RESUMO
BACKGROUND: The Duffy antigen receptor for chemokines (DARC) is an atypical receptor that regulates pro-inflammatory cytokines. However, the role of DARC in asthma pathophysiology is unknown. OBJECTIVE: To determine the role of DARC in allergic airways disease in mice, and the association between DARC single nucleotide polymorphisms (SNPs) and clinical outcomes in patients with asthma. METHODS: Mice with targeted disruption of the Darc gene (Darc∆E2 ) or WT mice were challenged over 3 weeks with house dust mite (HDM) antigen. Allergic airways disease was assessed 24 hours and 7 days following the final challenge. Additionally, associations between DARC SNPs and clinical outcomes were analysed in a cohort of poorly controlled asthmatics. RESULTS: Total airway inflammation following HDM did not differ between Darc∆E2 and WT mice. At 24 hours, Darc∆E2 mice had increased airway hyperresponsiveness; however, at 7 days airway hyperresponsiveness had completely resolved in Darc∆E2 but persisted in WT mice. In poorly controlled asthmatics, DARC SNPs were associated with worse asthma control at randomization and subsequent increased risk of healthcare utilization (odds ratio 3.13(1.37-7.27), P=.0062). CONCLUSIONS AND CLINICAL RELEVANCE: Our animal model and human patient data suggest a novel role for DARC in the temporal regulation in asthma pathophysiology and symptoms.
Assuntos
Asma , Quimiocinas , Sistema do Grupo Sanguíneo Duffy , Receptores de Superfície Celular , Animais , Feminino , Humanos , Masculino , Camundongos , Antígenos de Dermatophagoides/imunologia , Asma/diagnóstico , Asma/etiologia , Asma/metabolismo , Quimiocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Sistema do Grupo Sanguíneo Duffy/genética , Sistema do Grupo Sanguíneo Duffy/metabolismo , Expressão Gênica , Loci Gênicos , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Aceitação pelo Paciente de Cuidados de Saúde , Avaliação de Resultados da Assistência ao Paciente , Fenótipo , Polimorfismo de Nucleotídeo Único , Prognóstico , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Hipersensibilidade Respiratória/diagnóstico , Hipersensibilidade Respiratória/etiologia , Hipersensibilidade Respiratória/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Inhalational antigen tolerance typically protects against the development of allergic airway disease but may be overcome to induce allergic sensitization preceding the development of asthma. OBJECTIVES: We examined in vivo whether pre-existing inhalational antigen tolerance could be overcome by activation of the transcription factor NF-κB in conducting airway epithelial cells, and used a combination of in vivo and in vitro approaches to examine the mechanisms involved. METHODS: Wild-type and transgenic mice capable of expressing constitutively active IκB kinase ß (CAIKKß) in airway epithelium were tolerized to inhaled ovalbumin. Twenty-eight days later, the transgene was transiently expressed and mice were exposed to inhaled OVA on Day 30 in an attempt to overcome inhalational tolerance. RESULTS: Following ovalbumin challenge on days 40-42, CAIKKß mice in which the transgene had been activated exhibited characteristic features of allergic airway disease, including airway eosinophilia and methacholine hyper-responsiveness. Increases in the CD103(+) and CD11b(HI) lung dendritic cell populations were present in CAIKKß mice on Day 31. Bronchoalveolar lavage from mice expressing CAIKKß mice induced CD4(+) T cells to secrete T(H)2 and T(H)17 cytokines, an effect that required IL-4 and IL-1 signalling, respectively. CAIKKß mice on Dox demonstrated increased numbers of innate lymphoid type 2 cells (ILC2) in the lung, which also exhibited elevated mRNA expression of the T(H)2-polarizing cytokine IL-4. Finally, airway epithelial NF-kB activation induced allergic sensitization in CAIKKß mice on Dox that required IL-4 and IL-1 signalling in vivo. CONCLUSIONS: Our studies demonstrate that soluble mediators generated in response to airway epithelial NF-κB activation orchestrate the breaking of inhalational tolerance and allergic antigen sensitization through the effects of soluble mediators, including IL-1 and IL-4, on pulmonary dendritic cells as well as innate lymphoid and CD4(+) T cells.
Assuntos
Antígenos/imunologia , Tolerância Imunológica , NF-kappa B/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Administração por Inalação , Alérgenos , Animais , Antígenos/administração & dosagem , Diferenciação Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Ativação Enzimática , Imunidade Inata , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Interleucina-1/metabolismo , Interleucina-4/metabolismo , Cloreto de Metacolina/efeitos adversos , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Infectious pneumonias are the leading cause of death worldwide, particularly among immunocompromised patients. Therapeutic stimulation of the lungs' intrinsic defenses with a unique combination of inhaled Toll-like receptor (TLR) agonists broadly protects mice against otherwise lethal pneumonias. As the survival benefit persists despite cytotoxic chemotherapy-related neutropenia, the cells required for protection were investigated. The inducibility of resistance was tested in mice with deficiencies of leukocyte lineages due to genetic deletions and in wild-type mice with leukocyte populations significantly reduced by antibodies or toxins. Surprisingly, these serial reductions in leukocyte lineages did not appreciably impair inducible resistance, but targeted disruption of TLR signaling in the lung epithelium resulted in complete abrogation of the protective effect. Isolated lung epithelial cells were also induced to kill pathogens in the absence of leukocytes. Proteomic and gene expression analyses of isolated epithelial cells and whole lungs revealed highly congruent antimicrobial responses. Taken together, these data indicate that lung epithelial cells are necessary and sufficient effectors of inducible resistance. These findings challenge conventional paradigms about the role of epithelia in antimicrobial defense and offer a novel potential intervention to protect patients with impaired leukocyte-mediated immunity from fatal pneumonias.
Assuntos
Células Epiteliais Alveolares/metabolismo , Resistência à Doença/imunologia , Pneumonia/imunologia , Pneumonia/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Resistência à Doença/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/metabolismo , Lipopeptídeos/metabolismo , Lipopeptídeos/farmacologia , Camundongos , Camundongos Knockout , Pneumonia/genética , Pneumonia/mortalidade , Transdução de Sinais , Receptores Toll-Like/metabolismoRESUMO
Mediators produced by the airway epithelium control the activation, recruitment, and survival of pulmonary dendritic cells (DC) that present antigen to CD4(+) T cells during the genesis and exacerbation of allergic asthma. The epithelial-derived acute phase protein, serum amyloid A (SAA), induces DC maturation and TH17 polarization. TH17 responses are associated with severe forms of allergic asthma that are poorly controlled by corticosteroids. We sought to determine whether SAA would enhance the survival of DC during serum starvation and could then contribute to the development of a glucocorticoid-resistant phenotype in CD4(+) T cells. Bone marrow-derived dendritic cells (BMDC) that were serum starved in the presence of SAA were protected from activation of caspase-3 and released less lactate dehydrogenase. In comparison with untreated serum-starved BMDC, treatment with SAA downregulated mRNA expression of the pro-apoptotic molecule Bim, increased production of the pro-survival heat shock protein 70 (HSP70), and induced secretion of pro-inflammatory cytokines. SAA-treated BMDC that were serum starved for 48 h remained capable of presenting antigen and induced OTII CD4(+) T cells to secrete IL-17A, IL-17F, IL-21, IL-22, and IFNγ in the presence of ovalbumin. IL-17A, IL-17F, IL-21, and IFNγ production occurred even when the CD4(+) T cells were treated with dexamethasone (Dex), whereas glucocorticoid treatment abolished cytokine secretion by T cells cocultured with untreated serum-starved BMDC. Measurement of Dex-responsive gene expression demonstrated CD4(+) T cells as the target of glucocorticoid hyperresponsiveness manifest as a consequence of BMDC stimulation by SAA. Finally, allergic airway disease induced by SAA and antigen inhalation was unresponsive to Dex treatment. Our results indicate that apo-SAA affects DC to both prolong their viability and increase their inflammatory potential under apoptosis-inducing conditions. These findings reveal mechanisms through which SAA enhances the CD4(+) T-cell-stimulating capacity of antigen-presenting cells that may actively participate in the pathogenicity of glucocorticoid-resistant lung disease.
Assuntos
Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/citologia , Células Dendríticas/citologia , Glucocorticoides/farmacologia , Proteína Amiloide A Sérica/farmacologia , Animais , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Células da Medula Óssea/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Caspase 3/metabolismo , Meios de Cultura Livres de Soro , Citocinas/biossíntese , Citoproteção/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/enzimologia , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistência a Medicamentos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Imunização , Inflamação/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/metabolismo , SolubilidadeRESUMO
The transcription factor, nuclear factor-kappaB (NF-kappaB) has been studied extensively due to its prominent role in the regulation of immune and inflammatory genes, apoptosis, and cell proliferation. It has been known for more that a decade that NF-kappaB is a redox-sensitive transcription factor. The contribution of redox regulation and the location of potential redox-sensitive sites within the NF-kappaB activation pathway are subject to intense debate due to many conflicting reports. Redox regulation of NF-kappaB has been extensively addressed in this journal and the reader is referred to two comprehensive reviews on the subject [1,2]. With the identification of signaling intermediates proximal to the degradation of the inhibitor, IkappaB, the number of potential redox-sensitive sites is rapidly increasing. The purpose of this review is to address recent insights into the NF-kappaB signaling cascades that are triggered by proinflammatory cytokines such as TNF-alpha and IL-1beta. In addition, the role of nitrogen monoxide (.NO) in the regulation of NF-kappaB will be reviewed. Opportunities for redox regulation that occur upstream of IkappaB-alpha degradation, as well as the potential for redox control of phosphorylation of NF-kappaB subunits, will be discussed. Redox-sensitive steps are likely to depend on the nature of the NF-kappaB activator, the type of reactive oxygen or nitrogen species involved, the selectivity of signaling pathways activated, as well as the cell type under investigation. Lastly, it is discussed how redox regulation of NF-kappaB activation is likely to involve multiple subcellular compartments.
Assuntos
Regulação da Expressão Gênica/fisiologia , NF-kappa B/fisiologia , Transdução de Sinais/fisiologia , Transcrição Gênica , Animais , Dimerização , Perfilação da Expressão Gênica , Humanos , Proteínas I-kappa B/fisiologia , Inflamação , Sistema de Sinalização das MAP Quinases/fisiologia , Modelos Biológicos , NF-kappa B/química , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/fisiologia , Oxirredução , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/fisiologiaRESUMO
Alterations in transcription factor activities in aged mice may lead to the production of many inflammatory molecules in the absence of exogenous stimulation. Splenocytes from 22-month-old female C57BL/6 mice are dysregulated in their capacity to control the inducible nitric oxide synthase gene as a result of elevations in the endogenous levels and activity of interferon (IFN)-gamma. Splenocytes from aged mice produced high levels of IFN-gamma in vitro and active STAT-1 was found in nuclear extracts from these splenocytes. Administration to aged mice of neutralizing antibodies against IFN-gamma imposed appropriate regulation over nitric oxide production by stimulated splenocytes. Reestablishment of normal redox balance following dietary supplementation of aged mice with activators of the peroxisome proliferator-activated receptor alpha or the antioxidant alpha-tocopherol (vitamin E) restored appropriate regulation over both the production of IFN-gamma and the secretion of nitric oxide.
Assuntos
Envelhecimento/metabolismo , Interferon gama/metabolismo , Óxido Nítrico Sintase/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Baço/enzimologia , Fatores de Transcrição/agonistas , Vitamina E/farmacologia , Animais , Anticorpos/imunologia , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Indução Enzimática/efeitos dos fármacos , Feminino , Interferon gama/biossíntese , Interferon gama/imunologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico/urina , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Oxirredução , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Fator de Transcrição STAT1 , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/fisiologia , Vitamina E/administração & dosagemRESUMO
In addition to their capacity to damage macromolecules, oxidants play important roles in initiation of a number of signal transduction pathways. These include phosphorylation and dephosphorylation of members of the extracellular-regulated kinase (ERK) family of the mitogen-activated protein kinase (MAPK) cascade and events leading to activation of the transcription factor nuclear factor-kappaB (NF-kappaB). These cascades are key to transcriptional upregulation of genes important for cell survival, apoptosis, proliferation, transformation, and inflammation. To complement biochemical assays, cell-imaging approaches are necessary to detect the phosphorylated proteins of these cascades and their nuclear translocation, i.e., activation in cells. Protocols for these studies are presented, and the advantages of in situ microscopy-based techniques to detect oxidant-induced signaling pathways are discussed.
Assuntos
Oxidantes/farmacologia , Proteínas/metabolismo , Transdução de Sinais , Animais , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismoRESUMO
In aged mice, the redox-regulated transcription factor nuclear factor-kappaB (NF-kappaB) becomes constitutively active in many tissues, as well as in cells of the hematopoietic system. This oxidative stress-induced activity promotes the production of a number of pro-inflammatory cytokines, which can contribute to the pathology of many disease states associated with aging. The administration to aged mice of agents capable of activating the alpha isoform of the peroxisome proliferator-activated receptor (PPARalpha) was found to restore the cellular redox balance, evidenced by a lowering of tissue lipid peroxidation, an elimination of constitutively active NF-kappaB, and a loss in spontaneous inflammatory cytokine production. Aged animals bearing a null mutation in PPARalpha failed to elicit these changes following treatment with PPARalpha activators, but remained responsive to vitamin E supplementation. Aged C57BL/6 mice were found to express reduced transcript levels of PPARalpha and the peroxisome-associated genes acyl-CoA oxidase and catalase. Supplementation of these aged mice with PPARalpha activators or with vitamin E caused elevations in these transcripts to levels seen in young animals. Our results suggest that PPARalpha and the genes under its control play a role in the evolution of oxidative stress excesses observed in aging.
Assuntos
Envelhecimento/metabolismo , Citocinas/biossíntese , NF-kappa B/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Acil-CoA Oxidase , Animais , Sequência de Bases , Catalase/metabolismo , Primers do DNA , Sulfato de Desidroepiandrosterona/farmacologia , Mediadores da Inflamação/metabolismo , Peróxidos Lipídicos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Oxirredução , Oxirredutases/metabolismo , Fenótipo , Pirimidinas/farmacologiaRESUMO
The pathophysiology of many disease states observed in aged individuals has been linked to the dysregulated production of several pleiotropic cytokines. We have demonstrated that NF-kappa B, a major transcriptional regulator of these aberrantly expressed cytokines, exists in a constitutively activated state in cells obtained from the major lymphoid organs of aged animals. Therapeutic treatment with dietary antioxidants or with agents capable of activating the peroxisome proliferator-activated receptor (PPAR)-alpha was able to correct the abnormal nuclear NF-kappa B activity, reduce lipid peroxide levels, and eliminate the dysregulated expression of cytokines and other genes under NF-kappa B control. These results suggest that abnormal activation of NF-kappa B in aging contributes to the dysregulated expression of certain pleiotropic cytokines. Effective therapeutic regimens for aging and other inflammatory disease states might benefit from the administration of antioxidants or other agents which specifically activate PPAR-alpha.
Assuntos
Envelhecimento/imunologia , Envelhecimento/metabolismo , NF-kappa B/metabolismo , Envelhecimento/genética , Animais , Antioxidantes/farmacologia , Sequência de Bases , Citocinas/genética , Primers do DNA/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Reação em Cadeia da Polimerase , Receptores Citoplasmáticos e Nucleares/metabolismo , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Interleukin (IL)-12 or IL-4 produced early in an immune response directs the differentiation of naive antigen-activated CD4+ T cells down a Th1 or Th2 pathway. The NK1.1+ subset of T cells promptly produces IL-4 following activation in vivo. We demonstrate here that NK1.1+ T cells can be directly induced to produce IL-4 in vitro when activated under serum-free culture conditions. Platelet-derived growth factor in cell culture medium was inhibitory to the production of IL-4 by NK1.1+ T cells in vitro. Lymphocytes obtained from secondary lymphoid organs of aged mice produced greater quantities of IL-4 following stimulation than lymphocytes from mature adult animals. Aged mice expressed elevated percentages of NK1.1+ T cells in their secondary lymphoid organs and peripheral blood. While this cell type was responsible for the total early IL-4 produced by lymphocytes from mature adult mice, both NK1.1+ and memory phenotype (CD44high, CD45RBlow, NK1.1-) T cells from aged donors produced IL-4 following polyclonal T cell activation.
Assuntos
Envelhecimento/imunologia , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Interleucina-4/biossíntese , Ativação Linfocitária , Proteínas/imunologia , Actinas/genética , Animais , Antígenos Ly , Antígenos de Superfície , Becaplermina , Complexo CD3/imunologia , Células Cultivadas , Feminino , Receptores de Hialuronatos/imunologia , Interleucina-2/biossíntese , Interleucina-4/genética , Lectinas Tipo C , Antígenos Comuns de Leucócito/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Baço/citologia , Baço/imunologiaRESUMO
The results from the present study demonstrate that the innate defense mechanisms which control the progressive growth of Listeria monocytogenes in normal animals in vivo are dependent upon the active catabolism of endogenous glucocorticoids by the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). When 11 beta-HSD activity was pharmacologically inhibited in vivo, host susceptibility to progressive bacterial disease was markedly increased. Depressed natural resistance following 11 beta-HSD inhibition correlated with changes in the patterns of inducible cytokines by macrophages and T cells. Similar changes were observed when normal adult animals were treated with low doses of dexamethasone prior to experimental infection with Listeria.
Assuntos
Glucocorticoides/metabolismo , Hidroxiesteroide Desidrogenases/fisiologia , Listeriose/imunologia , 11-beta-Hidroxiesteroide Desidrogenases , Animais , Citocinas/biossíntese , Suscetibilidade a Doenças , Feminino , Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Imunidade Inata/efeitos dos fármacos , Listeriose/enzimologia , Listeriose/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BLRESUMO
Tooth wear has been studied in a dentally attending population, aged 46-85 years. The level of tooth wear was recorded for 100 persons using the tooth wear index of Smith and Knight. Aetiology was assessed using a history/questionnaire/examination. Erosion/attrition were postulated in 98 persons while abrasion was present in 82. All demonstrated some tooth wear and in 6.93% of 7,822 surfaces this was defined as pathological according to the threshold levels associated with the index. 84 persons had pathological wear on some surfaces but this was predominately cervical, only 12 persons showing pathological wear on the occlusal/incisal surfaces. Occlusal contact area was measured using imprints in soft opaque wax, transmitted light and a charge-coupled linear scanning array. The array is moved at 90 degrees to its axis by a linear translator to produce an image consisting of 1,600 lines, each of 2,048 pixels. This image is converted to hard copy using a DEC DDP 11/23 computer which will also give area measurements. Occlusal contact area ranged from 3.16 to 194.40 sq mm with a mean of 59.23 sq mm. Tooth wear is a significant clinical problem in this population. Wear on the occlusal/incisal surfaces is more common in older age groups and in males but could not be related to occlusal contact area or denture status by the methods used.