RESUMO
Non-activated macrophages express low levels of A(2A)Rs and lipopolysaccharides (LPS) upregulates A(2A)R expression in an NF-κB-dependent manner. The murine A(2A)R gene is encoded by three exons, m1, m2 and m3. Exons m2 and m3 are conserved, while m1 encodes the 5' untranslated UTR. Three m1 variants have been defined, m1A, m1B and m1C, with m1C being farthest from the transcriptional start site. LPS upregulates A(2A)Rs in primary murine peritoneal and bone-marrow-derived macrophages and RAW264.7 cells by selectively splicing m1C to m2, through a promoter located upstream of m1C. We have cloned â¼1.6 kb upstream of m1C into pGL4.16(luc2CP/Hygro) promoterless vector. This construct in RAW 264.7 cells responds to LPS, and adenosine receptor agonists augmented LPS responsiveness. The NF-κB inhibitors BAY-11 and triptolide inhibited LPS-dependent induction. Deletion of a key proximal NF-κB site (402-417) abrogated LPS responsiveness, while deletion of distal NF-κB and C/EBPß sites did not. Site-directed mutagenesis of CREB (309-320), STAT1 (526-531) and AP2 (566-569) sites had little effect on LPS and adenosine receptor agonist responsiveness; however, mutation of a second STAT1 site (582-588) abrogated this responsiveness. Further analysis of this promoter should provide valuable insights into regulation of A(2A)R expression in macrophages in response to inflammatory stimuli.
Assuntos
Lipopolissacarídeos/farmacologia , Regiões Promotoras Genéticas/genética , Receptor A2A de Adenosina/genética , Ativação Transcricional/efeitos dos fármacos , Processamento Alternativo , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Células Cultivadas , Diterpenos/farmacologia , Compostos de Epóxi/farmacologia , Éxons/genética , Feminino , Luciferases/genética , Luciferases/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Nitrilas/farmacologia , Fenantrenos/farmacologia , Isoformas de Proteínas/genética , Agonistas do Receptor Purinérgico P1/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/metabolismo , Sulfonas/farmacologiaRESUMO
Sepsis is a life-threatening organ dysfunction caused by a dysregulated inflammatory response to infection. To date, there is no specific treatment established for sepsis. In the extracellular compartment, purines such as adenosine triphosphate (ATP) and adenosine play essential roles in the immune/inflammatory responses during sepsis and septic shock. The balance of extracellular levels among ATP and adenosine is intimately involved in the signals related to immune stimulation/immunosuppression balance. Specialized enzymes, including CD39, CD73, and adenosine deaminase (ADA), are responsible to metabolize ATP to adenosine which will further sensitize the P2 and P1 purinoceptors, respectively. Disruption of the purinergic pathway had been described in the sepsis pathophysiology. Although purinergic signaling has been suggested as a potential target for sepsis treatment, the majority of data available were obtained using pre-clinical approaches. We hypothesized that, as a reflection of deregulation on purinergic signaling, septic patients exhibit differential measurements of serum, neutrophils and monocytes purinergic pathway markers when compared to two types of controls (healthy and ward). It was observed that ATP and ADP serum levels were increased in septic patients, as well as the A2a mRNA expression in neutrophils and monocytes. Both ATPase/ADPase activities were increased during sepsis. Serum ATP and ADP levels, and both ATPase and ADPase activities were associated with the diagnosis of sepsis, representing potential biomarkers candidates. In conclusion, our results advance the translation of purinergic signaling from pre-clinical models into the clinical setting opening opportunities for so much needed new strategies for sepsis and septic shock diagnostics and treatment.
Assuntos
Sepse , Choque Séptico , Humanos , Apirase/metabolismo , Adenosina , Trifosfato de Adenosina/metabolismo , Biomarcadores , Sepse/diagnóstico , Difosfato de Adenosina , Adenosina TrifosfatasesRESUMO
Ischaemia-reperfusion (I/R) is a pivotal mechanism of organ injury during stroke, myocardial infarction, organ transplantation and vascular surgeries. Ischaemic preconditioning (IPC) is a potent endogenous form of tissue protection against I/R injury. On the one hand, endocannabinoids have been implicated in the protective effects of IPC through cannabinoid CB1/CB2 receptor-dependent and -independent mechanisms. However, there is evidence suggesting that endocannabinoids are overproduced during various forms of I/R, such as myocardial infarction or whole body I/R associated with circulatory shock, and may contribute to the cardiovascular depressive state associated with these pathologies. Previous studies using synthetic CB1 receptor agonists or knockout mice demonstrated CB1 receptor-dependent protection against cerebral I/R injury in various animal models. In contrast, several follow-up reports have shown protection afforded by CB1 receptor antagonists, but not agonists. Excitedly, emerging studies using potent CB2 receptor agonists and/or knockout mice have provided compelling evidence that CB2 receptor activation is protective against myocardial, cerebral and hepatic I/R injuries by decreasing the endothelial cell activation/inflammatory response (for example, expression of adhesion molecules, secretion of chemokines, and so on), and by attenuating the leukocyte chemotaxis, rolling, adhesion to endothelium, activation and transendothelial migration, and interrelated oxidative/nitrosative damage. This review is aimed to discuss the role of endocannabinoids and CB receptors in various forms of I/R injury (myocardial, cerebral, hepatic and circulatory shock) and preconditioning, and to delineate the evidence supporting the therapeutic utility of selective CB2 receptor agonists, which are devoid of psychoactive effects, as a promising new approach to limit I/R-induced tissue damage.
Assuntos
Moduladores de Receptores de Canabinoides/fisiologia , Endocanabinoides , Precondicionamento Isquêmico , Receptores de Canabinoides/fisiologia , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/fisiopatologia , Animais , Agonistas de Receptores de Canabinoides , Moduladores de Receptores de Canabinoides/farmacologia , Moduladores de Receptores de Canabinoides/uso terapêutico , Humanos , Hepatopatias/tratamento farmacológico , Hepatopatias/fisiopatologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Choque/tratamento farmacológico , Choque/fisiopatologia , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
BACKGROUND AND PURPOSE: Vascular smooth muscle proliferation and migration triggered by inflammatory stimuli are involved in the development and progression of atherosclerosis and restenosis. Cannabinoids may modulate cell proliferation in various cell types through cannabinoid 2 (CB2) receptors. Here, we investigated the effects of CB2 receptor agonists on TNF-alpha-induced proliferation, migration and signal transduction in human coronary artery smooth muscle cells (HCASMCs). EXPERIMENTAL APPROACH: HCASMCs were stimulated with TNF-alpha. Smooth muscle proliferation was determined by the extent of BrdU incorporation and the migration was assayed by modified Boyden chamber. CB2 and/or CB1 receptor expressions were determined by immunofluorescence staining, western blotting, RT-PCR, real-time PCR and flow cytometry. KEY RESULTS: Low levels of CB2 and CB1 receptors were detectable in HCASMCs compared to the high levels of CB2 receptors expressed in THP-1 monocytes. TNF-alpha triggered up to approximately 80% increase (depending on the method used) in CB2 receptor mRNA and/or protein expression in HCASMCs, and induced Ras, p38 MAPK, ERK 1/2, SAPK/JNK and Akt activation, while increasing proliferation and migration. The CB2 agonists, JWH-133 and HU-308, dose-dependently attenuated these effects of TNF-alpha. CONCLUSIONS AND IMPLICATIONS: Since the above-mentioned TNF-alpha-induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis, our findings suggest that CB2 agonists may offer a novel approach in the treatment of these pathologies by decreasing vascular smooth muscle proliferation and migration.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Receptor CB2 de Canabinoide/agonistas , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Células Cultivadas , Citometria de Fluxo , Técnica Direta de Fluorescência para Anticorpo , Genes ras/efeitos dos fármacos , Humanos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Miócitos de Músculo Liso/metabolismo , Proteína Oncogênica v-akt/genética , Proteína Oncogênica v-akt/fisiologia , Receptor CB2 de Canabinoide/biossíntese , Receptor CB2 de Canabinoide/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Regulação para Cima/efeitos dos fármacosRESUMO
The amiloride-inhibitable Na(+)/H(+) antiporter plays an important role in macrophage activation. The intracellular pathways leading to interleukin (IL)-12 p40 production by activated macrophages are incompletely understood. In the present study, we examined the contribution of the Na(+)/H(+) antiporter to the production of IL-12 p40. Amiloride or its analogs decreased the production of IL-12 p40 in macrophages stimulated with bacterial lipopolysaccharide and interferon-gamma. The order of potency of amiloride analogs was consistent with the proposition that the effect of amiloride is mediated by the inhibition of the Na(+)/H(+) antiporter. The effect of amiloride was post-transcriptional, as IL-12 p40 mRNA levels induced by lipopolysaccharide and interferon-gamma were not affected by this inhibitor. Furthermore, the inhibitory effect of amiloride on IL-12 p40 production was not a result of interference with the activation of the p38 and p42/44 mitogen-activated protein kinases or c-Jun kinase. In summary, the production of IL-12 p40 requires a functional Na(+)/H(+) antiporter.
Assuntos
Amilorida/farmacologia , Interleucina-12/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Amilorida/análogos & derivados , Animais , Quimiocina CCL4 , Quimiocina CXCL2 , Quimiocinas/metabolismo , Diuréticos/farmacologia , Interações Medicamentosas , Ativação Enzimática/efeitos dos fármacos , Inativação Gênica , Proteínas Quinases JNK Ativadas por Mitógeno , Lipopolissacarídeos/farmacologia , Proteínas Inflamatórias de Macrófagos/metabolismo , Macrófagos Peritoneais/metabolismo , Masculino , Metacrilatos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Peroxynitrite-induced poly(ADP-ribose) polymerase activation has been implicated in the pathogenesis of various inflammatory conditions. Here we have investigated whether peroxynitrite and poly(ADP-ribose) polymerase may play a role in the pathophysiology of the elicitation phase of contact hypersensitivity. We have detected nitrotyrosine, DNA breakage, and poly(ADP-ribose) polymerase activation in the epidermis of mice in an oxazolone-induced contact hypersensitivity model. As tyrosine nitration is mostly mediated by peroxynitrite, a nitric-oxide-derived cytotoxic oxidant capable of causing DNA breakage, we have applied peroxynitrite directly on mouse skin and showed poly(ADP-ribose) polymerase activation in keratinocytes and in some scattered dermal cells. We have also investigated the cellular effects of peroxynitrite in HaCaT cells, a human keratinocyte cell line. We found that peroxynitrite inhibited cell proliferation and at higher concentrations also caused cytotoxicity. Peroxynitrite activates poly(ADP-ribose) polymerase in HaCaT cells and poly(ADP-ribose) polymerase activation contributes to peroxynitrite-induced cytotoxicity, as indicated by the cytoprotective effect of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. The cytoprotective effect of 3-aminobenzamide cannot be attributed to inhibition of apoptosis, as apoptotic parameters (caspase activation and DNA fragmentation) were not reduced in the presence of 3-aminobenzamide in peroxynitrite-treated cells. Moreover, poly(ADP-ribose) polymerase inhibition by 3-aminobenzamide dose-dependently reduced interferon-induced intercellular adhesion molecule 1 expression as well as interleukin-1beta-induced interleukin-8 expression. Our results indicate that peroxynitrite and poly(ADP-ribose) polymerase regulate keratinocyte function and death in contact hypersensitivity.
Assuntos
Dano ao DNA/fisiologia , Dermatite de Contato/metabolismo , Nitratos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Adjuvantes Imunológicos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Fragmentação do DNA/fisiologia , Dermatite de Contato/genética , Dermatite de Contato/imunologia , Feminino , Marcação In Situ das Extremidades Cortadas , Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-8/imunologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos , Necrose , Oxazolona , Pele/imunologia , Pele/metabolismo , Pele/patologia , Tirosina/metabolismoRESUMO
Poly(ADP-ribose) polymerase (PARP), or poly-(ADP-ribose) synthetase, is a nuclear enzyme that consumes NAD when activated by DNA damage. The role of PARP in the pathogenesis of traumatic brain injury (TBI) is unknown. Using a controlled cortical impact (CCI) model of TBI and mice deficient in PARP, the authors studied the effect of PARP on functional and histologic outcome after CCI using two protocols. In protocol 1, naive mice (n = 7 +/+, n = 6 -/-) were evaluated for motor and memory acquisition before CCI. Mice were then subjected to severe CCI and killed at 24 hours for immunohistochemical detection of nitrated tyrosine, an indicator of peroxynitrite formation. Motor and memory performance did not differ between naive PARP +/+ and -/- mice. Both groups showed nitrotyrosine staining in the contusion, suggest ing that peroxynitrite is produced in contused brain. In protoco 2, mice (PARP +/+, n = 8; PARP -/-, n = 10) subjected to CCI were tested for motor and memory function, and contusion volume was determined by image analysis. PARP -/- mice demonstrated improved motor and memory function after CC versus PARP +/+ mice (P < 0.05). However, contusion volume was not different between groups. The results suggest a detri mental effect of PARP on functional outcome after TBI.
Assuntos
Lesões Encefálicas/fisiopatologia , Lesões Encefálicas/psicologia , Encéfalo/fisiopatologia , Cognição/fisiologia , Aprendizagem em Labirinto , Memória/fisiologia , Atividade Motora/fisiologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Lesões Encefálicas/genética , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/fisiopatologia , Sinais (Psicologia) , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Poli(ADP-Ribose) Polimerases/deficiência , Percepção EspacialRESUMO
Transmitters and cotransmitters of the sympathetic nervous system are involved in the regulation of a variety of immune cell functions. However, it is not entirely clear what stimuli lead to the release of these molecules in immune organs. In this study, we investigated whether local ischemia can cause the parallel release of norepinephrine and its cotransmitter, ATP, in the spleen. Ischemic-like conditions, simulated by transient (15 min) O(2) and glucose deprivation, elicited a reversible increase in the release of both norepinephrine and purines from superfused spleen strips preloaded with [3H]norepinephrine or [3H]adenosine. HPLC analysis of the released tritium label revealed a net increase in the amount of ATP, ADP, AMP, adenosine, inosine, hypoxanthine and xanthine in response to ischemic-like condition. Selective O(2) or glucose deprivation, and Ca(2+)-free conditions differentially affected the outflow of [3H]norepinephrine and [3H]purines, indicating that they derived from different sources. The ABC transporter inhibitors glibenclamide (100 microM) and verapamil (100 microM) as well as low-temperature inhibited [3H]purine release evoked by ischemic-like conditions. Surgical denervation of the spleen reduced endogenous catecholamine content and [3H]norepinephrine uptake of the spleen, but not that of [3H]adenosine. In summary, these results demonstrate the release of norepinephrine and purines in response to an ischemic-like condition in an immune organ. Although both could provide an important source of extracellular catecholamines and purines involved at various levels of immunomodulation, the source and mechanism of norepinephrine and purine efflux seem different.
Assuntos
Adenosina/farmacocinética , Isquemia/imunologia , Isquemia/metabolismo , Norepinefrina/farmacocinética , Baço/metabolismo , Simpatomiméticos/farmacocinética , Vasodilatadores/farmacocinética , Adenina/farmacocinética , Difosfato de Adenosina/farmacocinética , Monofosfato de Adenosina/farmacocinética , Trifosfato de Adenosina/farmacocinética , Animais , Cálcio/farmacologia , Hipoxantina/farmacocinética , Inosina/farmacocinética , Masculino , Ratos , Ratos Wistar , Baço/irrigação sanguínea , Baço/inervação , Simpatectomia , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/imunologia , Sistema Nervoso Simpático/metabolismo , Tetrodotoxina/farmacologia , Trítio , Xantina/farmacocinéticaRESUMO
In a previous study we demonstrated that mice pretreated with the highly selective alpha 2-adrenoceptor antagonist CH-38083 showed blunting of the tumor necrosis factor-alpha (TNF-alpha) response induced by bacterial lipopolysaccharide (LPS). In the present study, the effect of a selective block of alpha 2-adrenoreceptors and the role of the sympathetic nervous system (SNS) in the regulation of LPS-induced TNF-alpha production was explored further using different selective adrenoceptor antagonists and agonists. While adrenalectomy did not prevent the effect of CH-38083, the block of the sympathetic transmission by chlorisondamine fully abolished the inhibitory effect of CH-38083 on LPS-induced TNF-alpha production, suggesting that the effect of the alpha 2-adrenoceptor blocking agent is corticosteroid-independent, but it requires intact sympathetic activity. Since the selective block of alpha 2-adrenoceptors results in an increased sympathetic activity and an increase of the release of noradrenaline (NA) in both the central and the peripheral nervous systems, and in our experiments propranolol, a non-selective beta-adrenoceptor antagonist, and atenolol, a selective antagonist of beta 1-adrenoceptors, prevented the effect of alpha 2-adrenoceptor blockade by CH-38083 of the TNF-alpha response induced by LPS, it seems likely that the excessive stimulation by NA of beta 1-adrenoceptors is responsible for this action. The role of beta-adrenoceptors and endogenous catecholamines is further substantiated by the finding that pretreatment of animals with propranolol alone resulted in a dose-dependent increase of the TNF-alpha response induced by LPS, and that isoproterenol, a non-selective beta-adrenoceptor agonist, decreased it.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Lipopolissacarídeos/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Adrenalectomia , Animais , Berberina/análogos & derivados , Berberina/farmacologia , Masculino , Camundongos , Propranolol/farmacologiaRESUMO
Stimulation of beta-adrenoceptors has been shown to regulate the production of various inflammatory mediators. In the present study, we investigated in mice whether ligation of beta-adrenoceptors, modulates lipopolysaccharide (LPS)-induced plasma levels of interleukin (IL)-12, interferon-gamma (IFN-gamma), and IL-10. In BALB/c mice, isoproterenol (1-10 mg kg(-1) , i.p.), a selective agonist of beta-adrenoceptors and also dexamethasone (10 mg kg(-1), i.p.) pretreatment 30 min before the administration of LPS suppressed plasma IL-12 (p40 and p70) concentrations as determined at various time points after the LPS challenge. The inhibition of IL-12 release by isoproterenol was prevented by the beta-adrenoceptor antagonist propranolol confirming the involvement of beta-adrenoceptors in the effect of isoproterenol. Furthermore, pretreatment of the animals with propranolol alone enhanced LPS-induced plasma IL-12, suggesting that endogenous catecholamines inhibit IL-12 production via the beta-adrenoceptors. In IL-10 deficient C57BL/6 IL-10(-/-) mice, plasma levels of IL-12 and IFN-gamma were significantly higher than in their counterparts, with more than 70-fold increase in IL-12. Furthermore, while augmenting the IL-10 response in C57BL/6 IL-10(+/+), isoproterenol inhibited the production of IL-12 in both the C57BL/6 IL-10(+/+) and C57BL/6 IL-10(-/-) mice, suggesting that the inhibition of IL-12 production by this compound is independent of the increased release of IL-10. Our results demonstrate, for the first time, that stimulation of beta-adrenoceptors by isoproterenol or endogenous catecholamines suppresses IL-12 production in vivo.
Assuntos
Endotoxinas/farmacologia , Interleucina-10/deficiência , Interleucina-12/biossíntese , Receptores Adrenérgicos beta/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Interleucina-12/sangue , Isoproterenol/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Valores de ReferênciaRESUMO
The object of these experiments was to investigate whether noradrenaline is the signal neurotransmitter between the sympathetic nervous system and rat thymocytes. Using immunocytochemistry, evidence was obtained that the rat thymus (thymic capsule, subcapsular region and connective tissue septa) is innervated by noradrenergic varicose axons terminals (tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunostained nerve fibres). This innervation is mainly associated with the vasculature and separately from vessels along the thymic tissue septa it branches into the thymic parenchyma. Using electron microscopy, classical synapses between thymocytes and neuronal elements were not observed. The neurochemical study revealed that these nerve terminals are able to take up, store and release noradrenaline upon axonal stimulation in a [Ca2+]o-dependent manner. The release was tetrodotoxin (1 microM)-sensitive, and reserpine pretreatment prevented axonal stimulation to release noradrenaline, indicating vesicular origin of noradrenaline. In addition, it was found that the release of noradrenaline was subjected to negative feedback modulation via presynaptic alpha 2-adrenoreceptors. Using a patch-clamp technique, electrophysiological evidence was obtained showing that noradrenaline inhibits in a concentration-dependent manner outward voltage-dependent potassium (k+) current recorded from isolated thymocytes. Since noradrenergic varicose axon terminals enter the parenchyma thymocytes and the boutons are not in close apposition to their target cells, noradrenaline released from these terminals diffuses away from release site to reach its targets, thymocytes, and to exert its inhibitory effect on voltage-dependent K+ -current. Since K+ channels are believed to be involved in T cell proliferation and differentiation, the modulation of K+ channel gating by noradrenaline released in response to axonal activity suggests that signals from blood-born or locally released hormones and cytokines. In this respect, noradrenaline released from non-synaptic neuronal varicosities and exerting its effect within the radius of diffusion may serve as a chemical link between the sympathetic nervous system and thymocytes and may have physiological and pathological importance in the thymus during stress and inflammatory/immune responses.
Assuntos
Norepinefrina/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Sistema Nervoso Simpático/fisiologia , Timo/inervação , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Sistema Cromafim/citologia , Sistema Cromafim/metabolismo , Estimulação Elétrica , Eletrofisiologia , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Norepinefrina/metabolismo , Sistema Nervoso Parassimpático/metabolismo , Sistema Nervoso Parassimpático/ultraestrutura , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/ultraestrutura , Timo/citologia , Timo/fisiologiaRESUMO
Here we examined the changes in NADPH diaphorase (NADPHd) and inducible nitric oxide synthetase (iNOS) positivity in the medulla of the mouse thymus in response to treatment with the superantigen, Staphylococcal enterotoxin B (SEB). A few NADPHd+ and iNOS+ cells scattered in the medulla were detected in the thymi of naive mice. SEB induced the appearance of a large number of NADPHd+- and iNOS-immunoreactive cells in the thymic medulla. In the thymus of iNOS-deficient mice, a total absence of these NADPHd+ and iNOS+ medullary cells was found both under basal conditions and after SEB stimulation. With the NADPHd reaction, only endothelial staining was detected in the thymi of iNOS-deficient mice. Our data indicate that NADPHd+ cells in the thymic medulla express iNOS and that SEB induces iNOS expression in the mouse thymus.
Assuntos
Histocitoquímica/métodos , NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase/metabolismo , Timo/enzimologia , Animais , Enterotoxinas/farmacologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Wistar , Superantígenos/farmacologia , Timo/efeitos dos fármacosRESUMO
In a previous study we have shown that, in response to electrical stimulation, there is a substantial release of noradrenaline (NA) from the sympathetic nerve terminals of the rat thymus which is of axonal, vesicular origin. In the present study neurochemical evidence was obtained that the release of NA is subject to presynaptic modulation. This modulation operates through stimulation of alpha 2B-adrenoreceptors, N-nicotinic, P1-purinergic and prostaglandin E2 presynaptic receptors. Through these receptors the release of NA, i.e., the message from the central nervous system to the thymus, can be affected by endogenous ligands or drugs. A novel, potent and highly selective competitive antagonist of the alpha 2-adrenoreceptor, CH-38083, significantly enhanced the release of NA, suggesting that its release in the thymus is under tonic inhibitory control exerted by endogenously released NA. Since adrenoreceptors on thymocytes involved in the modulation of certain thymocyte functions have recently been described, it is suggested that the presynaptic modulation of the release of NA in the thymus is involved in neuro-immune communication.
Assuntos
Terminações Nervosas/metabolismo , Norepinefrina/metabolismo , Receptores Pré-Sinápticos/fisiologia , Sistema Nervoso Simpático/metabolismo , Timo/inervação , Agonistas Adrenérgicos/farmacologia , Antagonistas Adrenérgicos/farmacologia , Animais , Técnicas In Vitro , Masculino , Terminações Nervosas/efeitos dos fármacos , Perfusão , Ratos , Ratos Wistar , Receptores Pré-Sinápticos/efeitos dos fármacos , Sistema Nervoso Simpático/efeitos dos fármacos , Timo/efeitos dos fármacos , Timo/metabolismoRESUMO
The effects of various agonist and antagonists of dopamine D1 and D2 receptors on lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production was investigated in mice. Pretreatment of animals with bromocryptine or quinpirole, agonists of dopamine D2 receptors caused a blunting of both the TNF-alpha and NO responses to LPS injected intraperitoneally. Sulpiride, an antagonist of dopamine D2 receptors, decreased the LPS-induced TNF-alpha plasma levels in a dose-dependent manner and inhibited the LPS-induced NO production by peritoneal macrophages. Bromocryptine or quinpirole blunted both the TNF-alpha and NO response to LPS. SCH-23390, an antagonist of dopamine D1 receptors did not alter LPS-induced TNF-alpha production, but inhibited LPS-induced NO production. These results indicate that while the D2 subtype of dopamine receptors is involve in the modulation of both LPS-induced TNF-alpha and NO production, dopamine D1 receptors only regulate the production of NO. Since several drugs possess effect on dopamine D2 receptors, the present observations may be of clinical relevance.
Assuntos
Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Lipopolissacarídeos/imunologia , Óxido Nítrico/biossíntese , Receptores de Dopamina D2/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Macrófagos Peritoneais/metabolismo , Masculino , CamundongosRESUMO
Interleukin-12 (IL-12) is a pivotal cytokine in driving the immune system towards a T helper (Th)1 type response and preventing a Th2 type immune profile. Therefore, IL-12 is indispensable in the defense against certain, mainly intracellular pathogens, but overproduction of this cytokine is crucially involved in the etiology of several inflammatory and autoimmune diseases. Hence, IL-12 is an ideal target for pharmacological intervention in the therapy of autoimmune and inflammatory diseases. The production of IL-12 and a resultant Th1 type immune response can be suppressed with several pharmacological approaches including modulation of intracellular cyclic AMP levels, glucocorticoids and nuclear factor-kappaB inhibition. IL-12 responsiveness may be inhibited using anti-IL-12 antibodies, soluble IL-12 receptors or the IL-12 p40 homodimer. Exploitation of these approaches may provide novel means for the experimental therapy of a variety of pathophysiological states.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Inflamação/tratamento farmacológico , Interleucina-12/antagonistas & inibidores , Animais , Doenças Autoimunes/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-12/biossíntese , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologiaRESUMO
Immune cell activation releases ATP into the extracellular space. ATP-sensitive P2 purinergic receptors are expressed on immune cells and activation of these receptors alters immune cell function. Furthermore, ATP is metabolized by ectonucleotidases to adenosine, which has also been shown to alter cytokine production. In the present study, we investigated how extracellular ATP affects interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha production in bacterial lipopolysaccharide (LPS)-treated murine peritoneal macrophages and we also examined whether extracellular ATP alters the production of the T helper 1 cytokine interferon (IFN)-gamma. Pretreatment of the peritoneal macrophages with ATP or various ATP analogues decreased both IL-12 and TNF-alpha production induced by LPS (10 microgram ml(-1)). The effect of ATP was partially reversed by cotreatment with adenosine deaminase (0.1 - 1 u ml(-1)), suggesting that the suppressive effect of ATP on cytokine production is, in part, due to its degradation products. Immunoneutralization with an anti-IL-10 antibody demonstrated that although ATP increases IL-10 production, the inhibition of IL-12 and TNF-alpha production is independent of the increased IL-10. The effect of ATP was pretranslational, as it suppressed steady state levels of mRNAs for IL-12 (both p35 and p40). In spleen cells stimulated with either LPS (10 microgram ml(-1)) or anti-CD3 (2 microgram ml(-1)) antibody, ATP suppressed, in a concentration-dependent manner, the production of IFN-gamma. These results suggest that extracellular ATP has multiple anti-inflammatory effects and that release of ATP into the extracellular space may play a role in blunting the overactive immune response in autoimmune diseases.
Assuntos
Trifosfato de Adenosina/farmacologia , Interleucina-12/metabolismo , Macrófagos Peritoneais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Complexo CD3/imunologia , Interferon gama/biossíntese , Interleucina-10/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , RNA/biossíntese , RNA/isolamento & purificação , Ribonucleases/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismoRESUMO
1. Intracellular calcium has been suggested to be an important mediator of the cellular response in endotoxaemia and shock. Dantrolene is an agent that interferes with intracellular calcium fluxes resulting in a decreased availability of calcium in the cytoplasm. Here we have investigated the effect of dantrolene on lipopolysaccharide (LPS)-induced production of interleukin-10 (IL-10), tumour necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) in mice and in cultured RAW 264.7 macrophages in vitro. 2. In BALB/c mice, LPS-induced plasma IL-10 levels were significantly enhanced by pretreatment with dantrolene (20 mg kg(-1), i.p.) (P < 0.005 at the 90 min time-point). On the other hand, dantrolene pretreatment suppressed circulating TNF-alpha and nitrite/nitrate (breakdown products of NO) concentrations. However, dantrolene had no effect on LPS-induced plasma interleukin-6 (IL-6) levels (67.22+/-5.51 ng ml(-1) in vehicle-pretreated mice and 62.22+/-3.66 ng ml(-1) in dantrolene-pretreated mice, n = 9). 3. Dantrolene inhibited TNF-alpha and NO production in C57BL/6 IL-10+/+ mice, as well as in their IL-10 deficient counterparts (C57BL/6 IL-10(0/0)). 4. In RAW 264.7 macrophages, dantrolene (10-300 microM) reduced IL-10, TNF-alpha, and nitrite (breakdown product of NO) production elicited by LPS (10 microg ml(-1)). Dantrolene (300 microM) did not affect the LPS-induced nuclear translocation of transcription factor nuclear factor kappaB in these cells. 5. Although LPS failed to alter the intracellullar concentration of calcium in single macrophages loaded with Fura-2, dantrolene caused a significant decrease of the basal calcium level as determined 30 min after dantrolene treatment (P < 0.005). ATP (1 mM) caused a rapid rise in intracellular calcium levels in both dantrolene-pretreated and vehicle-pretreated cells. 6. These results indicate that unlike the secretion of TNF-alpha and NO, IL-10 production is differentially regulated in vitro and in vivo. The decrease of plasma levels of the pro-inflammatory mediators TNF-alpha and NO, and increase in circulating IL-10 concentrations by dantrolene suggest that this drug might offer a new therapeutic approach in inflammatory diseases and septic shock.
Assuntos
Dantroleno/farmacologia , Interleucina-10/biossíntese , Lipopolissacarídeos/farmacologia , Óxido Nítrico/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Animais , Transporte Biológico , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Interleucina-10/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nitratos/sangue , Nitritos/sangueRESUMO
Activation of poly(ADP-ribose) synthetase (PARS, also termed polyADP-ribose polymerase or PARP) has been proposed as a major mechanism contributing to beta-cell destruction in type I diabetes. In the present study, we have investigated the role of PARS in mediating the induction of diabetes and beta-cell death in the multiple-low-dose-streptozotocin (MLDS) model of type I diabetes. Mice genetically deficient in PARS were found to be less sensitive to MLDS than wild type mice, with a lower incidence of diabetes and reduced hyperglycemia. A potent inhibitor of PARS, 5-iodo-6-amino-1,2-benzopyrone (INH(2)BP), was also found to protect mice from MLDS and prevent beta-cell loss, in a dose-dependent manner. Paradoxically, in the PARS deficient mice, the compound increased the onset of diabetes. In vitro the cytokine combination; interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma inhibited glucose-stimulated insulin secretion from isolated rat islets of Langerhans and decreased RIN-5F cell viability. The PARS inhibitor, INH(2)BP, protected both the rat islets and the beta-cell line, RIN-5F, from these cytokine-mediated effects. These protective effects were not mediated by inhibition of cytokine-induced nitric oxide formation. Inhibition of PARS by INH(2)BP was unable to protect rat islet cells from cytokine-mediated apoptosis. Cytokines, peroxynitrite and streptozotocin were all shown to induce PARS activation in RIN-5F cells, an effect suppressed by INH(2)BP. The present study provides evidence for in vivo PARS activation contributing to beta-cell damage and death in the MLDS model of diabetes, and indicates a role for PARS activation in cytokine-mediated depression of insulin secretion and cell viability in vitro.
Assuntos
Cumarínicos/farmacologia , Diabetes Mellitus Experimental/prevenção & controle , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Apoptose/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocinas/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Relação Dose-Resposta a Droga , Feminino , Genótipo , Técnicas In Vitro , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Mutação , Nitratos/farmacologia , Óxido Nítrico/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Ratos , Índice de Gravidade de Doença , Estreptozocina/administração & dosagem , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologiaRESUMO
Noradrenaline (NA) and adrenaline (Ad) are modulators of cytokine production. Here we investigated the role of these neurotransmitters in the regulation of macrophage inflammatory protein (MIP)-1alpha expression. Pretreatment of RAW 264.7 macrophages with NA or Ad decreased, in a concentration-dependent manner (1 nM-100 microM), MIP-1alpha release induced by bacterial lipopolysaccharide (LPS 10 ng ml(-1) LPS). The effect of NA was reversed by the selective beta-adrenoceptor antagonist propranolol (10 microM), but not by the alpha-adrenoceptor antagonist phentolamine (10 microM). In the concentration range of 10 nM-10 microM, isoproterenol, a beta-adrenoceptor agonist, but not phenylephrine (a selective alpha1-adrenoceptor agonist) or UK-14304 (a selective alpha2-adrenoceptor agonist) mimicked the inhibitory effects of catecholamines on MIP-1alpha production. Increases in intracellular cyclic adenosine monophosphate, elicited either by the selective type IV phosphodiesterase inhibitor rolipram (0.1 - 10 microM), or by prostaglandin E2, (10 nM-10 microM) decreased MIP-1alpha release, suggesting that increased cyclic AMP may contribute to the suppression of MIP-1alpha release by beta-adrenoceptor stimulation. Northern blot analysis demonstrated that NA (100 nM-10 microM), Ad, isoproterenol, as well as rolipram (100 nM-10 microM) decreased LPS-induced MIP-1alpha mRNA accumulation. NA and Ad (1-100 microM) also decreased MIP-1alpha production in thioglycollate-elicited murine peritoneal macrophages. Pretreatment of mice with either isoproterenol (10 mg kg(-1), i.p.) or rolipram (25 mg kg(-1), i.p.) decreased LPS-induced plasma levels of MIP-1alpha, while propranolol (10 mg kg(-1), i.p.) augmented the production of this chemokine, confirming the role of a beta-adrenoceptor mediated endogenous catecholamine action in the regulation of MIP-1alpha production in vivo. Thus, based on our data we conclude that catecholamines are important endogenous regulators of MIP-1alpha expression in inflammation.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Epinefrina/farmacologia , Epinefrina/fisiologia , Proteínas Inflamatórias de Macrófagos/biossíntese , Norepinefrina/farmacologia , Norepinefrina/fisiologia , Receptores Adrenérgicos beta/fisiologia , Animais , Formação de Anticorpos , Adesão Celular/fisiologia , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL4 , AMP Cíclico/metabolismo , AMP Cíclico/fisiologia , Isoproterenol/farmacologia , Lipopolissacarídeos/farmacologia , Proteínas Inflamatórias de Macrófagos/sangue , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Receptores Adrenérgicos beta/efeitos dos fármacos , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiologia , Tioglicolatos/farmacologiaRESUMO
1. Ligands of the various adenosine receptor subtypes modulate the production of pro-and anti-inflammatory cytokines. Here we evaluated the effect of adenosine and various ligands of the adenosine receptor subtypes (A1, A2, A3) on the chemokine macrophage inflammatory protein (MIP) 1alpha production in immunostimulated RAW macrophages in vitro. Furthermore, we studied whether a selected A3 adenosine receptor agonist inhibits MIP-1alpha production and affects the course of inflammation in collagen-induced arthritis. 2. In the cultured macrophages, the A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA), and, less potently, the A2 receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS; 1-200 micro) dose-dependently suppressed the production of MIP-1alpha. The selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 1-200 microM) was ineffective, and adenosine was a weak inhibitor. The inhibition of MIP-1alpha production by the A3 and A2 agonist was associated with suppression of its steady-state mRNA levels. 3. Based on the in vitro data, we concluded that activation of A3, and to a lesser extent A2 adenosine receptors suppresses MIP-1alpha expression. Since IB-MECA was the most potent inhibitor of MIP-1alpha expression, we next investigated whether it affects the production of other pro-inflammatory mediators. We observed that IB-MECA (1-300 microM) inhibited, in a dose-dependent manner, the production of IL-12, IL-6, and, to a lesser extent, nitric oxide in the immunostimulated cultured macrophages. 4. Since MIP-alpha is a chemokine which enhances neutrophil recruitment into inflammatory sites, we investigated whether the A3 agonist IB-MECA affects the course of inflammation, MIP-alpha production and the degree of neutrophil recruitment in arthritis. In a model of collagen-induced arthritis in mice, IB-MECA (0.5 mg/kg/day) reduced the severity of joint inflammation. IB-MECA inhibited the formation of MIP-1alpha, IL-12 and nitrotyrosine (an indicator of reactive nitrogen species) in the paws, and suppressed neutrophil infiltration. 5. We conclude that adenosine receptor agonists, most notably the A3 agonist IB-MECA suppress the production of MIP-alpha, and exert anti-inflammatory effects. Therefore, stimulation of adenosine receptor subtypes A3 and A2 may be a strategy worthy of further evaluation for the abrogation of acute or chronic inflammatory disorders.