RESUMO
Cardiovascular disease (CVD) is a major cause of morbidity and mortality in rheumatoid arthritis (RA). There are limited experimental data on vascular involvement in arthritis models. To study the link between CVD and inflammation in RA, we developed a model of vascular dysfunction and articular inflammation by collagen-induced arthritis (CIA) in C57Bl/6 (B6) mice. We studied the expression of vascular inflammatory markers in CIA with and without concomitant hyperlipidic diet (HD). Collagen-induced arthritis was induced with intradermal injection of chicken type-II collagen followed by a boost 21 days later. Mice with and without CIA were fed a standard diet or an HD for 12 weeks starting from the day of the boost. Arthritis severity was evaluated with a validated clinical score. Aortic mRNA levels of vascular cell adhesion molecule-1 (VCAM-1), inducible nitric oxide synthase (iNOS) and interleukin-17 were analysed by quantitative RT-PCR. Vascular cell adhesion molecule-1 localization in the aortic sinus was determined by immunohistochemistry. Atherosclerotic plaque presence was assessed in aortas. Collagen-induced arthritis was associated with increased expression of VCAM-1, independent of diet. VCAM-1 overexpression was detectable as early as 4 weeks after collagen immunization and persisted after 15 weeks. The HD induced atheroma plaque formation and aortic iNOS expression regardless of CIA. Concomitant CIA and HD had no additive effect on atheroma or VCAM-1 or iNOS expression. CIA and an HD diet induced a distinct and independent expression of large-vessel inflammation markers in B6 mice. This model may be relevant for the study of CVD in RA.
Assuntos
Aorta/imunologia , Artrite Experimental/imunologia , Aterosclerose/imunologia , Placa Aterosclerótica/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Aorta/patologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Aterosclerose/etiologia , Aterosclerose/genética , Aterosclerose/patologia , Biomarcadores/metabolismo , Colágeno/administração & dosagem , Dieta Hiperlipídica/efeitos adversos , Expressão Gênica , Humanos , Inflamação , Interleucina-17/genética , Interleucina-17/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Índice de Gravidade de Doença , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes inflammation and destruction of the joints. In the collagen-induced arthritis mouse model of RA, we developed a nonviral gene therapy method designed to block in situ the main cytokine tumour necrosis factor (TNF)-alpha METHODS: Electrotransfer was used to deliver a plasmid encoding extracellular domain of mouse soluble TNF-alpha receptor type I fused to the Fc fragment of mouse immunoglobulin (Ig)G1 (pTNFR-Is) corresponding to a dimeric TNF-alpha soluble receptor fusion protein (mTNFR-Is/Ig). RESULTS: Delivery of the plasmid into the knees at symptom onset improved the histological inflammation and destruction not only at the knees, but also at the ankles, indicating a local and a regional therapeutic effect. The plasmid was detected in synovial membrane and meniscus specimens from the injected joints. In the synovial membrane, 15 days post-injection, interleukin (IL)-17 and TNF-alpha mRNAs expression were increased, whereas IL-10 mRNA was unchanged. However, the empty plasmid exerted a pro-inflammatory effect 30 days post-injection. CONCLUSIONS: These data indicate that local nonviral gene therapy against TNF-alpha is effective, although further work is needed to decrease plasmid induced inflammation.
Assuntos
Artrite Reumatoide/terapia , Eletroporação , Terapia Genética/métodos , Receptores do Fator de Necrose Tumoral/genética , Animais , Técnicas de Transferência de Genes , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Camundongos , Modelos Animais , Plasmídeos/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
As highlighted in this review, the phosphoinositide-phospholipase C pathway is strongly implicated in the control of mouse oocyte meiosis. The pathway becomes progressively functional as oocyte growth advances, and it appears to play a role in the G2/M transition when meiosis resumes, at least in the in vitro spontaneous model. Even if the inositol 1,4,5-trisphosphate receptors are present from the beginning, they function and release Ca2+ when the follicular antrum appears. Phospholipase C beta1 (PLC beta 1) is first exclusively localized to the nucleus and then migrates to the cytoplasm when the oocyte is fully grown. During oocyte maturation PLC beta 1 is active in the cytoplasm before it migrates and becomes active in the nucleus just prior to germinal vesicle breakdown. Because a similar circuit is observed for protein kinase C alpha (PKC alpha), PKC beta 1, PKC beta 2, and active mitogen-activated protein kinase, it is tempting to envisage that a feedback loop occurs between these pathways as demonstrated in other cell types. The chronology of these molecular movements into the oocyte reveals the particular and important role of the nucleus phosphoinositide cycle during oocyte meiosis. It appears also that this chronology is crucial and that defects leading to an inappropriate intracellular localization can have dramatic consequences. Such anomalies can prevent the production of competent oocytes and lead to fertility problems.
Assuntos
Isoenzimas/metabolismo , Oócitos/enzimologia , Fosfatidilinositóis/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Cálcio/metabolismo , Feminino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfolipase C betaRESUMO
This study was conducted on human Jurkat T-cells to investigate the role of depletion of intracellular Ca(2+) stores in the phosphorylation of two mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK) 1 and ERK2, and their modulation by a polyunsaturated fatty acid, docosahexaenoic acid (DHA). We observed that thapsigargin (TG) stimulated MAPK activation by store-operated calcium (SOC) influx via opening of calcium release-activated calcium (CRAC) channels as tyrphostin-A9, a CRAC channel blocker, and two SOC influx inhibitors, econazole and SKF-96365, diminished the action of the former. TG-stimulated ERK1/ERK2 phosphorylation was also diminished in buffer containing EGTA, a calcium chelator, further suggesting the implication of calcium influx in MAPK activation in these cells. Moreover, TG stimulated the production of diacylglycerol (DAG) by activating phospholipase D (PLD) as propranolol (PROP) (a PLD inhibitor), but not U73122 (a phospholipase C inhibitor), inhibited TG-evoked DAG production in these cells. DAG production and protein kinase C (PKC) activation were involved upstream of MAPK activation as PROP and GF109203X, a PKC inhibitor, abolished the action of TG on ERK1/ERK2 phosphorylation. Furthermore, DHA seems to act by inhibiting PKC activation as this fatty acid diminished TG- and phorbol 12-myristate 13-acetate-induced ERK1/ERK2 phosphorylation in these cells. Together these results suggest that Ca(2+) influx via CRAC channels is implicated in PLD/PKC/MAPK activation which may be a target of physiological agents such as DHA.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfolipase D/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Tapsigargina/farmacologia , Cálcio/metabolismo , Diglicerídeos/biossíntese , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Humanos , Células Jurkat , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismoRESUMO
New micro-vessels formation within synovium and macro-vessels endothelial damage with atheroma are two major features of rheumatoid arthritis, the former related to the articular involvement of the disease, the latter to its main systemic complication. The similarities between pannus development and solid tumors growth, and the efficacy of anti-angiogenic treatments in oncology, opened the perspective of directly targeting angiogenesis in arthritis. Nevertheless, despite the success of different anti-angiogenic therapeutic strategies in many arthritis experimental models, the application in human disease is still lacking. Recent data suggest that synovial neoangiogenesis and macro-vessels endothelial damage might be two linked phenomena. While synovial angiogenesis seems to be detrimental to endothelial damage repair, even anti-angiogenic treatments might paradoxically aggravate macro-vascular disease, especially in the context of uncontrolled inflammation. These elements induce to further explore the interconnections between inflammation and angiogenesis on one side and between micro- and macro-vascular diseases on the other, in order to establish the proper way to therapeutically target blood vessels in rheumatoid arthritis.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/fisiopatologia , Vasos Sanguíneos/fisiopatologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/fisiopatologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/fisiopatologia , Membrana Sinovial/irrigação sanguíneaRESUMO
Modulation of the T-cell response depends chiefly on regulatory T cells (Treg), which express CD4 and CD25. Some Treg cells are present naturally, whereas others are induced in response to antigens. The immunomodulating effects of Treg cells are mediated by membrane molecules (e.g., CTLA4, GITR, and OX40) and cytokines. IL-35 seems to be a crucial mediator, although IL-10 and TGFbeta are also important. The role for Treg cells in rheumatoid arthritis (RA) has been established in both patients and animal models. Treg function is deficient in RA, whereas Treg counts vary. Treg counts increase in patients who are responding to TNFalpha antagonist therapy. Among current hypotheses, Treg expansion or transfer may hold promise for the treatment of RA.
Assuntos
Artrite Reumatoide/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD/metabolismo , Artrite Reumatoide/metabolismo , Antígenos CD4/metabolismo , Antígeno CTLA-4 , Contagem de Células , Modelos Animais de Doenças , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Humanos , Fatores Imunológicos/imunologia , Interleucina-10/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucinas/metabolismo , Ligante OX40/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Most autoimmune diseases have an unknown etiology, but all involve cytokines cascade in their development. At the present time, several cytokines have been identified as major targets in various autoimmune diseases, involving the development of monoclonal antibodies (MAbs) against those cytokines. Even if MAbs are indeed efficient, the passive immunotherapies also present some disadvantages and are expensive. To counter this, several strategies have been developed, including active immunotherapy, based on the vaccination principle. The aim of such a strategy is to induce a B cell response and to obtain autoantibodies able to neutralize the interaction of the self-cytokine with its receptor. To that purpose, cytokines (entire or peptide) are either coupled with a protein-carrier or virus-like particle, or modified with foreign Th cell epitopes. DNA vaccination can also be used with cytokine sequences. This review focuses on the different vaccination strategies with cytokines (including Tumor Necrosis Factor (TNF)alpha, Interleukin-1beta (IL-1beta), IL-17) in different autoimmune diseases in preclinical studies; the benefit/risk ratio of such a strategy and the present development of clinical trials in some autoimmune diseases are also discussed.
Assuntos
Doenças Autoimunes/terapia , Citocinas/imunologia , Citocinas/uso terapêutico , Vacinação , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Ensaios Clínicos como Assunto , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Humanos , Vacinas de DNA/imunologiaRESUMO
PKC modulators were used to investigate the role of the PKC pathway either on the maintenance of meiotic arrest or on FSH-induced maturation of mouse cumulus cell enclosed oocytes (CEOs). (1) Whereas PKC activation (PMA 8 microM) overcomed clearly the HX-maintained meiotic arrest (83.7 +/- 3.6% vs. 16.1 +/- 10.6% GVBD oocytes), PKC inhibition (Calphostin C 100 nM) did not. On the contrary, it better maintained the meiotic arrest than HX alone. (2) No significant effect of PKC activation or inhibition was observed. (3) HX alone maintained PKCbeta1 in the cytoplasm, whereas FSH and PKC activation induced partly its translocation into the nucleus. The results show that whereas the PKC pathway is clearly involved in maintenance of the meiotic arrest through PKCbeta1, it is not involved in FSH-induced meiosis of CEOs.
Assuntos
Células do Cúmulo/enzimologia , Meiose , Oócitos/enzimologia , Proteína Quinase C/metabolismo , Animais , Células do Cúmulo/efeitos dos fármacos , Feminino , Hormônio Foliculoestimulante/farmacologia , Meiose/efeitos dos fármacos , Camundongos , Oócitos/efeitos dos fármacos , Proteína Quinase C/análise , Proteína Quinase C betaRESUMO
We elucidated the mechanisms of action of two n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in Jurkat T-cells. Both DHA and EPA were principally incorporated into phospholipids in the following order: phosphatidylcholine < phosphatidylethanolamine < phosphatidylinositol/phosphatidylserine. Furthermore, two isoforms of phospholipase A(2) (i.e., calcium-dependent and calcium-independent) were implicated in the release of DHA and EPA, respectively, during activation of these cells. The two fatty acids inhibited the phorbol 12-myristate 13-acetate (PMA)-induced plasma membrane translocation of protein kinase C (PKC)-alpha and -epsilon. The two n-3 PUFAs also inhibited the nuclear translocation of nuclear factor kappaB (NF-kappaB) and the transcription of the interleukin-2 (IL-2) gene in PMA-activated Jurkat T-cells. Together, these results demonstrate that DHA and EPA, being released by two isoforms of phospholipase A(2), modulate IL-2 gene expression by exerting their action on two PKC isoforms and NF-kappaB in Jurkat T-cells.
Assuntos
Ácidos Graxos Ômega-3/farmacologia , Ativação Linfocitária/efeitos dos fármacos , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Linfócitos T/enzimologia , Linfócitos T/imunologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/metabolismo , Ácido Eicosapentaenoico/farmacologia , Ácidos Graxos Ômega-3/metabolismo , Humanos , Interleucina-2/genética , Isoenzimas/metabolismo , Células Jurkat , Ativação Linfocitária/imunologia , Ésteres de Forbol/farmacologia , Fosfolipases A/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Proteína Quinase C-alfa , Proteína Quinase C-épsilon , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
In order to investigate the implication of docosahexacnoic acid (DHA) and eicosapentaenoic acid (EPA) in T signalling, we assessed their effects on the activation of two mitogen activated protein (MAP) kinases, i.e. extracellularly-regulated kinases 1 and 2 (ERK1/ERK2) in Jurkat T-cells. The n-3 polyunsaturated fatty acids (PUFAs) alone failed to induce MAP kinase (MAPK) enzyme activity. To elucidate whether DHA and EPA act via protein kinase C (PKC) dependent and independent pathways, we employed their respective activators, i.e. phorbol 12-myristate 13-acetate (PMA) and antiCD3 antibodies. We observed that U0126, an inhibitor of MAPK kinase-ERK kinase 1/2 (MEK1/2), abolished the actions of these two agents on MAPK activation, suggesting that they act upstream of MEK1/2. Further EPA and DHA diminished both the PMA- and antiCD3 antibodies-induced enzyme activity of ERK1/ERK2 in Jurkat T-cells. Interestingly, okadaic acid (OA), a phosphatase inhibitor seems to act downstream of MEK1/2 as U0126 failed to inhibit the OA-induced MAPK activation. It is noteworthy that EPA and DHA not only failed to curtail the OA-induced MAPK activity but also these n-3 PUFAs at 20 microM potentiated the action of OA. Therefore, EPA and DHA seem to modulate MAPK activation upstream and downstream of MEK1/2. On the hand, arachidonic acid, an n-6 PUFA potentiated the MAPK enzyme activity. In conclusion, our study shows that EPA and DHA may regulate T-cells functions by modulating MAPK enzyme activity.