RESUMO
Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro-inflammatory mediators, including cytokines and lipid mediators of inflammation. Previous studies suggest that electromagnetic fields (EMFs) may represent a potential therapeutic approach to limit cartilage degradation and control inflammation associated to OA, and that they may act through the adenosine pathway. Therefore, we investigated whether EMFs might modulate inflammatory activities of human SFs from OA patients (OASFs) treated with interleukin-1ß (IL-1ß), and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. EMF exposure induced a selective increase in A(2A) and A(3) ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active also in EMF-exposed cells. Functional data obtained in the presence of selective A(2A) and A(3) adenosine agonists and antagonists showed that EMFs inhibit the release of prostaglandin E(2) (PGE(2)) and the proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), while stimulating the release of interleukin-10 (IL-10), an antinflammatory cytokine. These effects seem to be mediated by the EMF-induced upregulation of A(2A) and A(3) ARs. No effects of EMFs or ARs have been observed on matrix degrading enzyme production. In conclusion, this study shows that EMFs display anti-inflammatory effects in human OASFs, and that these EMF-induced effects are in part mediated by the adenosine pathway, specifically by the A(2A) and A(3) AR activation. Taken together, these results open new clinical perspectives to the control of inflammation associated to joint diseases.
Assuntos
Citocinas/metabolismo , Dinoprostona/metabolismo , Campos Eletromagnéticos , Fibroblastos/metabolismo , Mediadores da Inflamação/metabolismo , Osteoartrite do Quadril/metabolismo , Receptores Purinérgicos P1/metabolismo , Membrana Sinovial/metabolismo , Proteínas ADAM/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Osteoartrite do Quadril/genética , Osteoartrite do Quadril/imunologia , Osteoartrite do Quadril/patologia , Agonistas do Receptor Purinérgico P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , RNA Mensageiro/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Receptores Purinérgicos P1/efeitos dos fármacos , Receptores Purinérgicos P1/genética , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/imunologia , Membrana Sinovial/patologiaRESUMO
BACKGROUND AND PURPOSE: Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A(1), A(2A), A(2B) and A(3)) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes. EXPERIMENTAL APPROACH: mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-kappaB activation, tumour necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis. KEY RESULTS: mRNA and protein for A(1), A(2A), A(2B) and A(3) adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A(2A) and A(3) adenosine receptors inhibited p38 MAPK and NF-kappaB pathways, an effect abolished by selective adenosine antagonists. A(2A) and A(3) receptor agonists decreased TNF-alpha and IL-8 production. The phosphoinositide 3-kinase or G(s) pathways were involved in the functional responses of A(3) or A(2A) adenosine receptors. Synoviocyte A(1) and A(2B) adenosine receptors were not implicated in the inflammatory process whereas stimulation of A(2A) and A(3) adenosine receptors was closely associated with a down-regulation of the inflammatory status. CONCLUSIONS AND IMPLICATIONS: These results indicate that A(2A) and A(3) adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation.
Assuntos
Osteoartrite/metabolismo , Receptor A1 de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Receptores A2 de Adenosina/metabolismo , Membrana Sinovial/metabolismo , Agonistas do Receptor A1 de Adenosina , Antagonistas do Receptor A1 de Adenosina , Agonistas do Receptor A2 de Adenosina , Antagonistas do Receptor A2 de Adenosina , Agonistas do Receptor A3 de Adenosina , Antagonistas do Receptor A3 de Adenosina , Anti-Inflamatórios não Esteroides/uso terapêutico , Ligação Competitiva , Células Cultivadas , AMP Cíclico/biossíntese , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Humanos , Interleucina-8/metabolismo , Masculino , Pessoa de Meia-Idade , NF-kappa B/fisiologia , Osteoartrite/tratamento farmacológico , Osteoartrite/imunologia , Fosfatidilinositol 3-Quinases/fisiologia , Transdução de Sinais , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
OBJECTIVE: To investigate the role of adenosine analogs and electromagnetic field (EMF) stimulation on prostaglandin E(2) (PGE(2)) release and cyclooxygenase-2 (COX-2) expression in bovine synovial fibroblasts (SFs). METHODS: SFs isolated from synovia were cultured in monolayer. Saturation and binding experiments were performed by using typical adenosine agonists: N6-cyclohexyladenosine (CHA, A(1)), 2-[p-(2-carboxyethyl)-phenetyl-amino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, A(2A)), 5'-N-ethylcarboxamidoadenosine (NECA, non-selective), N6-(3-iodobenzyl)2-chloroadenosine-5'-N-methyluronamide (Cl-IB-MECA, A(3)). SFs were treated with TNF-alpha (10 ng/ml) and lipopolysaccharide (LPS) (1 microg/ml) to activate inflammatory response. Adenosine analogs were added to control and TNF-alpha- or LPS-treated cultures both in the absence and in the presence of adenosine deaminase (ADA) which is used to deplete endogenous adenosine. Parallel cultures were exposed to EMFs (75 Hz, 1.5 mT) during the period in culture (24h). PGE(2) release was measured by immunoassay. COX-2 expression was evaluated by RT-PCR. RESULTS: TNF-alpha and LPS stimulated PGE(2) release. All adenosine agonists, except for Cl-IB-MECA, significantly inhibited PGE(2) production. EMFs inhibited PGE(2) production in the absence of adenosine agonists and increased the effects of CHA, CGS 21680 and NECA. In ADA, the inhibition on PGE(2) release induced by CHA, CGS and NECA was stronger than in the absence of ADA and the EMF-inhibitory effect was lost. Changes in PGE(2) levels were associated to modification of COX-2 expression. CONCLUSIONS: This study supports anti-inflammatory activities of A(1) and A(2A) adenosine receptors and EMFs in bovine SFs. EMF activity appears mediated by an EMF-induced up-regulation of A(2A) receptors. Biophysical and/or pharmacological modulation of adenosine pathways may play an important role to control joint inflammation.