RESUMO
1. The protective layer formed by intestinal epithelial cells acts as a barrier preventing the adhesion of pathogenic bacteria, aids digestion and passage of nutrients and reduces damage caused from toxins on the gastrointestinal tract. This study was conducted to investigate the effects of a yeast cell wall-based product (YCW), on broiler intestinal integrity, digestive enzyme capacity and immune function.2. A 35-d trial involving 246, one-d-of-hatch male broiler chickens was carried out at a trial facility at Agri-Food Biosciences Institute (AFBI, Belfast, UK). Birds were randomly allocated into 6 pens at day of hatch (41 birds/pen; 123 birds/group). Pens were divided into two groups: (1) basal diet and (2) basal diet that incorporated YCW at the manufacturers' recommended inclusion levels (Alltech Inc., Lexington, Kentucky, USA).3. In this study, YCW supplementation affected broiler intestinal morphology resulting in greater crypt depth, villus height and surface area, goblet cell density and mucus layer thickness and lower muscularis mucosae thickness. The digestive enzymes, maltase, sucrase and alkaline phosphatase, were significantly higher in the YCW supplemented group compared to the control. The expression levels of pro-inflammatory cytokines, IL-1ß, IL-12 and IL-18, were significantly lower as was necroptotic cell death in YCW supplemented birds.4. In conclusion, under the conditions of this study, YCW supplementation positively affected intestinal health parameters in broilers following 35-d supplementation.
Assuntos
Galinhas , Saccharomyces cerevisiae , Ração Animal/análise , Animais , Parede Celular , Dieta/veterinária , Suplementos Nutricionais , MasculinoRESUMO
Circadian rhythms are recurring near-24 hour patterns driven by an endogenous circadian timekeeping system. The master pacemaker in this system is the hypothalamic suprachiasmatic nucleus (SCN). Recently interest has been drawn to how the SCN clock responds to immune system stimulation. A major signalling component in the immune system is nuclear factor (NF)-κB. In the present study we examined the role of NF-κB in SCN function. Whilst serum shocked fibroblasts showed rhythmic nuclear localisation of p65 and p65-dependent transcription, there were no circadian changes in the SCN in expression of the NF-κB components p65, c-Rel, p-IκB or p-IKK. Chronic treatment with the NF-κB inhibitor PDTC did not impact on circadian or diurnal rhythms. Phase-shifting light pulses did not impact on SCN expression of p65, and PDTC treatment did not attenuate the behavioural or molecular response to light pulses. Peripheral treatment with lipopolysaccharide resulted in increased NF-κB component expression in the SCN. In vitro experiments with SCN slice cultures showed that treatment with NF-κB inhibitors did not markedly alter rhythmic changes in PER2::LUC expression. Further, SCN slices from nf-κb::luc mice did not show any evidence for circadian rhythms in NF-κB-mediated transcription. Experiments utilising older mice (~16 months old) showed that SCN treatment in vitro with PDTC resulted in increased amplitude of rhythmic PER2::LUC expression, and LPS treatment resulted in altered PER2::LUC rhythm acrophase. Overall, we interpret our results as providing evidence for the involvement of NF-κB in the suprachiasmatic circadian clock following immune stimulation, but not under basal conditions.
Assuntos
Relógios Circadianos/fisiologia , NF-kappa B/metabolismo , Neuroimunomodulação/fisiologia , Núcleo Supraquiasmático/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Bovinos , Técnicas de Cultura de Células , Fármacos do Sistema Nervoso Central/farmacologia , Relógios Circadianos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/fisiologia , Meios de Cultura , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , NF-kappa B/antagonistas & inibidores , Células NIH 3T3 , Neuroimunomodulação/efeitos dos fármacos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Estimulação Luminosa/métodos , Soro , Núcleo Supraquiasmático/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
Innate immunity represents the first line of defence against invading pathogens. It consists of an initial inflammatory response that recruits white blood cells to the site of infection in an effort to destroy and eliminate the pathogen. Some pathogens replicate within host cells, and cell death by apoptosis is an important effector mechanism to remove the replication niche for such microbes. However, some microbes have evolved evasive strategies to block apoptosis, and in these cases host cells may employ further countermeasures, including an inflammatory form of cell death know as necroptosis. This review aims to highlight the importance of the RIP kinase family in controlling these various defence strategies. RIP1 is initially discussed as a key component of death receptor signalling and in the context of dictating whether a cell triggers a pathway of pro-inflammatory gene expression or cell death by apoptosis. The molecular and functional interplay of RIP1 and RIP3 is described, especially with respect to mediating necroptosis and as key mediators of inflammation. The function of RIP2, with particular emphasis on its role in NOD signalling, is also explored. Special attention is given to emphasizing the physiological and pathophysiological contexts for these various functions of RIP kinases.
Assuntos
Apoptose , Morte Celular , Imunidade Inata , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Expressão Gênica , Humanos , Inflamação , Transdução de Sinais , Receptores Toll-Like/imunologiaRESUMO
Dendritic cells (DCs) are key immune sentinels linking the innate and adaptive immune systems. DCs recognise danger signals and initiate T-cell tolerance, memory and polarisation. They are critical cells in responding to a viral illness. Obese individuals have been shown to have an impaired response to vaccinations against virally mediated conditions and to have an increased susceptibility to multi-organ failure in response to viral illness. We investigated if DCs are altered in an obese cohort (mean body mass index 51.7±7.3 kg m(-2)), ultimately resulting in differential T-cell responses. Circulating DCs were found to be significantly decreased in the obese compared with the lean cohort (0.82% vs 2.53%). Following Toll-like receptor stimulation, compared with lean controls, DCs generated from the obese cohort upregulated significantly less CD83 (40% vs 17% mean fluorescence intensity), a molecule implicated in the elicitation of T-cell responses, particularly viral responses. Obese DCs produced twofold more of the immunosuppressive cytokine interleukin (IL)-10 than lean controls, and in turn stimulated fourfold more IL-4-production from allogenic naive T cells. We conclude that obesity negatively impacts the ability of DCs to mature and elicit appropriate T-cell responses to a general stimulus. This may contribute to the increased susceptibility to viral infection observed in severe obesity.
Assuntos
Antígenos CD/metabolismo , Células Dendríticas/imunologia , Imunidade Inata/genética , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Obesidade Mórbida/imunologia , Linfócitos T/imunologia , Adulto , Suscetibilidade a Doenças , Feminino , Citometria de Fluxo , Anticorpos Anti-Hepatite B/sangue , Vacinas contra Hepatite B/imunologia , Humanos , Masculino , Obesidade Mórbida/complicações , Toxoide Tetânico/imunologia , Antígeno CD83RESUMO
AIMS/HYPOTHESIS: The innate immune cells, invariant natural killer T cells (iNKT cells), are implicated in the pathogenesis of psoriasis, an inflammatory condition associated with obesity and other metabolic diseases, such as diabetes and dyslipidaemia. We observed an improvement in psoriasis severity in a patient within days of starting treatment with an incretin-mimetic, glucagon-like peptide-1 (GLP-1) receptor agonist. This was independent of change in glycaemic control. We proposed that this unexpected clinical outcome resulted from a direct effect of GLP-1 on iNKT cells. METHODS: We measured circulating and psoriatic plaque iNKT cell numbers in two patients with type 2 diabetes and psoriasis before and after commencing GLP-1 analogue therapy. In addition, we investigated the in vitro effects of GLP-1 on iNKT cells and looked for a functional GLP-1 receptor on these cells. RESULTS: The Psoriasis Area and Severity Index improved in both patients following 6 weeks of GLP-1 analogue therapy. This was associated with an alteration in iNKT cell number, with an increased number in the circulation and a decreased number in psoriatic plaques. The GLP-1 receptor was expressed on iNKT cells, and GLP-1 induced a dose-dependent inhibition of iNKT cell cytokine secretion, but not cytolytic degranulation in vitro. CONCLUSIONS/INTERPRETATION: The clinical effect observed and the direct interaction between GLP-1 and the immune system raise the possibility of therapeutic applications for GLP-1 in inflammatory conditions such as psoriasis.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células T Matadoras Naturais/metabolismo , Psoríase/complicações , Psoríase/tratamento farmacológico , Receptores de Glucagon/metabolismo , Contagem de Células , Linhagem Celular , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/efeitos adversos , Peptídeo 1 Semelhante ao Glucagon/agonistas , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Fatores Imunológicos/efeitos adversos , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Liraglutida , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Obesidade/complicações , Psoríase/imunologia , Psoríase/metabolismo , RNA Mensageiro/metabolismo , Receptores de Glucagon/agonistas , Receptores de Glucagon/genética , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologiaRESUMO
Pro-inflammatory cytokines are known to be elevated in several neuropathological states that are associated with learning and memory. We have previously demonstrated in our laboratory that the inhibition of long-term potentiation (LTP) in the dentate gyrus region of the rat hippocampus, by tumor necrosis factor (TNF)-alpha, represents a biphasic response, an early phase dependent on p38 mitogen activated protein kinase (MAPK) activation and a later phase, possible dependent on protein synthesis. Many of the factors involved in the early modulation of LTP by TNF-alpha have yet to be elucidated. This study investigated if metabotropic glutamate receptors (mGluRs) are functionally linked to the inhibitory effect of TNF-alpha on LTP in the rat dentate gyrus in vitro. We report that the impairment of early-LTP by TNF-alpha is significantly attenuated by prior application of the group I/II mGluR antagonist MCPG and more specifically the mGluR5 antagonist MPEP. Since TNF-alpha is now known to cause transient increases in intracellular Ca(2+) levels from ryanodine-sensitive stores, we explored the possibility that disruption of intracellular Ca(2+) homeostasis could be involved. Ryanodine was found to significantly reverse the inhibition of LTP by TNF-alpha. From these studies we propose that the TNF-alpha inhibition of LTP is dependent upon the activation of TNFR1 and mGlu5-receptors. Importantly this study provides the first proof of the involvement of ryanodine-sensitive intracellular Ca(2+) stores in TNF-alpha mediated inhibition of LTP.
Assuntos
Potenciação de Longa Duração/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Giro Denteado/efeitos dos fármacos , Giro Denteado/fisiologia , Giro Denteado/efeitos da radiação , Interações Medicamentosas , Estimulação Elétrica/métodos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Técnicas In Vitro , Potenciação de Longa Duração/efeitos da radiação , Masculino , Modelos Biológicos , Ratos , Rianodina/farmacologia , Fatores de TempoRESUMO
The anti-inflammatory/immunoparalytic phase of the systemic inflammatory response syndrome (SIRS) following major insult (surgery, thermal/traumatic injury) is of major clinical importance in the neonate, during which the risk of infection is particularly great. Here, the mechanisms by which TNF-alpha production is suppressed in response to infection are largely unknown. We questioned whether TNF-alpha itself could be a critical mediator of this suppression. Monocytes, isolated from cord blood (n=3), were treated with LPS (100 ng/ml), TNF-alpha (10 ng/ml, +/- anti-TNF-alpha antibody) for 18 and 36 h. Cells were then restimulated with LPS (Gram -ve) or Pam-3-Cys (Gram +ve) for 24 h. This was also done in the presence of selective inhibitors of MAP kinases p38, MEK and JNK. TNF-alpha, IL-6, IL-10 and IL-8 were quantified by ELISA CD86 and HLA-DR expression were determined flow cytometrically. Cells stimulated with LPS for 24 h produced TNF-alpha (282 pg/ml), IL-10 (1,236 pg/ml), IL-6 (2,694 pg/ml) and IL-8 (2,144 pg/ml). In cells pre-exposed to TNF-alpha for 36 h, there was a significant suppression in TNF-alpha and IL-6 levels (9 and 221 pg/ml, respectively) (P<0.05) with minimal impact on IL-10 (1,206 pg/ml) and IL-8 levels (1,886 pg/ml). A similar effect was seen with Pam-3-Cys with a tenfold decrease in levels of TNF-alpha and IL-6 (86-->8.5 pg/ml and 458-->46 pg/ml, respectively) with no effect on IL-10 and IL-8 levels. Anti-TNF-alpha antibody negated this effect. Inhibition of p38 kinase reversed the TNF-alpha effect. Inhibition of the JNK and MEK kinases had no effect. A reduction in the expression of CD86 and HLA-DR was observed. This ex-vivo model of non-septic SIRS demonstrates that TNF-alpha, released during a major insult, can suppress subsequent monocyte responses to bacterial agents through p38 MAP kinase, making it a potential therapeutic target.
Assuntos
Tolerância Imunológica/imunologia , Monócitos/imunologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Fator de Necrose Tumoral alfa/imunologia , Citocinas/sangue , Citocinas/imunologia , Sangue Fetal , Humanos , Técnicas In Vitro , Recém-Nascido , Lipopolissacarídeos , Lipoproteínas , Estatísticas não Paramétricas , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
The pro-inflammatory cytokine tumor-necrosis factor-alpha (TNF-alpha) is elevated in several neuropathological states that are associated with learning and memory deficits. Previous work has reported that TNF-alpha inhibits the induction of LTP in areas CA1 [Neurosci Lett 146 (1992) 176] and dentate gyrus [Neurosci Lett 203 (1996) 17]. The mechanism(s) underlying this process of inhibition have not to date been addressed. Here, we show that perfusion of TNF-alpha prior to long-term potentiation (LTP) inducing stimuli inhibited LTP, and that in late-LTP (3 h post-tetanus) a depression in synaptic field recordings was observed (68 +/- 5%, n = 6 versus control 175 +/- 7%, n = 6, P < 0.001). We investigated the involvement of the mitogen-activated protein kinase (MAPK) p38 in the inhibition of LTP by TNF-alpha as p38 MAPK has previously been shown to be involved in interleukin-1beta inhibition of LTP in the dentate gyrus [Neuroscience 93 (1999b) 57]. Perfusion of TNF-alpha led to an increase in the levels of phosphorylated p38 MAPK detectable in the granule cells of the dentate gyrus. The p38 MAPK inhibitor SB 203580 (1 microM) was found by itself to have no significant effect on either early or late phase LTP in the dentate gyrus. SB 203580 was found to significantly reverse the inhibition of early LTP by TNF-alpha (SB/TNF-alpha 174 +/- 5%, n = 6 versus TNF-alpha 120 +/- 7%, n = 6, P < 0.001, 1 h post-tetanus) to values comparable to control LTP (control 175 +/- 7%, n = 6). Interestingly however, the depressive effects of TNF-alpha on late LTP (2-3 h) were clearly not attenuated by p38 MAPK inhibition (SB/TNF-alpha 132 +/- 5%, n = 6 versus control LTP 175 +/- 7%, n = 6, P < 0.001, 3 h post-tetanus). This work suggests that TNF-alpha inhibition of LTP represents a biphasic response, a p38 MAPK-dependent phase that coincides with the early phase of LTP and a p38 MAPK independent phase that temporally maps to late LTP.
Assuntos
Hipocampo/citologia , Potenciação de Longa Duração/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Interações Medicamentosas , Estimulação Elétrica/métodos , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Hipocampo/metabolismo , Imidazóis/farmacologia , Imuno-Histoquímica/métodos , Técnicas In Vitro , Potenciação de Longa Duração/fisiologia , Potenciação de Longa Duração/efeitos da radiação , Masculino , Piridinas/farmacologia , Ratos , Ratos Wistar , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/efeitos da radiação , Fatores de Tempo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Toll-like receptors (TLRs) play crucial roles in the induction of innate immune responses by recognising pathogen-associated molecular patterns. The engagement of TLRs by pathogens results in induction of co-stimulatory molecules that facilitate a specific immune response and also in the induction of pro-inflammatory proteins that will promote the elimination of pathogens from the body. TLRs employ many of the same signalling components as the type I interleukin (IL)-1 receptor (IL-1R). This is hardly surprising since the intracellular regions of TLRs and the IL-1R share a conserved Toll/IL-1R homology domain (TIR) that allows the receptors to recruit the intracellular TIR-containing adaptor protein Myd88. The latter then activates IL-1R-associated kinases that in turn recruit well-characterised downstream effectors culminating in activation of MAP kinases and transcription factors such as NFkappaB and AP-1. Since glucocorticoids are known to target the latter transcription factors and the MAP kinase cascades, this commentary highlights the likely crucial importance of Toll-like receptor signalling pathways as key targets for mediating the anti-inflammatory and immunosuppressive effects of steroids.
Assuntos
Anti-Inflamatórios/farmacologia , Glucocorticoides/farmacologia , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores Toll-LikeRESUMO
IgE switching requires the prior induction of C epsilon germline transcripts which is mediated by the concerted binding of STAT-6 and NF kappa B to the C epsilon promoter. These transcription factors are regulated by IL-4 and CD40, respectively. However the latter can effect other signaling pathways and the present study explores the role of p38 MAPK in induction of C epsilon germline transcripts. CD40 and IL-4, both alone and in synergy, were initially shown to activate the C epsilon promoter in a B cell lymphoma cell line. Under the same conditions CD40 caused activation of p38 MAPK, whereas IL-4 was ineffective. The p38 MAPK inhibitor, SB203580, and a dominant negative form of p38 MAPK decreased the CD40 activation of the C epsilon promoter by reducing the ability of CD40 to increase the transactivation potential of NF kappa B. This study suggests that p38 MAPK is crucially important in mediating CD40 activation of NF kappa B which acts to induce C epsilon germline transcripts, ultimately facilitating IgE switching.
Assuntos
Antígenos CD40/metabolismo , Imunoglobulina E/metabolismo , Isotipos de Imunoglobulinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Linhagem Celular , Genes Reporter , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Imunoglobulina E/genética , Isotipos de Imunoglobulinas/genética , Região de Troca de Imunoglobulinas , Luciferases/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição STAT6 , Transativadores/metabolismo , Ativação Transcricional , Transfecção , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
BACKGROUND: Type 2 T-helper cells (Th2) are involved in the regulation of the humoral immune response against antigens and allergens and directly affect which isotype will be produced. The mechanism that regulates antigen-specific IgE secretion and immune deviation is still not known. OBJECTIVES: To delineate mechanisms behind antigen-specific IgE secretion we have used in vitro immunization and focused on T-cell phenotype and the activation status of the transcription factor NFkappaB. METHODS: Peripheral blood lymphocytes (PBMC) from seronegative donors were immunized in vitro with a peptide consisting of both a T-cell and a B-cell epitope. RESULTS: Antigen-specific IgE antibodies could be detected after a primary immunization, during which T-helper cells secreted type 2 cytokines. Specific IgE was also detected in the secondary immunization, but due to a rapid polarization from Th2 to Th1 phenotype, exogenous IL-4 was required for the specific IgE secretion. Analysis of NFkappaB activation in B and T cells during primary and secondary immunization showed that NFkappaB could be detected in both B and T cells during primary immunization, but was dependent on exogenous IL-4 in the secondary immunization. CONCLUSION: This is the first evidence of antigen-specific IgE induction in vitro using naive B cells, demonstrating the involvement of T-helper cell phenotype and NFkappaB and demonstrates the usefulness of in vitro cultures to study the effect of antigens on human immunocytes.
Assuntos
Antígenos/imunologia , Imunoglobulina E/biossíntese , Peptídeos/imunologia , Células Th2/imunologia , Sequência de Aminoácidos , Antígenos/química , Linfócitos B/imunologia , Polaridade Celular , Células Cultivadas , Epitopos , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Imunização , Imunização Secundária , Imunoglobulina E/sangue , Interleucina-4/metabolismo , Leucócitos Mononucleares/imunologia , Dados de Sequência Molecular , NF-kappa B/metabolismo , Peptídeos/química , Linfócitos T/imunologia , Toxoide Tetânico/química , Toxoide Tetânico/imunologia , Células Th1/imunologiaRESUMO
The process of IgE switching requires the prior transcription of the unrearranged Cepsilon gene, which leads to its recombination with the VDJ region. The activation of NF-kappaB by CD40 is a key process in facilitating this transcription by promoting the activation of the Cepsilon promoter. The present study explores the uncharacterized signalling pathways employed by CD40 in activating NF-kappaB by the overexpression of genes encoding wild-type and dominant-negative forms of the signalling components tumour-necrosis-factor-receptor-associated factor 6 (TRAF-6), NF-kappaB-inducing kinase (NIK), IkappaB kinase (IKK)-1 and IKK-2 in the BJAB B-cell line. The overexpression of TRAF-6 or NIK was sufficient to activate NF-kappaB and the Cepsilon promoter, whereas their dominant-negative counterparts decreased the ability of CD40 to activate NF-kappaB and the Cepsilon promoter. The overexpression of wild-type IKK-1 or IKK-2 seemed to cause toxic effects on the cells, whereas the dominant-negative forms were selective in their blockade of NF-kappaB and the Cepsilon promoter. These results suggest that CD40 employs TRAF-6, which presumably recruits NIK, which in turn employs IKK-1/IKK-2 to activate NF-kappaB and the Cepsilon promoter, the prologue to IgE switching. Thus the findings define a crucially important pathway in the generation of allergic states.
Assuntos
Antígenos CD40/metabolismo , Proteínas de Transporte/metabolismo , Proteínas I-kappa B/metabolismo , Switching de Imunoglobulina , Imunoglobulina E/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Humanos , Quinase Induzida por NF-kappaBRESUMO
Nuclear factor-kappaB (NF-kappaB) is an inducible transcription factor central in the regulation of expression of a wide variety of genes and synthesis of several proteins involved in the generation of the immune response and inflammatory processes. In resting cells, NF-kappaB is maintained in an inactive state through cytoplasmic retention by IkappaB inhibitors. Stimulation of cells with a wide variety of inducers results in proteolytic degradation of these IkappaB proteins, leading to activation of NF-kappaB. The present study shows that interleukin-1 (IL-1) causes persistent activation of NF-kappaB in glial cells. Stimulation with IL-1 also causes rapid but transient degradation of IkappaB-alpha and IkappaB-epsilon. However, NF-kappaB remains active even after these IkappaB isoforms have returned to control levels. In contrast, the IkappaB-beta isoform fails to reappear following its initial degradation by IL-1, coincident with sustained activation of NF-kappaB. In addition, in vivo overexpression of the various IkappaB isoforms revealed that IkappaB-beta is the only isoform that has the ability to inhibit IL-1-induced NF-kappaB-driven transcription. The findings also suggest that the inability of IkappaB-alpha and IkappaB-epsilon to modulate NF-kappaB activity is due to their modification in vivo. These findings indicate that IkappaB-beta is the key regulator of the activity of NF-kappaB in human glial cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , NF-kappa B/metabolismo , Neuroglia/metabolismo , Sequência de Bases , Primers do DNA , Proteínas de Ligação a DNA/biossíntese , Humanos , Hidrólise , Interleucina-1/farmacologia , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Ativação Transcricional , Células Tumorais CultivadasRESUMO
Glucocorticoids are potent antiinflammatory drugs. They inhibit the expression of proinflammatory cytokines and adhesion molecules. It has recently been proposed that the underlying basis to such inhibition is the induction of the protein I kappa B, which inhibits the transcription factor NF-kappa B. The latter is a key activator of the genes encoding cytokines and adhesion molecules. The present study shows that the synthetic glucocorticoid, dexamethasone, inhibits the induction of the proinflammatory cytokine IL-8 and the adhesion molecules VCAM-1 and ICAM-1 in human 1321N1 astrocytoma and SK.N.SH neuroblastoma cells. However, dexamethasone failed to induce I kappa B or inhibit activation of NF-kappa B by IL-1 in the two cell types. EMSA confirmed the identity of the activated NF-kappa B by demonstrating that an oligonucleotide, containing the wild-type NF-kappa B-binding motif, inhibited formation of the NF-kappa B-DNA complexes whereas a mutated form of the NF-kappa B-binding motif was ineffective. In addition, supershift analysis showed that the protein subunits p50 and p65 were prevalent components in the activated NF-kappa B complexes. The lack of effect of dexamethasone on the capacity of IL-1 to activate NF-kappa B correlated with its inability to induce I kappa B and the ability of IL-1 to cause degradation of I kappa B, even in the presence of dexamethasone. The results presented in this paper strongly suggest that glucocorticoids may exert antiinflammatory effects in cells of neural origin by a mechanism(s) independent of NF-kappa B.
Assuntos
Anti-Inflamatórios/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Dexametasona/farmacologia , NF-kappa B/fisiologia , Astrocitoma/genética , Astrocitoma/metabolismo , Encéfalo/citologia , Genes Reporter/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-1/antagonistas & inibidores , Interleucina-1/fisiologia , Interleucina-8/antagonistas & inibidores , Interleucina-8/biossíntese , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Neuroblastoma/metabolismo , Células Tumorais Cultivadas , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
It has been proposed that reactive oxygen species, and in particular H2O2, may be involved in the activation of NF-kappaB by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-kappaB activation by interleukin-1 and tumor necrosis factor as well as the ability of H2O2 to activate NF-kappaB in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-kappaB and stimulation of IkappaBalpha degradation by H2O2 was only evident in the transformed cells and required much longer contact times than that observed with interleukin-1 or tumor necrosis factor. Furthermore, only H2O2 was sensitive to N-acetyl-L-cysteine, and no increase in H2O2 was detected in response to either cytokine. Pyrrolidine dithiocarbamate has been purported to be a specific antioxidant inhibitor of NF-kappaB that acts independently of activating agent or cell type. However, we found that tumor necrosis factor- but not interleukin-1-driven NF-kappaB activation and IkappaBalpha degradation were sensitive to pyrrolidine dithiocarbamate in transformed cells, while neither pathway was inhibited in primary cells. Phorbol ester-mediated activation was sensitive in both transformed and primary cells. Other antioxidants failed to inhibit either cytokine, while the iron chelators desferrioxamine and 2,2,6, 6-tetramethylpiperidine-1-oxyl mimicked the pattern of inhibition seen for the dithiocarbamate. This suggested that pyrrolidine dithiocarbamate was inhibiting NF-kappaB activation in endothelial cells primarily through its iron-chelating properties. Tumor necrosis factor, but not interleukin-1, was found to induce lipid peroxidation in ECV304 cells. This was inhibited by pyrrolidine dithiocarbamate and desferrioxamine. t-Butyl hydroperoxide, which induces lipid peroxidation, activated NF-kappaB. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-kappaB activation by tumor necrosis factor but not interleukin-1. Taken together, the results argue against a role for H2O2 in NF-kappaB activation by cytokines in endothelial cells. Furthermore, tumor necrosis factor and interleukin-1 activate NF-kappaB through different mechanisms in ECV304 cells, with the tumor necrosis factor pathway involving iron-catalyzed lipid peroxidation.
Assuntos
Endotélio Vascular/metabolismo , Peróxido de Hidrogênio/metabolismo , Interleucina-1/farmacologia , Peroxidação de Lipídeos , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Antioxidantes/farmacologia , Linhagem Celular Transformada , Óxidos N-Cíclicos/farmacologia , Desferroxamina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Células Jurkat , Cinética , NF-kappa B/antagonistas & inibidores , Pirrolidinas/farmacologia , Sideróforos/farmacologia , Marcadores de Spin , Acetato de Tetradecanoilforbol/farmacologia , Tiocarbamatos/farmacologiaRESUMO
Lindane, the gamma-isomer of hexachlorocyclohexane (HCH), and two other HCH-isomers, alpha and delta-HCH, inhibit steroidogenesis in a Y1 adrenocortical cell line. In determining the mechanism by which HCH isomers inhibit steroidogenesis, they were found not to act directly on the mitochondrial Cyt P-450scc enzyme, making likely that they act, instead, on intramitochondrial transport of cholesterol. gamma-HCH, but not alpha or delta-HCH, is a potent and selective inhibitor of ligand binding to the peripheral-type benzodiazepine binding site (PBBS) which, in turn, is reported to regulate the rate-limiting step in steroidogenesis. Although these results demonstrate that alpha and delta-HCH do not inhibit steroid production through the PBBS, the possibility that the interaction of gamma-HCH with the PBBS is responsible for its inhibitory effect on steroid ogenesis could not be excluded.
Assuntos
Glândulas Suprarrenais/efeitos dos fármacos , Hexaclorocicloexano/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Camundongos , Pregnenolona/metabolismo , Ensaio Radioligante , Receptores de GABA-A/efeitos dos fármacosAssuntos
Endotélio Vascular/metabolismo , Interleucina-1/farmacologia , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Antioxidantes/farmacologia , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Endotélio Vascular/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Prolina/análogos & derivados , Prolina/farmacologia , Espécies Reativas de Oxigênio , Acetato de Tetradecanoilforbol/farmacologia , Tiocarbamatos/farmacologiaAssuntos
Endotélio Vascular/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-1/farmacologia , NF-kappa B/metabolismo , Antioxidantes/farmacologia , Linhagem Celular , Endotélio Vascular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Modelos Biológicos , Pirrolidinas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tiocarbamatos/farmacologiaRESUMO
The effect of pretreatment with PK 11195, a ligand of the 'peripheral-type' benzodiazepine receptor (PBR), on convulsions induced by lindane (gamma-hexachlorocyclohexane, gamma-HCH) in rats was examined, to determine whether the mechanism of this convulsant activity may be mediated through the PBR. PK 11195 elicited a protective effect against the convulsant activity of orally administered lindane. It reduced the frequency of animals exhibiting convulsions and delayed the time to onset of these seizures. The concentration of lindane in the brain was found to be significantly lower in PK 11195 pretreated rats and a high correlation between blood and brain lindane concentrations was obtained. When similar experiments were repeated with alpha-HCH, a non-convulsant isomer of HCH, brain and blood concentrations were again found to be significantly reduced in PK 11195 pretreated animals. We conclude that the 'anticonvulsant' action of PK 11195 was not due to an interaction of PK 11195 and lindane on common CNS target sites, but by an action of PK 11195 on the gastrointestinal tract of the animal, delaying the absorption of lindane into the bloodstream.