RESUMO
Prolonged inappropriate inflammatory responses contribute to the pathogenesis of rheumatoid arthritis (RA) and to aspects of osteoarthritis (OA). The orphan nuclear receptor, NR4A2, is a key regulator and potential biomarker for inflammation and represents a potentially valuable therapeutic target. Both salmon calcitonin (sCT) and hyaluronic acid (HA) attenuated activated mRNA expression of NR4A1, NR4A2, NR4A3, and matrix metalloproteinases (MMPs) 1, 3 and 13 in three human cell lines: SW1353 chondrocytes, U937 and THP-1 monocytes. Ad-mixtures of sCT and HA further down-regulated expression of NR4A2 compared to either agent alone at specific concentrations, hence the rationale for their formulation in nanocomplexes (NPs) using chitosan. The sCT released from NP stimulated cAMP production in human T47D breast cancer cells expressing sCT receptors. When NP were injected by the intra-articular (I.A.) route to the mouse knee during on-going inflammatory arthritis of the K/BxN serum transfer model, joint inflammation was reduced together with NR4A2 expression, and local bone architecture was preserved. These data highlight remarkable anti-inflammatory effects of sCT and HA at the level of reducing NR4A2 mRNA expression in vitro. Combining them in NP elicits anti-arthritic effects in vivo following I.A. delivery.
Assuntos
Anti-Inflamatórios/administração & dosagem , Artrite Experimental/tratamento farmacológico , Calcitonina/administração & dosagem , Ácido Hialurônico/administração & dosagem , Nanopartículas/administração & dosagem , Animais , Anti-Inflamatórios/química , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Calcitonina/química , Linhagem Celular , Linhagem Celular Tumoral , Quitosana/química , AMP Cíclico/metabolismo , Humanos , Ácido Hialurônico/química , Metaloproteinase 13 da Matriz/genética , Camundongos , Nanopartículas/química , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , RNA Mensageiro/metabolismoRESUMO
OBJECTIVES: To establish the role and effect of glucocorticoids and the endogenous annexin A1 (AnxA1) pathway in inflammatory arthritis. METHODS: Ankle joint mRNA and protein expression of AnxA1 and its receptors were analysed in naive and arthritic mice by real-time PCR and immunohistochemistry. Inflammatory arthritis was induced with the K/BxN arthritogenic serum in AnxA1(+/+) and AnxA1(-/-) mice; in some experiments, animals were treated with dexamethasone (Dex) or with human recombinant AnxA1 or a protease-resistant mutant (termed SuperAnxA1). Readouts were arthritic score, disease incidence, paw oedema and histopathology, together with pro-inflammatory gene expression. RESULTS: All elements of the AnxA1 pathway could be detected in naive joints, with augmentation during ongoing disease, due to the infiltration of immune cells. No difference in arthritis intensity of profile could be observed between AnxA1(+/+) and AnxA1(-/-) mice. Treatment of mice with Dex (10 µg intraperitoneally daily from day 2) afforded potent antiarthritic effects highly attenuated in the knockouts: macroscopic changes were mirrored by histopathological findings and pro-inflammatory gene (eg, Nos2) expression. Presence of proteinase 3 mRNA in the arthritic joints led the authors to test AnxA1 and the mutant SuperAnxA1 (1 µg intraperitoneally daily in both cases from day 2), with the latter one being able to accelerate the resolving phase of the disease. CONCLUSION: AnxA1 is an endogenous determinant for the therapeutic efficacy of Dex in inflammatory arthritis. Such an effect can be partially mimicked by application of SuperAnxA1 which may represent the starting point for novel antiarthritic therapeutic strategies.
Assuntos
Anexina A1/fisiologia , Antirreumáticos/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Dexametasona/uso terapêutico , Glucocorticoides/uso terapêutico , Animais , Anexina A1/química , Anexina A1/farmacologia , Artrite Experimental/genética , Artrite Experimental/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Edema/tratamento farmacológico , Edema/patologia , Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteínas Mutantes/química , Proteínas Mutantes/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
Synthetic and natural melanocortin (MC) peptides afford inhibitory properties in inflammation and tissue injury, but characterization of receptor involvement is still elusive. We used the agonist AP214 to test MC-dependent anti-inflammatory effects. In zymosan peritonitis, treatment of mice with AP214 (400 to 800 µg/kg) inhibited cell infiltration, an effect retained in MC receptor type 1, or MC(1), mutant mice but lost in MC(3) null mice. In vitro, cytokine release from zymosan-stimulated macrophages was affected by AP214, with approximately 80%, 30%, and 40% reduction in IL-1ß, tumor necrosis factor-α, and IL-6, respectively. Inhibition of IL-1ß release was retained in MC(1) mutant cells but was lost in MC(3) null cells. Furthermore, AP214 augmented uptake of zymosan particles and human apoptotic neutrophils by wild-type macrophages: this proresolving property was lost in MC(3) null macrophages. AP214 displayed its pro-efferocytotic effect also in vivo. Finally, in a model of inflammatory arthritis, AP214 evoked significant reductions in the clinical score. These results indicate that AP214 elicits anti-inflammatory responses, with a preferential effect on IL-1ß release. Furthermore, we describe for the first time a positive modulation of an MC agonist on the process of efferocytosis. In all cases, endogenous MC(3) is the receptor that mediates these novel properties of AP214. These findings might clarify the tissue-protective properties of AP214 in clinical settings and may open further development for novel MC agonists.
Assuntos
Artrite Experimental/tratamento farmacológico , Inflamação/tratamento farmacológico , Melanocortinas/agonistas , Peritonite/tratamento farmacológico , Receptor Tipo 1 de Melanocortina/fisiologia , Receptor Tipo 3 de Melanocortina/fisiologia , alfa-MSH/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Western Blotting , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Peritonite/metabolismo , Peritonite/patologia , Fagocitose , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , alfa-MSH/farmacologiaRESUMO
Gouty arthritis is a form of acute joint inflammation provoked by joint deposition of urate crystals. Although this acute pathology resolves after a few days, the marked degree of inflammation in the joint and--possibly more important to the patient--the excruciating pain it causes require proper therapeutic management. Often deemed a "poor sibling" of chronic joint pathologies such as rheumatoid arthritis and psoriatic arthritis, the increasing incidence of gout makes it a more palatable disease for novel drug discovery programs. This fact, associated with novel insights into the molecular mechanisms activated by the urate crystal deposition, is at the basis of new therapeutics under clinical development for gout, a valid example being the effective targeting of the proinflammatory cytokine interleukin-1. Here we briefly review the current status of antigout drug development and propose another target; our focus is on melanocortin receptor agonists as novel therapeutics for gout and inflammatory arthritides, a prototype of which, the adrenocorticotropic hormone, is already used in clinical settings.
Assuntos
Artrite Gotosa/tratamento farmacológico , Supressores da Gota/uso terapêutico , Melanocortinas/metabolismo , Receptores de Melanocortina/metabolismo , Alopurinol/uso terapêutico , Animais , Artrite Gotosa/metabolismo , Colchicina/uso terapêutico , Cristalização , Modelos Animais de Doenças , Humanos , Injeções Intra-Articulares , Melanocortinas/farmacologia , Camundongos , Receptores de Melanocortina/efeitos dos fármacos , Ácido Úrico/administração & dosagem , Ácido Úrico/metabolismoRESUMO
Macrophages have crucial functions in initiating the inflammatory reaction in a strict temporal and spatial manner to provide a "clear-up" response required for resolution. Hormonal peptides such as melanocortins modulate macrophage reactivity and attenuate inflammation ranging from skin inflammation to joint disease and reperfusion injury. The melanocortins (e.g., adrenocorticotrophin, ACTH and αMSH) elicit regulatory properties through activation of a family of GPCRs, the melanocortin (MC) receptors; MC1-MC5. Several studies have focused on MC1 and MC3 as anti-inflammatory receptors expressed on cells of the macrophage lineage. We review here elements of the melanocortin pathway with particular attention to macrophage function in anti-inflammatory and pro-resolving inflammatory settings. Evidence shows that ACTH, αMSH, and other MC agonists can activate MC1 and MC3 on macrophage through cAMP and/or NFκB-dependent mechanisms to abrogate pro-inflammatory cytokines, chemokines, and NO and enhance anti-inflammatory mediators such as IL-10 and HO-1. Melanocortins and their receptors regulate inflammation by inhibiting leukocyte recruitment to and interaction with inflamed tissue. An intensely exciting addition to this field of research has been the ability of an αMSH analog; AP214 to activate MC3 expressed on macrophage to enhance their clearance of both zymosan particles and apoptotic neutrophils thus putting melanocortins in line with other pro-resolving mediators. The use of mouse colonies mutated or nullified for MC1 or MC3, respectively as well as availability of selective MC receptor agonist/antagonists have been key to deciphering mechanisms by which elements of the melanocortin system play a role in these phenomena. We review here melanocortin pathway components with attention to the macrophage, reiterating receptor targets required for pro-resolving properties. The overall outcome will be identification of selective MC agonists as a strategy for innovative anti-inflammatory therapeutics.
RESUMO
The development of biological therapies has improved management of rheumatoid arthritis. However, costs and unresponsiveness to therapy in a sizeable proportion of patients limit their use, making it imperative to identify new targets for drug development programs. Here we investigated the melanocortin-receptor type 3 (MC(3)) pathway. Gene-deficient mice were subjected to a model of serum-transfer-induced arthritis and joints analyzed for gene expression (cytokines, MCs) and morphology. Pharmacological analyses were also conducted in this model. Osteoclastogenesis was studied from bone marrow cells. Mc(3)(-/-) mice displayed an exacerbated inflammatory arthritis, associated with prominent bone erosion and higher articular expression of Rankl. Osteoclastogenesis studied from Mc(3)(-/-) bone marrow cells revealed a higher degree of responsiveness to Rankl, linked to prolonged NF-κB activation compared to wild types. Up-regulation of a discrete set of inflammatory genes, including Il-1ß, Il-6, and Nos2, was measured in Mc(3)(-/-) mice, and a marked up-regulation of joint Mc(3) accompanied arthritis resolution in wild-type mice. Administration of an MC(3) agonist, D[Trp8]-γ-MSH, attenuated disease incidence and severity in wild-type but not Mc(3)(-/-) mice. Overall, these findings identify MC(3)-mediated signaling as a beneficial pathway in experimental arthritis; hence this receptor is a novel target for the development of therapeutics for arthritis.
Assuntos
Artrite Experimental/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Receptor Tipo 3 de Melanocortina/metabolismo , Animais , Artrite Experimental/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Osteogênese/genética , Osteogênese/fisiologia , Reação em Cadeia da Polimerase , Receptor Tipo 3 de Melanocortina/genéticaRESUMO
Beta-defensins are antimicrobial peptides with an essential role in the innate immune response. In addition beta-defensins can also chemoattract cells involved in adaptive immunity. Until now, based on evidence from dendritic cell stimulation, human beta defensin-3 (hBD3) was considered pro-inflammatory. We present evidence here that hBD3 lacks pro-inflammatory activity in human and mouse primary Mphi. In addition, in the presence of LPS, hBD3 and the murine orthologue Defb14 (but not hBD2), effectively inhibit TNF-alpha and IL-6 accumulation implying an anti-inflammatory function. hBD3 also inhibits CD40/IFN-gamma stimulation of Mphi and in vivo, hBD3 significantly reduces the LPS-induced TNF-alpha level in serum. Recent work has revealed that hBD3 binds melanocortin receptors but we provide evidence that these are not involved in hBD3 immunomodulatory activity. This implies a dual role for hBD3 in antimicrobial activity and resolution of inflammation.
Assuntos
Tolerância Imunológica/imunologia , Inflamação/imunologia , beta-Defensinas/imunologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Sequência de Aminoácidos , Animais , Ligante de CD40/farmacologia , Células Cultivadas/imunologia , Endotoxemia/imunologia , Humanos , Interferon gama/farmacologia , Interleucina-10/fisiologia , Lipopolissacarídeos/toxicidade , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Dados de Sequência Molecular , Receptores de Melanocortina/deficiência , Receptores de Melanocortina/genética , Receptores de Melanocortina/fisiologia , Fator de Necrose Tumoral alfa/análise , beta-Defensinas/farmacologiaRESUMO
The human formyl-peptide receptor (FPR)-2 is a G protein-coupled receptor that transduces signals from lipoxin A(4), annexin A1, and serum amyloid A (SAA) to regulate inflammation. In this study, we report the creation of a novel mouse colony in which the murine FprL1 FPR2 homologue, Fpr2, has been deleted and describe its use to explore the biology of this receptor. Deletion of murine fpr2 was verified by Southern blot analysis and PCR, and the functional absence of the G protein-coupled receptor was confirmed by radioligand binding assays. In vitro, Fpr2(-/-) macrophages had a diminished response to formyl-Met-Leu-Phe itself and did not respond to SAA-induced chemotaxis. ERK phosphorylation triggered by SAA was unchanged, but that induced by the annexin A1-derived peptide Ac2-26 or other Fpr2 ligands, such as W-peptide and compound 43, was attenuated markedly. In vivo, the antimigratory properties of compound 43, lipoxin A(4), annexin A1, and dexamethasone were reduced notably in Fpr2(-/-) mice compared with those in wild-type littermates. In contrast, SAA stimulated neutrophil recruitment, but the promigratory effect was lost following Fpr2 deletion. Inflammation was more marked in Fpr2(-/-) mice, with a pronounced increase in cell adherence and emigration in the mesenteric microcirculation after an ischemia-reperfusion insult and an augmented acute response to carrageenan-induced paw edema, compared with that in wild-type controls. Finally, Fpr2(-/-) mice exhibited higher sensitivity to arthrogenic serum and were completely unable to resolve this chronic pathology. We conclude that Fpr2 is an anti-inflammatory receptor that serves varied regulatory functions during the host defense response. These data support the development of Fpr2 agonists as novel anti-inflammatory therapeutics.
Assuntos
Inflamação/metabolismo , Leucócitos/metabolismo , Ligantes , Receptores de Formil Peptídeo/fisiologia , Sequência de Aminoácidos , Animais , Anexina A1/metabolismo , Anexina A1/farmacologia , Carragenina , Quimiotaxia/efeitos dos fármacos , Edema/induzido quimicamente , Edema/genética , Edema/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Citometria de Fluxo , Immunoblotting , Inflamação/induzido quimicamente , Inflamação/genética , Leucócitos/citologia , Lipoxinas/metabolismo , Lipoxinas/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Dados de Sequência Molecular , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Receptores de Formil Peptídeo/genética , Receptores de Formil Peptídeo/metabolismo , ZimosanRESUMO
Over 20 years of research based upon application of experimental models of inflammation and tissue injury have revealed exquisite controlling functions for melanocortin hormones and, subsequently, their synthetic derivatives. More recent discoveries have shed light on the receptor targets responsible for these effects, leading to what could be the next step-change for this line of research, the development of novel therapeutics for the control of human inflammatory pathologies. Here we review some of this work with particular emphasis on more recent studies that have substantiated the activities of melanocortin peptides to reveal important regulatory functions for their receptors in vascular inflammation and disease models. Moreover, we summarise the drug discovery activities (for what is published knowledge) attempting to capitalise on this wealth of research on melanocortins, though we should not forget the successful employment of ACTH to treat human gouty arthritis. Altogether, this chapter would corroborate and flare the enthusiasm for this line of research, as we are confident that the right times might have arrived to develop novel anti-arthritic and tissue-protective compounds that will be acting by mimicking the way our endogenous melanocortins would act to exert their homeostatic and check-point functions.
Assuntos
Melanocortinas/metabolismo , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia , Sequência de Aminoácidos , Animais , Descoberta de Drogas , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Inflamação/fisiopatologia , Microcirculação/efeitos dos fármacos , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/fisiopatologiaRESUMO
The existence of anti-inflammatory circuits centered on melanocortin receptors (MCRs) has been supported by the inhibitory properties displayed by melanocortin peptides in models of inflammation and tissue injury. Here we addressed the pathophysiological effect that one MCR, MCR type 3 (MC3R), might have on vascular inflammation. After occlusion (35 min) and reopening of the superior mesenteric artery, MC3R-null mice displayed a higher degree of plasma extravasation (45 min postreperfusion) and cell adhesion and emigration (90 min postreperfusion). These cellular alterations were complemented by higher expression of mesenteric tissue CCL2 and CXCL1 (mRNA and protein) and myeloperoxydase, as compared with wild-type animals. MC1R and MC3R mRNA and protein were both expressed in the inflamed mesenteric tissue; however, no changes in vascular responses were observed in a mouse colony bearing an inactive MC1R. Pharmacological treatment of animals with a selective MC3R agonist ([D-Trp(8)]-gamma-melanocyte-stimulating hormone; 10 microg i.v.) produced marked attenuation of cell adhesion, emigration, and chemokine generation; such effects were absent in MC3R-null mice. These new data reveal the existence of a tonic inhibitory signal provided by MC3R in the mesenteric microcirculation of the mouse, acting to down-regulate cell trafficking and local mediator generation.