RESUMO
BACKGROUND: Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory cytokine, which is closely associated with the pathogenesis of various types of cutaneous vasculitis (CV). AIM: To investigate the therapeutic effects of an anti-TWEAK monoclonal antibody (mAb) in a mouse model of cutaneous reverse passive Arthus (RPA) reaction. METHODS: Cutaneous RPA reaction was induced in BALB/c mice by intradermal injection of anti-ovalbumin IgG into the left ear followed immediately by intravenous injection of chicken ovalbumin. After treatment, haemorrhagic lesions in the mouse skin were scored semiquantitatively. The amount of extravasated fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) in the ears was detected spectrophotometrically. Expression of myeloperoxidase (MPO) was detected by immunohistochemical staining, while mRNA expression of TNF-α and interleukin (IL)-6 in lesional skin was detected by real-time quantitative (q)PCR. RESULTS: Our results indicated that anti-TWEAK mAb significantly attenuated the clinical and histopathological changes in immune complex (IC)-induced mice, and also reduced the semiquantitative haemorrhage score, FITC-labelled BSA extravasation and MPO activity. Real-time qPCR showed that anti-TWEAK mAb significantly inhibited mRNA expression of TNF-α and IL-6 in lesional skin from IC-induced mice. CONCLUSION: These data suggest that anti-TWEAK mAb can block vascular damage and leucocyte infiltration in IC-induced mice. TWEAK might be a candidate immunotherapeutic medicine for suppression of IC-induced CV.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Reação de Arthus/tratamento farmacológico , Citocina TWEAK/antagonistas & inibidores , Dermatopatias/tratamento farmacológico , Animais , Reação de Arthus/metabolismo , Reação de Arthus/patologia , Citocina TWEAK/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peroxidase/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Dermatopatias/metabolismo , Dermatopatias/patologiaRESUMO
The complement system is important for the host defence against infection as well as for the development of inflammatory diseases. Here we show that C1q/TNF-related protein 6 (CTRP6; gene symbol C1qtnf6) expression is elevated in mouse rheumatoid arthritis (RA) models. C1qtnf6(-/-) mice are highly susceptible to induced arthritis due to enhanced complement activation, whereas C1qtnf6-transgenic mice are refractory. The Arthus reaction and the development of experimental autoimmune encephalomyelitis are also enhanced in C1qtnf6(-/-) mice and C1qtnf6(-/-) embryos are semi-lethal. We find that CTRP6 specifically suppresses the alternative pathway of the complement system by competing with factor B for C3(H2O) binding. Furthermore, treatment of arthritis-induced mice with intra-articular injection of recombinant human CTRP6 cures the arthritis. CTRP6 is expressed in human synoviocytes, and CTRP6 levels are increased in RA patients. These results indicate that CTRP6 is an endogenous complement regulator and could be used for the treatment of complement-mediated diseases.
Assuntos
Adipocinas/imunologia , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Via Alternativa do Complemento/imunologia , Adipocinas/genética , Adulto , Animais , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Reação de Arthus/genética , Reação de Arthus/imunologia , Reação de Arthus/metabolismo , Western Blotting , Colágeno/imunologia , Colágeno/metabolismo , Convertases de Complemento C3-C5/imunologia , Complemento C3a/imunologia , Complemento C5a/imunologia , Via Alternativa do Complemento/genética , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunoprecipitação , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/citologia , Membrana Sinovial/metabolismoRESUMO
Paeoniflorin (PF) extracted from the root of Paeonia lactiflora pall, displays anti-inflammation properties in several animal models. Adhesion molecules are important for the recruitment of leucocyte to the vessel wall and involved in the pathogenesis of various autoimmune and inflammatory diseases. Herein, we investigate the effects of PF on adhesion molecule expression in a mouse model of cutaneous Arthus reaction and cultured human dermal microvascular endothelial cells (HDMECs). We showed that PF significantly ameliorated the immune complex (IC) induced vascular damage, leucocyte infiltrates and adhesion molecules expression. Furthermore, PF markedly blocked tumor necrosis factor-α (TNF-α)-induced E-selectin and intercellular adhesion molecule-1 (ICAM-1) expression in HDMECs at both mRNA and protein levels. PF also suppressed TNF-α-induced adhesion of polymorphonuclear leucocytes (PMNs) to HDMECs. Finally, western blot data revealed that PF can inhibit the phosphorylation of p38, JNK in TNF-α-treated HDMECs. These data suggest that PF, as an anti-inflammatory agent, can downregulate adhesion molecules expression. PF may be a candidate medicine for the treatment of IC-induced inflammatory response.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Reação de Arthus/metabolismo , Benzoatos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Selectina E/metabolismo , Glucosídeos/farmacologia , Molécula 1 de Adesão Intercelular/metabolismo , Pele/metabolismo , Animais , Complexo Antígeno-Anticorpo/metabolismo , Reação de Arthus/tratamento farmacológico , Autoimunidade , Adesão Celular , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação , Leucócitos/citologia , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microcirculação , Monoterpenos , Reação em Cadeia da Polimerase em Tempo Real , Fator de Necrose Tumoral alfa/metabolismo , Doenças Vasculares/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The deposition of immune complexes (IC) induces an acute inflammatory response with tissue injury, for which the involvement of nitric oxide (NO) and carbon monoxide (CO) has been suggested. NO is induced by NO synthase (NOS) and CO is generated by haeme oxygenase (HO). Among HO isoenzymes, HO-1 is an induced type. To assess the role of NO and CO in the pathogenic process, the cutaneous reverse passive Arthus reaction was examined using NOS inhibitor, HO-1 stimulator and HO-1 inhibitor. To evaluate the reaction we considered oedema, tumour necrosis factor-alpha, interleukin-6, and neutrophil number. The values of these four parameters were significantly reduced in mice treated with HO-1 stimulator as compared with the positive control mice. Quite the reverse was observed in mice treated with HO-1 inhibitor. These results suggest that the HO-1/CO signalling pathway is a therapeutic target for human IC-mediated disease.
Assuntos
Reação de Arthus/metabolismo , Monóxido de Carbono/metabolismo , Crioprotetores/metabolismo , Pele/imunologia , Animais , Reação de Arthus/imunologia , Biomarcadores/análise , Feminino , Gases , Heme Oxigenase-1/análise , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/metabolismo , Hemina/farmacologia , Imuno-Histoquímica , Interleucina-6/análise , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , NG-Nitroarginina Metil Éster/farmacologia , Neutrófilos/imunologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Nitritos/análise , Protoporfirinas/farmacologia , Espectrofotometria , Fator de Necrose Tumoral alfa/análiseRESUMO
Leukocyte adhesion via beta(2) integrins (CD11/CD18) activates the tyrosine kinase Syk. We found that Syk was enriched at the lamellipodium during N-formyl-Met-Leu-Phe-induced migration of neutrophil-like differentiated HL-60 cells. Here, Syk colocalized with Vav, a guanine nucleotide exchange factor for Rac and Cdc42. The enrichment of Syk at the lamellipodium and its colocalization with Vav were absent upon expression of a Syk kinase-dead mutant (Syk K402R) or a Syk mutant lacking the binding site of Vav (Syk Y348F). Live cell imaging revealed that both mutations resulted in excessive lamellipodium formation and severely compromised migration compared with control cells. Similar results were obtained upon down-regulation of Syk by RNA interference (RNAi) technique as well as in Syk(-/-) neutrophils from wild-type mice reconstituted with Syk(-/-) bone marrow. A pivotal role of Syk in vivo was demonstrated in the Arthus reaction, where neutrophil extravasation, edema formation, and hemorrhage were profoundly diminished in Syk(-/-) bone marrow chimeras compared with those in control animals. In the inflamed cremaster muscle, Syk(-/-) neutrophils revealed a defect in adhesion and migration. These findings indicate that Syk is critical for beta(2) integrin-mediated neutrophil migration in vitro and plays a fundamental role in neutrophil recruitment during the inflammatory response in vivo.
Assuntos
Antígenos CD11/metabolismo , Antígenos CD18/metabolismo , Movimento Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neutrófilos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Animais , Reação de Arthus/genética , Reação de Arthus/metabolismo , Sítios de Ligação , Antígenos CD11/genética , Antígenos CD18/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células HL-60 , Humanos , Técnicas In Vitro , Inflamação/genética , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Camundongos , Camundongos Knockout , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Proteínas de Neoplasias , Infiltração de Neutrófilos/genética , Neutrófilos/citologia , Proteínas Tirosina Quinases/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Pseudópodes/genética , Pseudópodes/metabolismo , Quinase Syk , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Complement and FcgammaR effector pathways are central triggers of immune inflammation; however, the exact mechanisms for their cooperation with effector cells and their nature remain elusive. In this study we show that in the lung Arthus reaction, the initial contact between immune complexes and alveolar macrophages (AM) results in plasma complement-independent C5a production that causes decreased levels of inhibitory FcgammaRIIB, increased levels of activating FcgammaRIII, and highly induced FcgammaR-mediated TNF-alpha and CXCR2 ligand production. Blockade of C5aR completely reversed such changes. Strikingly, studies of pertussis toxin inhibition show the essential role of G(i)-type G protein signaling in C5aR-mediated control of the regulatory FcgammaR system in vitro, and analysis of the various C5aR-, FcgammaR-, and G(i)-deficient mice verifies the importance of Galpha(i2)-associated C5aR and the FcgammaRIII-FcgammaRIIB receptor pair in lung inflammation in vivo. Moreover, adoptive transfer experiments of C5aR- and FcgammaRIII-positive cells into C5aR- and FcgammaRIII-deficient mice establish AM as responsible effector cells. AM lacking either C5aR or FcgammaRIII do not possess any such inducibility of immune complex disease, whereas reconstitution with FcgammaRIIB-negative AM results in an enhanced pathology. These data suggest that AM function as a cellular link of C5a production and C5aR activation that uses a Galpha(i2)-dependent signal for modulating the two opposing FcgammaR, FcgammaRIIB and FcgammaRIII, in the initiation of the inflammatory cascade in the lung Arthus reaction.
Assuntos
Reação de Arthus/imunologia , Reação de Arthus/patologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Mediadores da Inflamação/metabolismo , Pulmão/patologia , Macrófagos Alveolares/imunologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Complemento/metabolismo , Receptores Fc/metabolismo , Animais , Reação de Arthus/metabolismo , Linhagem Celular , Complemento C5a/fisiologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Temperatura Alta , Imunoglobulina G/farmacologia , Mediadores da Inflamação/fisiologia , Pulmão/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/fisiologia , Receptor Cross-Talk/imunologia , Receptor da Anafilatoxina C5a , Receptores de Complemento/biossíntese , Receptores de Complemento/deficiência , Receptores de Complemento/genética , Receptores de IgG/deficiência , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores de IgG/fisiologiaRESUMO
Immune complex (IC)-induced tissue injury is mediated by inflammatory cell infiltration that is highly regulated by various adhesion molecules. To assess the contribution of P-selectin glycoprotein ligand-1 (PSGL-1) and selectins in the pathogenetic process, the cutaneous reverse-passive Arthus reaction was examined in mice treated with monoclonal antibodies (mAb) to PSGL-1 or P- and/or E-selectin. Edema and hemorrhage were significantly reduced in mice treated with anti-P-selectin mAb compared with control mice while they were not inhibited in mice treated with anti-E-selectin mAb. It is remarkable that blocking PSGL-1 by mAb resulted in significant, further reduction in edema and hemorrhage compared with blocking anti-P- or anti-E-selectin. However, blockade of E- and P-selectins exhibited more significant reduction relative to PSGL-1 blockade. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells. Reduced infiltration of neutrophils and mast cells was observed in the peritoneal Arthus reaction and was associated with the decreased production of tumor necrosis factor alpha and interleukin-6. The results of this study indicate that PSGL-1 contributes to the Arthus reaction mainly as a ligand of P-selectin and partly as a ligand of E- and/or L-selectin by regulating neutrophil and mast-cell recruitment and that PSGL-1 would be a therapeutic target for human IC-mediated diseases.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Doenças do Complexo Imune/imunologia , Glicoproteínas de Membrana/metabolismo , Vasculite Leucocitoclástica Cutânea/metabolismo , Animais , Reação de Arthus/imunologia , Reação de Arthus/metabolismo , Movimento Celular/imunologia , Citocinas/metabolismo , Edema/imunologia , Hemorragia/imunologia , Doenças do Complexo Imune/metabolismo , Leucócitos/imunologia , Camundongos , Peritônio/imunologia , Peritônio/metabolismo , Fatores de Tempo , Vasculite Leucocitoclástica Cutânea/imunologiaRESUMO
To elucidate the anti-inflammatory action of nicotine-induced corticosterone elevation on the passive skin Arthus reaction (PSAR), we investigated the inflammatory process in the PSAR. The polymorphonuclear leukocyte (PMNs) infiltration was observed just before as well as after elicitation by measuring extractable myeloperoxidase. The plasma exudation was significantly inhibited by anti-rat tumor necrosis factor (TNF)-alpha antibody (5 microg/site, i.d.) at the time of sensitization or by superoxide dismutase (52500 units/kg, i.p.) 1 h before elicitation or N(G)-nitro-L-arginine-methyl ester (100 mg/kg, i.v.) just at elicitation. Pretreatment with a single injection of nicotine (0.8 mg/kg, i.p.) 30 min before elicitation suppressed the plasma exudation but not the PMNs infiltration. This nicotine-induced decreasing effect was abolished in animals supplemented with L-arginine (300 mg/kg, i.v.) just at elicitation. The production of nitric oxide (NO) in peritoneal PMNs derived from an animal injected peritoneally with oyster glycogen was significantly suppressed by pretreatment with nicotine (0.8 mg/kg, i.v.) 30 min prior to harvesting. This inhibitory action of nicotine was abolished in animals pretreated with mifepristone (30 mg/kg, s.c.), a glucocorticoid receptor antagonist. These findings indicate that a single systematic administration of nicotine may attenuate the plasma exudation in the PSAR by suppressing the production of NO in the PMNs primed with TNF-alpha via nicotine-induced endogenous glucocorticoid.
Assuntos
Reação de Arthus/metabolismo , Corticosterona/sangue , Nicotina/farmacologia , Óxido Nítrico/biossíntese , Animais , Anticorpos/farmacologia , Arginina/farmacologia , Permeabilidade Capilar , Inibidores Enzimáticos/farmacologia , Exsudatos e Transudatos/citologia , Exsudatos e Transudatos/metabolismo , Leucócitos/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Cavidade Peritoneal/citologia , Peroxidase/metabolismo , Ratos , Ratos Wistar , Pele/efeitos dos fármacos , Pele/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Complement C2 receptor inhibitor trispanning (CRIT) is a Schistosoma protein that binds the human complement protein, C2. We recently showed that peptides based on the ligand binding region of CRIT inhibit the classical pathway (CP) of complement activation in human serum, using hemolytic assays and so speculated that on the parasite surface CRIT has the function of evading human complement. We now show that in vitro the C2-binding 11-aa C terminus of the first extracellular domain of CRIT, a 1.3-kDa peptide termed CRIT-H17, inhibits CP activation in a species-specific manner, inhibiting mouse and rat complement but not that from guinea pig. Hitherto, the ability of CRIT to regulate complement in vivo has not been assessed. In this study we show that by inhibiting the CP, CRIT-H17 is able to reduce immune complex-mediated inflammation (dermal reversed passive Arthus reaction) in BALB/c mice. Upon intradermal injection of CRIT-H17, and similarly with recombinant soluble complement receptor type 1, there was a 41% reduction in edema and hemorrhage, a 72% reduction in neutrophil influx, and a reduced C3 deposition. Furthermore, when H17 was administered i.v. at a 1 mg/kg dose, inflammation was reduced by 31%. We propose that CRIT-H17 is a potential therapeutic agent against CP complement-mediated inflammatory tissue destruction.
Assuntos
Complexo Antígeno-Anticorpo/farmacologia , Antígenos de Helmintos , Antígenos de Protozoários/farmacologia , Proteínas Inativadoras do Complemento/farmacologia , Proteínas de Helminto , Imunossupressores/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas de Protozoários/farmacologia , Receptores de Complemento 3d/antagonistas & inibidores , Pele/imunologia , Pele/patologia , Animais , Antígenos de Protozoários/administração & dosagem , Reação de Arthus/imunologia , Reação de Arthus/metabolismo , Reação de Arthus/patologia , Complemento C3/metabolismo , Proteínas Inativadoras do Complemento/administração & dosagem , Orelha Externa , Feminino , Cobaias , Imunossupressores/administração & dosagem , Imunossupressores/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Injeções Intravenosas , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/imunologia , Fragmentos de Peptídeos/administração & dosagem , Proteínas de Protozoários/administração & dosagem , Ratos , Receptores de Superfície Celular/administração & dosagem , Receptores de Complemento 3d/metabolismo , Schistosoma/imunologia , Especificidade da Espécie , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The participation of lipid mediators and tumor necrosis factor (TNF) on an experimental model of immune-complex arthritis was investigated. Male Wistar rats received intraarticular injection of rabbit antibodies to bovine serum albumin into the knee joint followed by i.v. injection of the antigen. The levels of eicosanoids and TNF released into the synovial exudates were then assessed using ELISA and the L929 lytic cell assay, respectively. Increase in the levels of LTB4, TXB2 and PGE2 were detected 5 min, 5 min, and 6 h after arthritis induction, respectively. Pretreatment with the PAF receptor antagonist WEB 2170 decreased the levels of PGE2 and increased those of LTB4, without altering TXB2 levels. Increase in the levels of TNF was detected at 3 h of arthritis. Pretreatment with either the cycloxygenase inhibitor indomethacin or the 5-lipoxygenase inhibitor L-663,536 had no effect on TNF levels. Pretreatment with WEB 2170 significantly decreased TNF levels. These results are the first demonstration of eicosanoids and TNF release in immune-complex arthritis. The data also suggest that PAF had both a positive and negative modulatory role on the release of PGE2 and LTB4, respectively. Moreover, TNF release into the synovial exudate did not depend on eicosanoids whereas platelet activating factor (PAF) appeared to mediate the release of this cytokine in the model.