RESUMO
Observations so far obtained in experimental autoimmune encephalomyelitis (EAE) have revealed the promising neuroprotective effects exerted by progesterone (PROG). The findings suggest that this neuroactive steroid may potentially represent a therapeutic tool for multiple sclerosis (MS). However, up to now, the efficacy of PROG has been only tested in the acute phase of the disease, whereas it is well known that MS expresses different features depending on the phase of the disease. Accordingly, we have evaluated the effect of PROG treatment in EAE induced in Dark Agouti rats (i.e. an experimental model showing a protracted relapsing EAE). Data obtained 45 days after EAE induction show that PROG treatment exerts a beneficial effect on clinical score, confirming surrogate parameters of spinal cord damage in chronic EAE (i.e. reactive microglia, cytokine levels, activity of the Na(+) ,K(+) -ATPase pump and myelin basic protein expression). An increase of the levels of dihydroprogesterone and isopregnanolone (i.e. two PROG metabolites) was also observed in the spinal cord after PROG treatment. Taken together, these results indicate that PROG is effective in reducing the severity of chronic EAE and, consequently, may have potential with respect to MS treatment.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Progesterona/farmacologia , Medula Espinal/efeitos dos fármacos , Animais , Doença Crônica , Modelos Animais de Doenças , Masculino , Progesterona/uso terapêutico , RatosRESUMO
Several European Union legislations request the use of in vitro methods for toxicological evaluations, including sensitization, in order to increase consumer safety but also to reduce the use of animals. The EU project SENS-IT-IV addresses the need of developing predictive in vitro tests to assess contact and respiratory hypersensitivity reactions. In this context, we have recently reported the possibility to use IL-18 production in the human keratinocyte cell line NCTC 2544 to discriminate contact sensitizer from irritants and low molecular weight respiratory allergens. The aims of the present study were to further develop this assay in order to optimize experimental conditions; to develop a 96-well plate format to establish a high throughput assay; to test the performance of other available keratinocyte cell lines, and to understand the signal transduction pathway involved in p-phenylenediamine (PPD)-induced IL-18 production. If cells reach confluence at the moment of treatment, the ability to identify contact allergens is lost; therefore a careful check for the optimal cell density using PPD as reference contact allergen is critical. In our hands, a cell density of 1-2.5 × 10(5)cells/ml gave optimal stimulation. In order to develop a high throughput test, cells seeded in 96-well plate were exposed to contact allergens (2,4-dinitrochlorobenzene, p-phenylenediamine, isoeugenol, cinnamaldehyde, tetramethylthiuram disulfite, resorcinol, cinnamic alcohol and eugenol), irritants (phenol, sodium laurel sulphate, lactic acid and salicylic acid) and respiratory allergens (hexachloroplatinate, diphenylmethane diisocyanate, trimellitic anhydride). A selective increase in total (intracellular plus released) IL-18 was observed 24h later in cells treated with contact allergens, whereas no changes were observed following treatment with respiratory allergens and irritants, confirming previous results obtained in a 24-well format assay. A selective induction of IL-18 was also obtained testing with PPD other keratinocyte cell lines, namely HPKII and HaCaT, with the HPKII showing the highest stimulation index. Regarding the signal transduction pathway, we could demonstrate using selective inhibitors a role for oxidative stress, NF-κB and p38 MAPK activation in PPD-induced IL-18 production. In conclusion, results obtained suggest that the production of IL-18 represents a promising endpoint for the screening of potential contact allergens. The assay can be performed in a 96-well plate format, different keratinocyte cell lines can be used, and a role for oxidative stress in contact allergen-induced IL-18 was demonstrated.
Assuntos
Alérgenos/toxicidade , Dermatite Alérgica de Contato/etiologia , Interleucina-18/metabolismo , Irritantes/toxicidade , Queratinócitos/efeitos dos fármacos , Xenobióticos/toxicidade , Alérgenos/classificação , Alternativas aos Testes com Animais , Dermatite Alérgica de Contato/imunologia , Dermatite Alérgica de Contato/metabolismo , Determinação de Ponto Final , Ensaios de Triagem em Larga Escala/métodos , Humanos , Técnicas In Vitro , Irritantes/classificação , Queratinócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Testes de Toxicidade , Xenobióticos/classificaçãoRESUMO
The local lymph node assay (LLNA) has been developed to assess skin sensitization, and based on the EC3 value, it can also be used to evaluate allergen potency. Therefore, in the development of in vitro alternatives to the LLNA assay, one should not only consider the hazard identification but also the possibility to classify allergens relatively to their potency. We have recently described a selective release of interleukin-8 (IL-8) by chemical allergens in THP-1 cell line, and identified the activation of p38 mitogen-activated protein kinase (p38 MAPK) as a common pathway. Therefore, the purpose of this study was to expand the number of chemicals tested and to investigate whether IL-8 production and p38 MAPK activation can be used to classify allergens according to their potency. THP-1 cells were exposed to the contact allergens (p-benzoquinone, 2-aminophenol, isoeugenol, diethyl maleate, citral and imidazolidinyl urea), selected according to their potency in the LLNA, and to lactic acid and propylene glycol as non-sensitizers. p38 MAPK activation was evaluated 5-15 min after treatment by FACS analysis, while IL-8 release was assed by ELISA following 24h of incubation. p38 MAPK was activated by all contact allergens, including the pro-apten isoeugenol, whereas IL-8 release was significantly increased after stimulation with all allergens tested, except for isoeugenol. The failure of isoeugenol may be due to decrease in the stability of IL-8 mRNA. Irritants exposure, as expected, failed to induce both p38 MAPK activation and IL-8 release. A significant correlation between IL-8 release and the LLNA EC(3) was found (Pearson correlation r=0.743, p=0.0036, n=12). On the contrary, the activation of p38 MAPK showed no significant correlation between LLNA data and vigor of p38 MAPK activation. Overall, data presented confirm our previous observations and reveal IL-8 as potential tool not only to identify sensitizers, with the exception of pro-haptens, but also to classify them according to their potency, while p38 MAPK activation allows the identification of all sensitizers, including pro-haptens, but was not useful for potency classification.
Assuntos
Alérgenos/toxicidade , Interleucina-8/metabolismo , Monócitos/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Alérgenos/classificação , Alternativas aos Testes com Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocinas , Relação Dose-Resposta Imunológica , Ativação Enzimática , Expressão Gênica , Humanos , Monócitos/enzimologia , Monócitos/imunologia , RNA Mensageiro/metabolismoRESUMO
Cholesterol is required in the brain for synaptogenesis and its turnover is critical for cerebral functions. Several proteins involved in cholesterol handling and metabolism are transcriptionally regulated by the nuclear liver X receptor (LXR) alpha and beta. Sterol 27-hydroxylase (CYP27) is a ubiquitously expressed enzyme involved in cholesterol metabolism. Notably, its deficiency causes a disease characterized by progressive neurologic impairment. With the final goal to understand the pathophysiological role of CYP27A1 in the CNS, we studied the expression pattern of Cyp27a1 and other related genes in primary cultures of rat glia and neurons. Secondly, given the pivotal role of LXR in the regulation of cholesterol homeostasis, we investigated the effects of its activation on the expression of Cyp27a1.We found that primary astrocytes express different sterol hydroxylases and are able to uptake exogenous 27-hydroxycholesterol. We found that both microglia and astrocytes express preferentially Lxrbeta. However, despite this similarity, we observed cell-specific responsiveness of known and novel (including Cyp27a1) target genes to LXR activation. The increase of mRNA and protein levels in treated astrocytes is paralleled by transactivation of the proximal Cyp27a1 promoter in transfected astrocytes. We suggest that the astrocyte-restricted up-regulation of Cyp27a1 may be ascribable to differential expression of transcriptional co-activators. Given the role of astrocytes in maintaining brain homeostasis, we hypothesize that impairment of CYP27 activity in these cells may alter critical features of the astrocytes, from the handling and delivery of cholesterol to neurons to the release of signaling molecules.
Assuntos
Encéfalo/metabolismo , Colestanotriol 26-Mono-Oxigenase/metabolismo , Microglia/metabolismo , Neuroglia/metabolismo , Receptores Nucleares Órfãos/metabolismo , Animais , Encéfalo/enzimologia , Células Cultivadas , Colestanotriol 26-Mono-Oxigenase/genética , Colesterol/metabolismo , Regulação da Expressão Gênica , Homeostase/genética , Hidroxicolesteróis/metabolismo , Receptores X do Fígado , Microglia/enzimologia , Neuroglia/enzimologia , Neurônios/enzimologia , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Esteroide Hidroxilases/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Skin immunosenescence accounts for increased susceptibility in the elderly to cutaneous infections and malignancies, and decreased contact hypersensitivity and response to vaccination. We have recently shown in immune cells that decreased expression of the receptor for activated C kinase (RACK)-1 underlies defective protein kinase C (PKC) activation and functional immune impairment with ageing. OBJECTIVES: This study was designed to determine if an age-related decline in skin RACK-1 expression was present and whether it correlated with defective tumour necrosis factor (TNF)-alpha production. METHODS: PKC isoforms and RACK-1 expression were evaluated by Western blot analysis and by immunofluorescence in skin obtained from Sprague-Dawley rats of different ages. TNF-alpha release by epidermal cells induced by lipopolysaccharide, 12-O-tetradecanoyl-phorbol-13-acetate and the contact allergen dinitrochlorobenzene was assessed by the L929 biological assay. RESULTS: Skin obtained from old rats (> 18 months) showed decreased RACK-1 immunoreactivity if compared with young rats (< 3 months). RACK-1 preferentially interacts with PKC beta. Despite a similar total skin content of this isoform, the reduced expression of RACK-1 was associated with a decreased translocation of PKC beta in the membrane compartment. The defective PKC beta translocation associated with ageing correlated with decreased TNF-alpha release from epidermal cells following treatment with different inflammatory stimuli. CONCLUSIONS: Overall, we demonstrated for the first time a decrease in RACK-1 expression, defective PKC beta translocation and reduced TNF-alpha release in epidermal cells with ageing. These alterations might be mechanistically significant, and provide a new understanding of the consequences of ageing on skin immunology.
Assuntos
Proteína Quinase C/metabolismo , Receptores de Superfície Celular/metabolismo , Envelhecimento da Pele/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fatores Etários , Animais , Células Cultivadas , Células Epidérmicas , Epiderme/metabolismo , Imuno-Histoquímica , Masculino , Proteína Quinase C/imunologia , Ratos , Ratos Sprague-Dawley , Receptores de Quinase C Ativada , Receptores de Superfície Celular/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Interleukin (IL)-1beta is a proinflammatory cytokine implicated in various pathophysiological conditions of the CNS involving NMDA receptor activation. Circumstantial evidence suggests that IL-1beta and NMDA receptors can functionally interact. Using primary cultures of rat hippocampal neurons, we investigated whether IL-1beta affects NMDA receptor function(s) by studying (1) NMDA receptor-induced [Ca2+]i increase and (2) NMDA-mediated neurotoxicity. IL1beta (0.01-0.1 ng/ml) dose-dependently enhances NMDA-induced [Ca2+]i increases with a maximal effect of approximately 45%. This effect occurred only when neurons were pretreated with IL-1beta, whereas it was absent if IL-1beta and NMDA were applied simultaneously, and it was abolished by IL-1 receptor antagonist (50 ng/ml). Facilitation of NMDA-induced [Ca2+]i increase by IL-1beta was prevented by both lavendustin (LAV) A (500 nm) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (1 microm), suggesting an involvement of tyrosine kinases. Increased tyrosine phosphorylation of NMDA receptor subunits 2A and 2B and coimmunoprecipitation of activated Src tyrosine kinase with these subunits was observed after exposure of hippocampal neurons to 0.05 ng/ml IL-1beta. Finally, 0.05 ng/ml IL-1beta increased by approximately 30% neuronal cell death induced by NMDA, and this effect was blocked by both lavendustin A and PP2. These data suggest that IL-1beta increases NMDA receptor function through activation of tyrosine kinases and subsequent NR2A/B subunit phosphorylation. These effects may contribute to glutamate-mediated neurodegeneration.
Assuntos
Cálcio/metabolismo , Interleucina-1/farmacologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Quinases da Família src/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteína Antagonista do Receptor de Interleucina 1 , Líquido Intracelular/metabolismo , N-Metilaspartato/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos , Sialoglicoproteínas/farmacologia , Quinases da Família src/efeitos dos fármacosRESUMO
Human immunodeficiency virus infection is often followed by neurodegeneration, the cause of motor and cognitive impairment in some patients affected by acquired immunodeficiency. Several in vitro data indicate glycoprotein (gp) 120 as one of the substances responsible for the neurodegenerative event that takes place only if non-neuronal cells (glial cells) are present. Our purpose was to investigate the molecular mechanisms through which glial cells could affect neuron viability after exposure to gp120 protein. We used a sandwich co-culture of primary hippocampal neurons and primary glial cells, where the two cell populations face each other but are separable. Exposure of 1-week-old rat hippocampal neurons in co-culture with glia to 600 pM gp120 protein resulted in the death of 30% of neurons after 6 days of treatment. A significant increase of intracellular calcium ([Ca2+]i), evident 72 h after gp120 exposure (control 45.8+/-7.6 nM, gp120 176.5+/-43.6 nM), preceded neuron death. The gp120 protein affected neither the viability nor the morphology or [Ca2+]i of glial cells. However, a significant amount of reactive oxygen species as well as of interleukin-1beta was produced. Treatment of the co-culture with an antibody against interleukin-1beta prevented neuron increase of [Ca2+]i and cell death but not glial production of reactive oxygen species, whereas prior incubation of glial cells with Trolox, an antioxidant analog of vitamin E, down-regulated interleukin-1beta expression and completely prevented neuron cell death. Our results indicate that reactive oxygen species produced in glial cells by gp120 exposure cause neurodegeneration by inducing the synthesis of interleukin-1beta.
Assuntos
Complexo AIDS Demência/metabolismo , Sistema Nervoso Central/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Degeneração Neural/metabolismo , Neuroglia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Complexo AIDS Demência/patologia , Complexo AIDS Demência/fisiopatologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Sistema Nervoso Central/fisiopatologia , Sistema Nervoso Central/virologia , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Proteína gp120 do Envelope de HIV/toxicidade , HIV-1/patogenicidade , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/virologia , Humanos , Interleucina-1/antagonistas & inibidores , Interleucina-1/metabolismo , Degeneração Neural/fisiopatologia , Degeneração Neural/virologia , Neuroglia/efeitos dos fármacos , Neuroglia/virologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/virologia , Estresse Oxidativo/fisiologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Cloricromene decreases myocardial infarct size after ischemic-reperfusion injury in vivo, and it has been suggested that this is due to inhibition of tumor necrosis factor-alpha (TNF-alpha). The purpose of this work was to characterize the mechanism of cloricromene-induced inhibition of TNF-alpha in rat macrophages. Cloricromene inhibited lipopolysaccharide-induced TNF-alpha release in a dose-dependent manner (IC(50)=5.9 +/- 0.8 microM). This was not due to cytotoxicity, as cloricromene was well tolerated up to 500 microM. Cloricromene inhibited lipopolysaccharide-induced expression of TNF-alpha mRNA, which suggests a pre-transcriptional effect. We then investigated the early signal transduction pathway triggered by lipopolysaccharide. The binding of lipopolysaccharide to its receptor CD14 activates protein kinase C and nuclear factor-kappaB (NF-kappaB). Cloricromene inhibited NF-kappaB activation in a dose-dependent manner, but affected protein kinase C translocation only slightly. We then established that cloricromene inhibited lipopolysaccharide-induced cellular oxidative activity, which is important for NF-kappaB activation. Our results show that cloricromene interferes with the early signal transduction pathway triggered by lipopolysaccharide.
Assuntos
Cromonar/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Animais , Cromonar/análogos & derivados , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Exposure of a primary culture of glial cells to the classical neurotoxicant trimethyltin (TMT) results in the release of prostaglandin (PG)E(2) and tumor necrosis factor (TNF)-alpha. Prior treatment of glial cells with either the nonspecific inhibitor of cyclooxygenase and lypoxygenase eicosatetraynoic acid (ETYA) or the cyclooxygenase inhibitor indomethacin completely prevented TMT-induced PGE(2) production and TNF-alpha release, suggesting a role for cyclooxygenase metabolites in TMT-induced TNF-alpha release. Exposure of glial cells to increasing concentrations of PGE(2) or other prostanoids did not increase TNF-alpha synthesis, while the presence of exogenous PGE(2) during treatment of glial cells with TMT actually suppressed TNF-alpha release. The activation of arachidonic acid metabolism produces reactive oxygen species (ROS). Scavenging of ROS by means of the antioxidant trolox prevented the TMT-induced release of TNF-alpha from glial cells, while indomethacin was found to suppress ROS formation induced by 1 microM TMT in glial cells. These results suggest that activation of arachidonic acid metabolism causes TNF-alpha release through the production of ROS rather than PGE(2). Indeed, PGE(2) may exert negative feedback on the release of TNF-alpha.
Assuntos
Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Compostos de Trimetilestanho/toxicidade , Fator de Necrose Tumoral alfa/metabolismo , Animais , Ácido Araquidônico/metabolismo , Doenças do Sistema Nervoso Central/induzido quimicamente , Doenças do Sistema Nervoso Central/metabolismo , Dinoprostona/biossíntese , Ativação Enzimática/efeitos dos fármacos , Feminino , Neuroglia/citologia , Gravidez , Ratos , Estimulação Química , Fator de Necrose Tumoral alfa/biossínteseRESUMO
After neuronal injury and in several neurodegenerative diseases, activated microglia secrete proinflammatory molecules that can contribute to the progressive neural damage. The recent demonstration of a protective role of estrogen in neurodegenerative disorders in humans and experimental animal models led us to investigate whether this hormone regulates the inflammatory response in the CNS. We here show that estrogen exerts an anti-inflammatory activity on primary cultures of rat microglia, as suggested by the blockage of the phenotypic conversion associated with activation and by the prevention of lipopolysaccharide-induced production of inflammatory mediators: inducible form of NO synthase (iNOS), prostaglandin-E(2) (PGE(2)), and metalloproteinase-9 (MMP-9). These effects are dose-dependent, maximal at 1 nm 17beta-estradiol, and can be blocked by the estrogen receptor (ER) antagonist ICI 182,780. The demonstration of ERalpha and ERbeta expression in microglia and macrophages and the observation of estrogen blockade of MMP-9 mRNA accumulation and MMP-9 promoter induction further support the hypothesis of a genomic activity of estrogen via intracellular receptors. This is the first report showing an anti-inflammatory activity of estrogen in microglia. Our study proposes a novel explanation for the protective effects of estrogen in neurodegenerative and inflammatory diseases and provides new molecular and cellular targets for the screening of ER ligands acting in the CNS.
Assuntos
Estradiol/análogos & derivados , Estrogênios/farmacologia , Inflamação/prevenção & controle , Lipopolissacarídeos/farmacologia , Microglia/fisiologia , Animais , Células Cultivadas , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Fulvestranto , Humanos , Inflamação/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genéticaRESUMO
Alveolar macrophages are the resident airway cells primarily responsible for the protection of the lungs against inhaled toxins and other biologically active materials. The purpose of this study was to investigate the maturation with age of alveolar macrophage functional responses. We characterised the ontogenesis of PKC betaII and its anchoring protein RACK1 in correlation with PKC-dependent immune functions, such as TNF-alpha, hydrogen peroxide production and lysozyme release in resident alveolar macrophages obtained from rats 2, 4 and 12 weeks old. Our results show an age-associated increase in the expression of PKC betaII and RACK1, which correlated with a maturation of alveolar macrophage functional responses.
Assuntos
Envelhecimento/imunologia , Isoenzimas/biossíntese , Macrófagos Alveolares/imunologia , Peptídeos/metabolismo , Proteína Quinase C/biossíntese , Animais , Diferenciação Celular , Células Cultivadas , Peróxido de Hidrogênio/metabolismo , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Masculino , Proteína Quinase C beta , Ratos , Ratos Sprague-Dawley , Receptores de Quinase C Ativada , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In searching for pharmacologic agents able to reduce xenobiotic-induced skin irritation, we found that cyclosporine A exacerbates the skin irritation induced by tributyltin. We previously demonstrated the involvement of interleukin-1 alpha and tumor necrosis factor alpha in tributyltin-induced skin irritation. Here, we show that cyclosporine A (28 mg per kg), at a dose that results in systemic immunosuppression, potentiates tributyltin-induced skin irritation through increased tumor necrosis factor alpha production, associated with increased tributyltin-induced activation of transcription factor nuclear factor kappa B in cyclosporine-A-treated mice. On the other hand, under the same experimental conditions, cyclosporine A prevented the elicitation phase of oxazolone-induced contact allergy, but was ineffective in preventing benzalkonium-chloride-induced skin irritation. Using a murine keratinocyte cell line (HEL30) we demonstrated, also in vitro, that the cyclosporine A potentiates tributyltin-induced nuclear factor kappa B activation and cytokine production, this being preceded by an increase in cellular oxidative activity, essential for nuclear factor kappa B activation, that is time and dose (0.1-10 microM) dependent. This effect was not exclusive to tributyltin but could be extended to other mitochondrial poisons such as sodium arsenate. It has been reported that cyclosporine A binds to cyclophilins. An 18-mer antisense phosphorothioate oligodeoxynucleotide was used to target mitochondrial cyclophilin D mRNA. After 24 h exposure to the oligonucleotide, the amount of cyclophilin D in the cells was decreased by 54% as judged by Western blot analysis. Cyclophilin D suppression prevented cyclosporine A potentiation of tributyltin-induced cellular oxidative activity, indicating the key role of the binding of cyclosporine A to mitochondrial cyclophilin D in mediating this effect.
Assuntos
Ciclosporina/farmacologia , Dermatite Irritante/metabolismo , Fármacos Dermatológicos/farmacologia , NF-kappa B/metabolismo , Compostos de Trialquitina/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Anti-Infecciosos Locais/farmacologia , Compostos de Benzalcônio/farmacologia , Células Cultivadas , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Ciclofilinas/metabolismo , Dermatite Alérgica de Contato/metabolismo , Dermatite Alérgica de Contato/prevenção & controle , Feminino , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Oxazolona/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) cause thymus atrophy in rodents. At low doses, antiproliferative modes of action have been shown to be involved, whereas at higher doses apoptosis seems to be the mechanism of thymotoxicity by these chemicals. In vitro, a similar concentration-dependency has been observed. The purpose of the present research was to investigate the mechanisms underlying DNA fragmentation induced by these organotin compounds in freshly isolated rat thymocytes. As previously shown for TBTC, DBTC is also able to significantly increase intracellular Ca(2+) level ([Ca(2+)](i)). The rise in [Ca(2+)](i), already evident 5 min after treatment, was followed by a dose- and time-dependent generation of reactive oxygen species (ROS) at the mitochondrial level. Simultaneously, organotins induced the release of cytochrome c from the mitochondrial membrane into the cytosol. ROS production and the release of cytochrome c were reduced by BAPTA, an intracellular Ca(2+) chelator, or rotenone, an inhibitor of the electron entry from complex I to ubiquinone, indicating the important role of Ca(2+) and mitochondria during these early intracellular events. Furthermore, we demonstrated that rotenone prevents apoptosis induced by 3 microM DBTC or TBTC and, in addition, that both BAPTA and Z-DEVD FMK (mainly a caspase-3 inhibitor) decreased apoptosis by DBTC (already shown for TBTC). Taken together these data show the apoptotic pathway followed by organotin compounds starts with an increase of [Ca(2+)](i), then continues with release of ROS and cytochrome c from mitochondria, activation of caspases, and finally results in DNA fragmentation.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Caspases/metabolismo , Mitocôndrias/metabolismo , Compostos Orgânicos de Estanho/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Western Blotting , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Mitocôndrias/efeitos dos fármacos , Oxirredução , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Compostos de Trialquitina/toxicidadeRESUMO
The close relationship between neurodegeneration and gliosis could play a relevant role in propagating the degenerative event in the brain. Although there is evidence of the neurotoxicity of activated glia, the ability of damaged neurons to modulate glial response remains unexplored. Exposure of primary glial cells to damaged or dead hippocampal neurons was followed by glial release of tumor necrosis factor-alpha (TNF-alpha). This release was reduced by a partial prevention of neural death. By contrast, no TNF-alpha was released when glial cells were exposed to damaged murine fibroblasts. Exposure of glial cells to the cerebrospinal fluid (CSF) of patients with Alzheimer's disease was also followed by TNF-alpha release, while the CSF of subjects with nondegenerative brain disorders evoked no response. These data suggest that damaged neurons both in vitro and in vivo release factor(s) that activate glial response. Heat treatment of sonicated neurons or use of a mixture of protease inhibitors, among them the caspase inhibitors Z-DEVD-FMK and Z-YVAD-FMK, prevented TNF-alpha release from glial cells. We conclude that a primary neurodegenerative event may induce glial response by releasing a neurospecific protein factor via activation of a caspase.
Assuntos
Endopeptidases/metabolismo , Gliose/enzimologia , Gliose/fisiopatologia , Degeneração Neural/enzimologia , Degeneração Neural/fisiopatologia , Neuroglia/enzimologia , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Animais Recém-Nascidos , Biomarcadores/análise , Inibidores de Caspase , Caspases/efeitos dos fármacos , Caspases/metabolismo , Extratos Celulares/farmacologia , Células Cultivadas , Proteínas do Líquido Cefalorraquidiano/metabolismo , Proteínas do Líquido Cefalorraquidiano/farmacologia , Endopeptidases/efeitos dos fármacos , Gliose/patologia , Hipocampo/citologia , Hipocampo/enzimologia , Lipopolissacarídeos/farmacologia , Degeneração Neural/patologia , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Inibidores de Proteases/farmacologia , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The recent epidemiologic studies report extremely varied rates for social phobia (SP). One of the reasons for this may be the difficulty in diagnosing SP, the boundaries of which are uncertain. A community survey was carried out using doctors with experience in clinical psychiatry as interviewers, and a clinical diagnostic instrument. Two thousand three hundred and fifty-five people (out of the 2,500 randomly selected from the population) living in Sesto Fiorentino, a suburb of Florence, Italy, were interviewed by their own general practitioner, using the MINI plus six additional questions. Six hundred and ten of the 623 subjects that were found positive for any form of psychopathology at the screening interview, and 57 negative subjects, were re-interviewed by residents in psychiatry using the Florence Psychiatric Interview (FPI). The FPI is a validated composite instrument that has the format of a structured clinical research record. It was found that 6.58% of subjects showed social anxiety not attributable to other psychiatric or medical conditions during their life. Social or occupational impairments meeting DSM-IV diagnostic requirements for SP was detected in 76 subjects (lifetime prevalence = 3.27%). Correction for age raises the lifetime expected prevalence to 4%. Sex ratio was approximately (F:M) 2:1. The most common fear was speaking in public (89.4%), followed by entering a room occupied by others (63.1%) and meeting with strangers (47.3%). Eighty-six point nine percent of subjects with SP complained of more than one fear. The mean age of onset (when the subjects first fully met DSM-IV criteria for SP) was 28.8 years, but the first symptoms of SP usually occurred much earlier, with a mean age of onset at 15.5 years. Ninety-two percent of cases with SP also showed at least one other co-morbid psychiatric disorder during their life. Lifetime prevalence of avoidant personality disorder (APD) was 3.6%. Forty-two point nine percent of cases with SP also had APD, whereas 37.9% of cases with APD developed SP.
Assuntos
Uso de Medicamentos/estatística & dados numéricos , Medicina de Família e Comunidade/estatística & dados numéricos , Transtornos Mentais/epidemiologia , Transtornos Fóbicos/tratamento farmacológico , Transtornos Fóbicos/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Idade de Início , Ansiolíticos/uso terapêutico , Antidepressivos/uso terapêutico , Comorbidade , Feminino , Humanos , Itália/epidemiologia , Masculino , Transtornos da Personalidade/epidemiologia , Transtornos Fóbicos/diagnóstico , Transtornos Fóbicos/psicologia , Vigilância da População , Prevalência , Escalas de Graduação Psiquiátrica , Estudos de Amostragem , Índice de Gravidade de Doença , Distribuição por SexoRESUMO
It has recently been demonstrated that arsenic induces overexpression of keratinocyte-derived growth factors, which are likely to have a significant role in arsenic-induced skin hyperkeratoses and cancer. The mechanism(s) involved in this induction are, however, still elusive. The purpose of this study was to investigate the early intracellular events that follow in vitro treatment with sodium arsenate in a murine keratinocyte cell line (HEL30), which leads to cytokine overproduction. First, we observed that sodium arsenate induced a concentration-dependent production of interleukin-1alpha and a significant increase in cell proliferation, that could be suppressed by the addition of a neutralizing antibody against murine interleukin-1alpha, confirming the ability of arsenic to induce keratinocyte growth-promoting cytokines. Electron microscopic analysis revealed that arsenate induced a dramatic alteration in keratinocyte mitochondria. This effect could be prevented by rotenone pretreatment, which suggests the possible involvement of mitochondria-derived reactive oxygen species. Arsenic induced a concentration- and time-dependent increase in cellular oxidative activity, which was followed by activation of redox-sensitive transcription factors such as nuclear factor-kappaB and activator protein-1, that are essential for interleukin-1alpha synthesis. Prior treatment with rotenone or prolonged treatment with ethidium bromide, an inhibitor of mitochondrial DNA and RNA synthesis, to deplete cells of functional mitochondria, completely prevented sodium arsenate-induced interleukin-1alpha production, this indicates the pivotal role of these organelles in sodium arsenate-induced keratinocyte growth factors.
Assuntos
Arseniatos/farmacologia , Interleucina-1/biossíntese , Queratinócitos/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos C3H , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Rotenona/farmacologia , Transdução de Sinais/fisiologia , Desacopladores/farmacologiaRESUMO
This study characterizes the role of glial cells in chemically induced neurodegeneration. We evaluated the effect of trimethyltin, a trisubstituted organotin compound that elicits distinct lesions in the central nervous system in vivo, on a sandwich co-culture of neurons and glia. Exposure of a 98% pure culture of rat hippocampal neurons to 0.1-1 microM trimethyltin for 24 h caused neural cell death and nuclear changes typical of apoptosis; at these doses glial cells viability was not affected but the cells released significant amounts of tumor necrosis factor-alpha (TNF-alpha). Neuronal apoptosis and TNF-alpha release from glial cells both increased when the two cell types were exposed together to trimethyltin, which indicates synergy. Treatment of a neuron-glia co-culture with TNF-alpha antibody prevented the increase in neuronal apoptosis, and TNF-alpha administration induced apoptosis in hippocampal cells. We conclude that glial cells and TNF-alpha both modulate trimethyltin-induced neurodegeneration.
Assuntos
Hipocampo/patologia , Degeneração Neural/induzido quimicamente , Neuroglia/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Comunicação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Inibidores Enzimáticos/farmacologia , Hipocampo/efeitos dos fármacos , Microglia/efeitos dos fármacos , Microglia/patologia , Degeneração Neural/patologia , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Antagonistas de Prostaglandina/farmacologia , Ratos , Ratos Sprague-Dawley , Compostos de Trimetilestanho/toxicidade , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Tributyltin (TBT) salts are potent skin irritants both in humans and rodents. Data in the literature indicate mitochondria as target of TBT effects. The purpose of this study was to investigate the early intracellular molecular events that follow TBT treatment and mitochondrial impairment and the relevance of mitochondria in gene-regulatory signalling pathways. The results obtained indicate the pivotal role of Ca(2+) as the starting event following TBT treatment and confirm the important role of mitochondria as the source of second messenger molecules essential for TBT-induced NF-kappaB activation and IL-1alpha production.
RESUMO
Tributyltin (TBT) salts are potent skin irritants both in humans and rodents. Data in the literature indicate mitochondria as target of TBT effects. Here, we investigate the early intracellular molecular events that follow TBT treatment and the relevance of calcium ions and mitochondria in gene-regulatory signaling pathways. Confluent HEL30 cells were treated with increasing doses of TBT (0-5 microM). At different times thereafter, the level of intracellular Ca2+, the cellular oxidative activity, nuclear factor-kappaB (NF-kappaB) activation, and IL-1alpha production were measured. TBT induced a dose-related increase of intracellular Ca2+ that reached the plateau 4 min following treatment. The increase of intracellular Ca2+ was followed by an increase in cellular oxidative activity as measured by DCFH oxidation (15 min) that preceded NF-kappaB activation (30 min) and IL-1alpha production (4 hr). All these events can be almost completely abrogated by BAPTA, an intracellular Ca2+ chelator. Furthermore, the modulation of cellular oxidative activity induced by TBT observed with rotenone, an inhibitor of the electron entry from complex I to ubiquinone, or after prolonged treatment with ethidium bromide, an inhibitor of mitochondrial DNA and RNA synthesis, indicates mitochondria as an important intracellular source of reactive oxygen species. These findings indicate the rise in intracellular Ca2+ as the starting event and indicate the role of mitochondria as the source of second messenger molecules essential for TBT-induced NF-kappaB activation and IL-1alpha production.