RESUMO
CONTEXT: High concentrations of inspired oxygen contribute to the pathogenesis of neonatal bronchopulmonary dysplasia and adult acute respiratory distress syndrome. Animal models of hyperoxia-associated lung injury (HALI) are characterized by enhanced generation of reactive oxygen species (ROS) and an adaptive antioxidant response. ROS contribute to pathogenesis, partly through enhancing pro-inflammatory activity in macrophages. Uncoupling protein-2 (UCP2) is an inner mitochondrial membrane protein whose expression lowers mitochondrial superoxide (O2â±â») production. UCP2, therefore, has potential to contribute to antioxidant response. It is inducible in macrophages. OBJECTIVES AND METHODS: We hypothesized that induction of UCP2 occurred in response to pulmonary hyperoxia in vivo and that expression localized to pulmonary macrophages. We then investigated mechanisms of UCP2 regulation in hyperoxia-exposed macrophages in vitro and correlated changing UCP2 expression with mitochondrial membrane potential (Δψm) and O2â±â» production. RESULTS: UCP2 is induced in lungs of mice within 1 h of hyperoxia exposure. Induction occurs in pulmonary alveolar macrophages in vivo, and can be replicated in vitro in isolated macrophages. UCP2 mRNA does not change. UCP2 increases quickly after the first hyperoxia-induced burst of mitochondrial O2â±â» generation. Suppression of Δψm and mitochondrial O2â±â» production follow and persist while UCP2 is elevated. DISCUSSION AND CONCLUSIONS: Induction of UCP2 is an early response to hyperoxia in pulmonary macrophages. The mechanism is post-transcriptional. UCP2 induction follows a transient rise in mitochondrial ROS generation. The subsequent falls in Δψm and mitochondrial O2â±â» support the notion that regulable UCP2 expression in macrophages acts to contain mitochondrial ROS generation. That, in turn, may limit inappropriate pro-inflammatory activation in HALI.
Assuntos
Hiperóxia/metabolismo , Canais Iônicos/metabolismo , Lesão Pulmonar/metabolismo , Macrófagos/fisiologia , Proteínas Mitocondriais/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células da Medula Óssea/citologia , Líquido da Lavagem Broncoalveolar/citologia , Linhagem Celular , Células Cultivadas , Hiperóxia/complicações , Canais Iônicos/genética , Pulmão/metabolismo , Lesão Pulmonar/etiologia , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Mitocondriais/genética , RNA Mensageiro/metabolismo , Superóxidos/metabolismo , Proteína Desacopladora 2RESUMO
Interleukin-17 (IL-17) and tumour necrosis factor-α (TNF) are critical in the pathogenesis of arthritis but their relationship during inflammatory pain has received limited attention. We aimed to establish whether IL-17 can induce hyperalgesia in acute conditions, and investigated the role of TNF in mediating the pain response. Hyperalgesia was elicited in C57BL/6 mice by injection of recombinant IL-17, TNF or vehicle into the plantar tissue. Elevated pain was measured by the Hargreaves test for thermal hyperalgesia and Linton incapacitance tester for weight-bearing change. Cellular infiltration during hyperalgesia was determined by histological analysis and myeloperoxidase assay. IL-17 was found to induce hyperalgesia, but this was dependent on neutrophil migration and TNF binding to TNF receptor 1 (TNFR1). Because TNF-induced hyperalgesia was also dependent on neutrophil migration, the relationship between the resident fibroblasts, the cytokines and the migrating neutrophils was further investigated. By means of an air pouch model of cell migration, it was established that IL-17-induced neutrophil infiltration was dependent of TNF/TNFR1 as this interaction was required for the induction of the chemokine keratinocyte chemoattractant. These findings suggest that IL-17 causes acute hyperalgesia indirectly by inducing TNF from resident cells. The subsequent production of keratinocyte chemoattractant then triggers neutrophil chemotaxis to the plantar tissue, releasing algesic mediators locally to sensitise the nerve.
Assuntos
Hiperalgesia/induzido quimicamente , Interleucina-17/efeitos adversos , Infiltração de Neutrófilos/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ensaio de Imunoadsorção Enzimática , Hiperalgesia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores do Fator de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Fatores de Tempo , Fator de Necrose Tumoral alfa/efeitos adversosRESUMO
OBJECTIVE: In vivo optical imaging can delineate at the macroscopic level processes that are occurring at the cellular and molecular levels. E-selectin, a leukocyte adhesion molecule expressed on endothelium, is induced by tumor necrosis factor α (TNFα) and other cytokines involved in the pathogenesis of rheumatoid arthritis (RA). Collagen-induced arthritis (CIA) in mice is widely used to study the disease mechanisms and identify new treatments for RA. The purpose of this study was to demonstrate E-selectin-targeted fluorescence imaging in vivo in a mouse model of paw edema generated by local injection of TNFα as well as in mice with CIA. METHODS: Animals with either CIA or TNFα-induced paw edema were injected with anti-E-selectin or control antibodies labeled with a DyLight 750-nm near-infrared (NIR) probe. In vivo imaging studies were undertaken using an NIR optical imaging system, and images were coregistered with plain radiographic images. RESULTS: The mean fluorescence intensity measured over the time-course of TNFα-induced edema demonstrated a 1.97-fold increase (P<0.001) in signal in inflamed paws at 8 hours following injection of anti-E-selectin antibody, as compared to that in the isotype control. In the CIA model, a 2.34-fold increase in E-selectin-targeted signal was demonstrated (P<0.01). Furthermore, significant E-selectin-targeted signal was observed in the paws of animals immunized with collagen that did not display overt signs of arthritis. CONCLUSION: E-selectin-targeted fluorescence in vivo imaging is a quantifiable method of detecting endothelial activation in arthritis and can potentially be applied to the quantification of disease and the investigation of the effects of new therapies. Importantly, this approach may also be useful for the detection of subclinical disease in RA.
Assuntos
Artrite Experimental/metabolismo , Selectina E/metabolismo , Endotélio Vascular/metabolismo , Análise de Variância , Animais , Artrite Experimental/imunologia , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Selectina E/imunologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Imuno-Histoquímica , Masculino , Camundongos , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase activity and hence PG production. However, the ability of NSAIDs to ameliorate pain and tenderness does not prevent disease progression in rheumatoid arthritis, a disease whose pathogenesis is linked to the presence of proinflammatory cytokines, such as TNF-alpha. To understand this observation, we have examined the effect of NSAIDs on the production of clinically validated proinflammatory cytokines. We show that a variety of NSAIDs superinduce production of TNF from human peripheral blood monocytes and rheumatoid synovial membrane cultures. A randomized, double-blinded, crossover, placebo-controlled trial in healthy human volunteers also revealed that the NSAID drug celecoxib increased LPS-induced TNF production in whole blood. NSAID-mediated increases in TNF are reversed by either the addition of exogenous PGE(2) or by a PGE(2) EP2 receptor agonist, revealing that PGE(2) signaling via its EP2 receptor provides a valuable mechanism for controlling excess TNF production. Thus, by reducing the level of PGE(2), NSAIDs can increase TNF production and may exacerbate the proinflammatory environment both within the rheumatoid arthritis joint and the systemic environment.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antirreumáticos/farmacologia , Artrite Reumatoide/imunologia , Membrana Sinovial/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/sangue , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Adulto , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Celecoxib , Células Cultivadas , Estudos Cross-Over , Citocinas/biossíntese , Citocinas/sangue , Método Duplo-Cego , Humanos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
INTRODUCTION: Type 4 phosphodiesterases (PDE4) play an important role in immune cells through the hydrolysis of the second messenger, cAMP. Inhibition of PDE4 has previously been shown to suppress immune and inflammatory responses, demonstrating PDE4 to be a valid therapeutic target for immune-mediated pathologies. We assessed the anti-inflammatory effects of a novel PDE4 inhibitor, apremilast, in human synovial cells from rheumatoid arthritis (RA) patients, as well as two murine models of arthritis. METHODS: Cells liberated from tissue excised from arthritic joints of RA patients were cultured in the presence of increasing concentrations of apremilast for 48 hours and spontaneous tumour necrosis factor-alpha (TNFalpha) production was analysed in culture supernatants by ELISA. In addition, arthritis was induced in BALB/c and DBA/1 mice by passive transfer of anti-type II collagen mAb and immunisation with type II collagen, respectively. Mice with established arthritis received 5 or 25 mg/kg apremilast and disease severity was monitored relative to mice receiving vehicle alone. At the end of the study, paws were removed and processed for histopathological assessment. Behavioural effects of apremilast, relative to rolipram, were assessed in naïve DBA/1 mice using an automated activity monitor (LABORAS). RESULTS: Apremilast dose dependently inhibited spontaneous release of TNFalpha from human rheumatoid synovial membrane cultures. Furthermore, apremilast significantly reduced clinical score in both murine models of arthritis over a ten day treatment period and maintained a healthy joint architecture in a dose-dependent manner. Importantly, unlike rolipram, apremilast demonstrated no adverse behavioural effects in naïve mice. CONCLUSIONS: Apremilast is an orally available PDE4 inhibitor that reduces TNFalpha production from human synovial cells and significantly suppresses experimental arthritis. Apremilast appears to be a potential new agent for the treatment of rheumatoid arthritis.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/metabolismo , Inibidores da Fosfodiesterase 4 , Membrana Sinovial/metabolismo , Talidomida/análogos & derivados , Fator de Necrose Tumoral alfa/metabolismo , Animais , Artrite Reumatoide/patologia , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Inibidores de Fosfodiesterase/efeitos adversos , Inibidores de Fosfodiesterase/uso terapêutico , Rolipram/efeitos adversos , Rolipram/uso terapêutico , Índice de Gravidade de Doença , Membrana Sinovial/efeitos dos fármacos , Talidomida/farmacologia , Talidomida/uso terapêuticoRESUMO
The origin of pain in osteoarthritis is poorly understood, but it is generally thought to arise from inflammation within the innervated structures of the joint, such as the synovium, capsule and bone. We investigated the role of nerve growth factor (NGF) in pain development in murine OA, and the analgesic efficacy of the soluble NGF receptor, TrkAD5. OA was induced in mice by destabilisation of the medial meniscus and pain was assessed by measuring hind-limb weight distribution. RNA was extracted from joints, and NGF and TNF expressions were quantified. The effect of tumour necrosis factor (TNF) and neutrophil blockade on NGF expression and pain were also assessed. NGF was induced in the joints during both post-operative (day 3) and OA (16weeks) pain, but not in the non-painful stage of disease (8weeks post-surgery). TrkAd5 was highly effective at suppressing pain in both phases. Induction of NGF in the post-operative phase of pain was TNF-dependent as anti-TNF reduced NGF expression in the joint and abrogated pain. However, TNF was not regulated in the late OA joints, and pain was not affected by anti-TNF therapy. Fucoidan, by suppressing cellular infiltration into the joint, was able to suppress post-operative, but not late OA pain. These results indicate that NGF is an important mediator of OA pain and that TrkAd5 represents a potent novel analgesic in this condition. They also suggest that, unlike post-operative pain, induction of pain in OA may not necessarily be driven by classical inflammatory processes.
Assuntos
Analgésicos/farmacologia , Artralgia/tratamento farmacológico , Fator de Crescimento Neural/agonistas , Osteoartrite do Joelho/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Receptor trkA/uso terapêutico , Analgésicos/síntese química , Animais , Artralgia/metabolismo , Artralgia/fisiopatologia , Modelos Animais de Doenças , Etanercepte , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/inervação , Articulação do Joelho/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/fisiopatologia , Medição da Dor/métodos , Fragmentos de Peptídeos/síntese química , Peptídeos/síntese química , Polissacarídeos/farmacologia , Receptores do Fator de Necrose Tumoral/uso terapêutico , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Non-malignant musculoskeletal pain is the most common clinical symptom that causes patients to seek medical attention and is a major cause of disability in the world. Musculoskeletal pain can arise from a variety of common conditions including osteoarthritis, rheumatoid arthritis, osteoporosis, surgery, low back pain and bone fracture. A major problem in designing new therapies to treat musculoskeletal pain is that the underlying mechanisms driving musculoskeletal pain are not well understood. This lack of knowledge is largely due to the scarcity of animal models that closely mirror the human condition which would allow the development of a mechanistic understanding and novel therapies to treat this pain. To begin to develop a mechanism-based understanding of the factors involved in generating musculoskeletal pain, in this review we present recent advances in preclinical models of osteoarthritis, post-surgical pain and bone fracture pain. The models discussed appear to offer an attractive platform for understanding the factors that drive this pain and the preclinical screening of novel therapies to treat musculoskeletal pain. Developing both an understanding of the mechanisms that drive persistent musculoskeletal pain and novel mechanism-based therapies to treat these unique pain states would address a major unmet clinical need and have significant clinical, economic and societal benefits.
Assuntos
Artralgia/fisiopatologia , Doenças Musculoesqueléticas/fisiopatologia , Animais , Artralgia/tratamento farmacológico , Artralgia/etiologia , Osso e Ossos/inervação , Osso e Ossos/fisiopatologia , Modelos Animais de Doenças , Fraturas Ósseas/fisiopatologia , Humanos , Hiperalgesia/etiologia , Hiperalgesia/fisiopatologia , Inflamação/fisiopatologia , Camundongos , Osteoartrite/fisiopatologia , Dor Pós-Operatória/fisiopatologia , Ratos , Toracotomia/efeitos adversosRESUMO
IL-17 is implicated in the pathogenesis of rheumatoid arthritis (RA) and has previously been shown to be induced by tumor necrosis factor (TNF) in vitro. The aim of this study was to assess the impact of TNF inhibition on IL-17 production in collagen-induced arthritis, a model of RA. TNF blockade using TNFR-Fc fusion protein or anti-TNF monoclonal antibody reduced arthritis severity but, unexpectedly, expanded populations of Th1 and Th17 cells, which were shown by adoptive transfer to be pathogenic. Th1 and Th17 cell populations were also expanded in collagen-immunized TNFR p55(-/-) but not p75(-/-) mice. The expression of IL-12/IL-23 p40 was up-regulated in lymph nodes (LN) from p55(-/-) mice, and the expansion of Th1/Th17 cells was abrogated by blockade of p40. Treatment of macrophages with rTNF also inhibited p40 production in vitro. These findings indicate that at least one of the ways in which TNF regulates Th1/Th17 responses in arthritis is by down-regulating the expression of p40. Finally, although TNF blockade increased numbers of Th1 and Th17 cells in LN, it inhibited their accumulation in the joint, thereby providing an explanation for the paradox that anti-TNF therapy ameliorates arthritis despite increasing numbers of pathogenic T cells.
Assuntos
Artrite Experimental/imunologia , Imunoglobulina G/farmacologia , Interferon gama/metabolismo , Interleucina-17/metabolismo , Células Th1/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Transferência Adotiva , Animais , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Etanercepte , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Receptores do Fator de Necrose Tumoral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: There is a disparity in the animal models used to study pain in rheumatoid arthritis (RA), which tends to be acute in nature, and models used to assess the pathogenesis of RA. The latter models, like human RA, are lymphocyte-driven and polyarthritic. We assessed pain behavior and mechanisms in collagen-induced arthritis (CIA), the model of preclinical arthritis used most commonly in the field of immunology. We then validated the model using anti-tumor necrosis factor (anti-TNF) therapy, which has analgesic effects in models of inflammation as well as in human RA. METHODS: CIA was induced in DBA/1 mice by immunization with type II collagen at the base of the tail. Swelling and mechanical and thermal hyperalgesia were assessed before and for 28 days after the onset of arthritis. Spontaneous behavior was assessed using an automated activity monitor. Glial activity was assessed by glial fibrillary acidic protein expression, and nerve damage was evaluated by activating transcription factor 3 expression. The actions of anti-TNF therapy on nociception were then evaluated. RESULTS: Arthritis resulted in a decrease in the threshold for thermal and mechanical stimuli, beginning on the day of onset. Decreased spontaneous activity was also observed. A significant increase in the number of hyperplasic spinal cord astrocytes was observed beginning 10 days after the onset of arthritis. Anti-TNF therapy was profoundly analgesic, with an efficacy similar to that of cyclooxygenase 2 inhibition, and reduced astrocyte activity in CIA. CONCLUSION: This study shows that the CIA model is suitable for testing not only antiinflammatory but also analgesic drugs for potential use in RA, and highlights the importance of using appropriate disease models to assess relevant pain pathways.
Assuntos
Artrite/fisiopatologia , Modelos Animais de Doenças , Hiperalgesia/fisiopatologia , Dor/fisiopatologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Astrócitos/patologia , Comportamento Animal/fisiologia , Celecoxib , Colágeno , Inibidores de Ciclo-Oxigenase/uso terapêutico , Proteína Glial Fibrilar Ácida/metabolismo , Hiperalgesia/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos DBA , Microglia/metabolismo , Dor/tratamento farmacológico , Pirazóis/uso terapêutico , Sulfonamidas/uso terapêuticoRESUMO
Type II collagen-induced arthritis (CIA) is an animal model of rheumatoid arthritis that has been used extensively to address questions of disease pathogenesis and to validate novel therapeutic targets. Susceptibility to CIA is strongly associated with major histocompatibility complex class II genes, and the development of arthritis is accompanied by a robust T- and B-cell response to type II collagen. The main pathological features of CIA include proliferative synovitis with infiltration of inflammatory cells, pannus formation, cartilage degradation, erosion of bone and fibrosis. Pro-inflammatory cytokines, such as tumour necrosis factor alpha and interleukin-1beta, are expressed in the arthritic joints in both murine CIA and human rheumatoid arthritis, and blockade of these molecules results in amelioration of disease. Hence, there is a great deal of interest in the development of small-molecular-weight inhibitors of pro-inflammatory cytokines. There is also interest in the development and testing of drugs with the capacity to modulate the immune pathways involved in driving the inflammatory response in arthritis. For these reasons, there is a need to monitor the effect of novel treatments on cytokine expression in vivo. In this review, we outline the various techniques used to detect cytokines in experimental arthritis and describe how these techniques have been used to quantify changes in cytokine expression following therapeutic intervention.
Assuntos
Artrite Experimental/imunologia , Artrite Experimental/terapia , Artrite Reumatoide/imunologia , Citocinas/análise , Imunoterapia/métodos , Membrana Sinovial/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Colágeno Tipo II , Humanos , Camundongos , Modelos Animais , Receptores do Fator de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Therapies directed against tumour necrosis factor (TNF) are effective for the treatment of rheumatoid arthritis and reduce pain scores in this condition. In this study, we sought to explore mechanisms by which TNF contributes to inflammatory pain in an experimental model of arthritis. The effects of an anti-TNF agent, etanercept, on behavioural pain responses arising from rat monoarthritis induced by complete Freund's adjuvant were assessed and compared with expression of TNF receptors (TNFRs) by dorsal root ganglion (DRG) cells at corresponding time points. Etanercept had no effect on evoked pain responses in normal animals but exerted a differential effect on the thermal and mechanical hyperalgesia associated with rat arthritis induced by complete Freund's adjuvant (CFA). Joint inflammation was associated with increased TNFR1 and TNFR2 expression on DRG cells, which was maintained throughout the time course of the model. TNFR1 expression was increased in neuronal cells of the DRG bilaterally after arthritis induction. In contrast, TNFR2 expression occurred exclusively on non-neuronal cells of the macrophage-monocyte lineage, with cell numbers increasing in a TNF-dependent fashion during CFA-induced arthritis. A strong correlation was observed between numbers of macrophages and the development of mechanical hyperalgesia in CFA-induced arthritis. These results highlight the potential for TNF to play a vital role in inflammatory hyperalgesia, both by a direct action on neurons via TNFR1 and by facilitating the accumulation of macrophages in the DRG via a TNFR2-mediated pathway.