabolismo , Macrófagos/enzimologia , Macrófagos/imunologia , Mastócitos/enzimologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/enzimologia , Neutrófilos/imunologia , Serina Endopeptidases , Líquido Sinovial/metabolismo , Membrana Sinovial/química , Membrana Sinovial/metabolismoRESUMO
OBJECTIVE: To examine the role of tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha), and IL-1 beta in collagen-induced arthritis (CIA), immediately after onset and during the phase of established arthritis. METHODS: Male DBA/1 mice with collagen-induced arthritis were treated with antibodies against murine TNF alpha and IL-1 alpha/beta at different time points of the disease. IL-1 receptor antagonist (IL-1Ra) was administered using Alzet osmotic minipumps. The effect of anticytokine treatment was monitored by visual scoring. Histology and cytokine reverse transcription polymerase chain reaction (RT-PCR) analyses were performed at the end of the treatment period. RESULTS: Anti-TNF alpha treatment showed efficacy shortly after onset of the disease, but had little effect on fully established CIA. Histologic analysis after early treatment revealed that anti-TNF alpha significantly reduced joint pathology, as determined by infiltration of inflammatory cells and cartilage damage. Anti-IL-1 alpha/beta treatment ameliorated both early and full-blown CIA. This clear suppression of established arthritis was confirmed by administration of high doses of IL-1Ra. Dose-response experiments showed that a continuous supply of 1 mg/day was needed for optimal suppression. Histologic analysis showed markedly reduced cartilage destruction both in the knee and the ankle joints. Autoradiography demonstrated full recovery of chondrocyte synthetic function of articular cartilage. In addition, we found that the IL-1 beta isoform plays a dominant role in established CIA. Profound suppression of CIA was observed with anti-IL-1 beta, although elimination of both IL-1 alpha and IL-1 beta still gave better protection. Analysis of messenger RNA with RT-PCR revealed that IL-1 beta was highly upregulated in synovium and cartilage at late stages of CIA, whereas anti-IL-1 beta treatment markedly reduced IL-1 beta message in the synovium. CONCLUSION: The present study identified different TNF alpha/IL-1 dependencies in various stages of CIA and revealed that blocking of TNF alpha does not necessarily eliminate IL-1. Continuous, high doses of IL-1Ra are needed to block CIA.
Assuntos
Antirreumáticos/história , Artrite Experimental/história , Citocinas/história , Animais , Antirreumáticos/farmacologia , Artrite Experimental/tratamento farmacológico , Citocinas/imunologia , Modelos Animais de Doenças , História do Século XX , Interleucina-1alfa/história , Interleucina-1alfa/imunologia , Interleucina-1beta/história , Interleucina-1beta/imunologia , Camundongos , Camundongos Endogâmicos DBA , Fator de Necrose Tumoral alfa/história , Fator de Necrose Tumoral alfa/imunologiaRESUMO
OBJECTIVE: The pathogenesis of rheumatoid arthritis is often linked to bacterial infections. The present study was undertaken to develop a mouse model of chronic destructive arthritis induced by repeated intraarticular (IA) exposure to bacterial cell wall fragments and to investigate the cytokine dependence of this model. METHODS: Mice that were deficient in various cytokines were injected IA with cell wall fragments of Streptococcus pyogenes on days 0, 7, 14, and 21. The development of chronic destructive arthritis was compared between groups of mice lacking different cytokines, to assess which cytokines were crucial for development of chronic destructive arthritis. RESULTS: Repeated exposure of a joint to S pyogenes cell wall fragments resulted in the development of chronic destructive arthritis. In mice deficient in recombination-activating gene 2, streptococcal cell wall (SCW)-directed T cell reactivity was found and chronic arthritis did not develop, implicating T cells in the generation of chronic SCW-induced arthritis. Interleukin-17 (IL-17) receptor-deficient mice showed a reduction of joint destruction in the chronic stage, implicating a detrimental role of the recently discovered IL-17-producing T helper cells (Th17 cells). IL-23 expression was apparent during the late stages of arthritis. Joint swelling was no longer dependent on tumor necrosis factor alpha (TNFalpha) after the last flare, and pronounced cartilage damage was found after 28 days in TNFalpha-deficient mice. In contrast, IL-1beta-deficient mice were fully protected against joint swelling and cartilage and bone destruction during the late stages of disease. CONCLUSION: These findings indicate that the TNFalpha dependence of arthritis is lost during the erosive stage, when Th17 cells become crucial. IL-1beta dependence remains strong, consistent with its pivotal role in the generation of Th17 cells.
Assuntos
Artrite Infecciosa/imunologia , Interleucina-17/imunologia , Interleucina-1beta/imunologia , Linfócitos T/imunologia , Receptores Toll-Like/imunologia , Doença Aguda , Animais , Artrite Infecciosa/patologia , Linfócitos B/imunologia , Linfócitos B/patologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Cartilagem/imunologia , Cartilagem/patologia , Doença Crônica , Modelos Animais de Doenças , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Streptococcus pyogenes/imunologia , Linfócitos T/patologia , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The IL-1R/Toll-like receptor (TLR) superfamily of receptors has a key role in innate immunity and inflammation. In this study, we report that streptococcal cell wall (SCW)-induced joint inflammation is predominantly dependent on TLR-2 signaling, since TLR-2-deficient mice were unable to develop either joint swelling or inhibition of cartilage matrix synthesis. Myeloid differentiation factor 88 (MyD88) is a Toll/IL-1R domain containing adaptor molecule known to have a central role in both IL-1R/IL-18R and TLR signaling. Mice deficient for MyD88 did not develop SCW-induced arthritis; both joint swelling and disturbance of cartilage chondrocyte anabolic function was completely abolished. Local levels of proinflammatory cytokines and chemokines in synovial tissue washouts were strongly reduced in MyD88-deficient mice. Histology confirmed the pivotal role of MyD88 in acute joint inflammation. TLR-2-deficient mice still allow influx of inflammatory cells into the joint cavity, although the number of cells was markedly reduced. No influx of inflammatory cells was seen in joints of MyD88-deficient mice. In addition, cartilage matrix proteoglycan loss was completely absent in MyD88 knockout mice. These findings clearly demonstrated that MyD88 is a key component in SCW-induced joint inflammation. Since agonists of the Toll-like pathway are abundantly involved in both septic and rheumatoid arthritis, targeting of MyD88 may be a novel therapy in inflammatory joint diseases.
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Antígenos de Diferenciação/fisiologia , Artrite Experimental/imunologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos/fisiologia , Streptococcus pyogenes/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Diferenciação/genética , Artrite Experimental/genética , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Cartilagem Articular/imunologia , Cartilagem Articular/metabolismo , Parede Celular/imunologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Interleucina-1/fisiologia , Interleucina-18/fisiologia , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Família Multigênica/imunologia , Fator 88 de Diferenciação Mieloide , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Proteoglicanas/imunologia , Proteoglicanas/metabolismo , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/biossíntese , Receptores de Interleucina-1/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 2 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
OBJECTIVE: Antibodies directed to citrulline-containing proteins are highly specific for rheumatoid arthritis (RA) and can be detected in up to 80% of patients with RA. Citrulline is a nonstandard amino acid that can be incorporated into proteins only by posttranslational modification of arginine by peptidylarginine deiminase (PAD) enzymes. The objective of this study was to investigate the presence of anticitrulline antibodies, PAD enzymes, and citrullinated antigens in mouse models of both acute and chronic destructive arthritis: streptococcal cell wall (SCW)-induced arthritis and collagen-induced arthritis (CIA), respectively. METHODS: Synovial tissue biopsy specimens were obtained from naive mice, mice with CIA, and mice with SCW-induced arthritis. The expression of messenger RNA (mRNA) for PAD enzymes was analyzed by reverse transcriptase-polymerase chain reaction; the presence of PAD proteins and their products (citrullinated proteins) was analyzed by Western blotting and by immunolocalization. The presence of anticitrullinated protein antibodies was investigated by an anti-cyclic citrullinated peptide (anti-CCP) enzyme-linked immunosorbent assay (ELISA) and an ELISA using in vitro citrullinated fibrinogen. RESULTS: In both mouse models, PAD type 2 (PAD2) mRNA was present in the synovium but was not translated into PAD2 protein. In contrast, PAD4 mRNA, although absent from healthy synovium, was readily transcribed and translated by polymorphonuclear neutrophils infiltrating the synovial tissue during inflammation. As a consequence, several synovial proteins were subjected to citrullination. One of these proteins was identified as fibrin, which has been reported to be citrullinated also in synovium of patients with RA. Although generation of citrullinated antigens during synovial inflammation in the mice was eminent, no anti-CCP antibodies could be detected. CONCLUSION: Citrullination of synovial antigens is an active process during joint inflammation in both mice and humans, but the induction of autoantibodies directed to these proteins is a more specific phenomenon, detectable only in human RA patients.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Citrulina/metabolismo , Membrana Sinovial/imunologia , Animais , Especificidade de Anticorpos , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Autoanticorpos/sangue , Biópsia , Citrulina/imunologia , Modelos Animais de Doenças , Epitopos , Regulação Enzimológica da Expressão Gênica/imunologia , Hidrolases/genética , Hidrolases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Proteína-Arginina Desiminase do Tipo 2 , Proteína-Arginina Desiminase do Tipo 3 , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Proteínas/imunologia , Proteínas/metabolismo , RNA Mensageiro/análise , Líquido Sinovial/imunologia , Membrana Sinovial/enzimologia , Membrana Sinovial/patologiaRESUMO
OBJECTIVE: To determine whether local overexpression of interleukin-12 (IL-12), a pleiotropic cytokine that promotes the development of naive T cells into Th1 cells, could aggravate murine streptococcal cell wall (SCW)-induced arthritis, a model of acute arthritis. METHODS: C57BL/6 mice were injected intraarticularly with saline or with 10(7) plaque-forming units of control vector (Ad5del70-3) or IL-12 vector (AdmIL-12.1) into the right knee joint 1 day before intraarticular injection of 25 microg of SCW fragments. The development of joint swelling, changes in chondrocyte proteoglycan (PG) synthesis, and joint destruction were examined thereafter. RESULTS: In normal joints, high levels of IL-12 (20 ng/ml on day 1) could be detected after application of the AdmIL-12.1 vector. After 14 days, expression of IL-12 was still found locally, but IL-12 alone did not induce protracted inflammation. Local expression of IL-12, in combination with SCW, markedly aggravated SCW-induced arthritis, as determined by enhanced joint swelling and prolonged inhibition of chondrocyte PG synthesis. Histologic examination on day 21 showed a chronic inflammatory process, with persistent cartilage PG depletion, cartilage erosion, and VDIPEN neoepitope expression (indicative of metalloproteinase activation). The mixture of IL-12 with SCW fragments did not lead to a chronic destructive process in mice deficient for recombination-activating gene 2, indicating the involvement of lymphocytes. In addition, systemic flare of smoldering SCW arthritis, produced by intravenous injection of SCW fragments, was only seen in the AdmIL-12/SCW group. CONCLUSION: These results indicate that local overexpression of IL-12 promotes conversion of an acute arthritis to a chronic destructive immune-mediated process, which is more susceptible to flares.
Assuntos
Artrite Infecciosa/imunologia , Artrite Infecciosa/patologia , Expressão Gênica/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Doença Aguda , Adenoviridae/genética , Animais , Cartilagem/patologia , Doença Crônica , Técnicas de Transferência de Genes , Articulação do Joelho/patologia , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Interleukin (IL)-18 is a member of the IL-1 family of proteins that exerts proinflammatory effects and is a pivotal cytokine for the development of Th1 responses. The goal of the present study was to investigate whether IL-18 induces joint inflammation and joint destruction directly or via induction of other cytokines such as IL-1 and tumor necrosis factor (TNF). To this end we performed both in vitro and in vivo kinetic studies. For in vivo IL-18 exposure studies C57BL/6, TNF-deficient, and IL-1-deficient mice were injected intra-articularly with 1.10(7) pfu mIL-18 adenovirus followed by histopathological examination. Local overexpression of IL-18 resulted in pronounced joint inflammation and cartilage proteoglycan loss in control mice. Of high interest, IL-18 gene transfer in IL-1-deficient mice did not show cartilage damage, although joint inflammation was similar to that in wild-type animals. Overexpression of IL-18 in TNF-deficient mice showed that TNF was partly involved in IL-18-induced joint swelling and influx of inflammatory cells, but cartilage proteoglycan loss occurred independent of TNF. In vitro cartilage degradation by IL-18 was found after a 72-hour culture period. Blocking of IL-1 with IL-1Ra or an ICE-inhibitor resulted in complete protection against IL-18-mediated cartilage degradation. The present study demonstrated that IL-18 induces joint inflammation independently of IL-1. In addition, we showed that IL-1beta generation, because of IL-18 exposure, was essential for marked cartilage degradation both in vitro and in vivo. These findings implicate that IL-18, in contrast to TNF, contributes through separate pathways to joint inflammation and cartilage destruction.