RESUMO
RATIONALE: Polysensitization of patients who are allergic is a common feature. The underlying immunologic mechanism is not clear. The maturation status of dendritic cells (DCs) is considered to be important for priming naive T cells in the draining lymph nodes. We hypothesized that chronic airway inflammation can induce an enhanced maturation of airway DCs and facilitate subsequent priming to neoallergens. OBJECTIVES: To investigate whether chronic airway inflammation could induce an altered activation of airway DCs in mice and whether this influences the development of allergic sensitization. METHODS: Balb/c mice were repeatedly challenged with DCs to induce a chronic airway inflammation. We evaluated (1) the induction of the main characteristic features of human asthma including persistent remodeling, (2) the maturation status of airway DCs 1 month after inflammation resolved, (3) whether this influences tolerance to inhaled neoallergen, and (4) what type of T helper response would be induced by DCs. MEASUREMENTS AND MAIN RESULTS: Airway DCs displayed a mature phenotype after complete resolution of airway eosinophilia. Inhalation of a neoallergen without any adjuvant was able to induce airway inflammation in postinflammation lungs but not in control lungs. One month after inflammation, airway DCs were able to induce Th2 polarization in naive T cells consistent with the up-regulation of the Th2 skewing molecules Ym1/2 and OX-40L compared with DCs of control airways. CONCLUSIONS: This study provides evidence that sustained maturation of DCs after resolution of Th2-mediated inflammation can contribute to polysensitization.
Assuntos
Alérgenos/imunologia , Células Dendríticas/imunologia , Inflamação/imunologia , Hipersensibilidade Respiratória , Administração por Inalação , Alérgenos/administração & dosagem , Alérgenos/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos BALB C , FenótipoRESUMO
Although dendritic cells (DCs) play an important role in sensitization to inhaled allergens, their function in ongoing T helper (Th)2 cell-mediated eosinophilic airway inflammation underlying bronchial asthma is currently unknown. Here, we show in an ovalbumin (OVA)-driven murine asthma model that airway DCs acquire a mature phenotype and interact with CD4(+) T cells within sites of peribronchial and perivascular inflammation. To study whether DCs contributed to inflammation, we depleted DCs from the airways of CD11c-diphtheria toxin (DT) receptor transgenic mice during the OVA aerosol challenge. Airway administration of DT depleted CD11c(+) DCs and alveolar macrophages and abolished the characteristic features of asthma, including eosinophilic inflammation, goblet cell hyperplasia, and bronchial hyperreactivity. In the absence of CD11c(+) cells, endogenous or adoptively transferred CD4(+) Th2 cells did not produce interleukin (IL)-4, IL-5, and IL-13 in response to OVA aerosol. In CD11c-depleted mice, eosinophilic inflammation and Th2 cytokine secretion were restored by adoptive transfer of CD11c(+) DCs, but not alveolar macrophages. These findings identify lung DCs as key proinflammatory cells that are necessary and sufficient for Th2 cell stimulation during ongoing airway inflammation.
Assuntos
Asma/imunologia , Antígeno CD11c/imunologia , Células Dendríticas/imunologia , Pulmão/imunologia , Células Th2/imunologia , Transferência Adotiva , Aerossóis/administração & dosagem , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Asma/induzido quimicamente , Asma/genética , Antígeno CD11c/genética , Citocinas/imunologia , Toxina Diftérica/genética , Toxina Diftérica/imunologia , Eosinófilos/imunologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Pulmão/citologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Células Th2/transplanteRESUMO
Tolerance is the usual outcome of inhalation of harmless antigen, yet T helper (Th) type 2 cell sensitization to inhaled allergens induced by dendritic cells (DCs) is common in atopic asthma. Here, we show that both myeloid (m) and plasmacytoid (p) DCs take up inhaled antigen in the lung and present it in an immunogenic or tolerogenic form to draining node T cells. Strikingly, depletion of pDCs during inhalation of normally inert antigen led to immunoglobulin E sensitization, airway eosinophilia, goblet cell hyperplasia, and Th2 cell cytokine production, cardinal features of asthma. Furthermore, adoptive transfer of pDCs before sensitization prevented disease in a mouse asthma model. On a functional level, pDCs did not induce T cell division but suppressed the generation of effector T cells induced by mDCs. These studies show that pDCs provide intrinsic protection against inflammatory responses to harmless antigen. Therapies exploiting pDC function might be clinically effective in preventing the development of asthma.
Assuntos
Antígenos/imunologia , Asma/imunologia , Células Dendríticas/imunologia , Pulmão/citologia , Hipersensibilidade Respiratória/imunologia , Administração por Inalação , Animais , Antígenos/administração & dosagem , Asma/patologia , Células Dendríticas/citologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologiaRESUMO
Inhalation of iloprost, a stable prostacyclin (PGI(2)) analog, is a well-accepted and safe treatment for pulmonary arterial hypertension. Although iloprost mainly acts as a vasodilator by binding to the I prostanoid (IP) receptor, recent evidence suggests that signaling via this receptor also has antiinflammatory effects through unclear mechanisms. Here we show in a murine model of asthma that iloprost inhalation suppressed the cardinal features of asthma when given during the priming or challenge phase. As a mechanism of action, iloprost interfered with the function of lung myeloid DCs, critical antigen-presenting cells of the airways. Iloprost treatment inhibited the maturation and migration of lung DCs to the mediastinal LNs, thereby abolishing the induction of an allergen-specific Th2 response in these nodes. The effect of iloprost was DC autonomous, as iloprost-treated DCs no longer induced Th2 differentiation from naive T cells or boosted effector cytokine production in primed Th2 cells. These data should pave the way for a clinical effectiveness study using inhaled iloprost for the treatment of asthma.
Assuntos
Antiasmáticos/administração & dosagem , Asma/tratamento farmacológico , Asma/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Iloprosta/administração & dosagem , Administração por Inalação , Animais , Asma/patologia , Diferenciação Celular , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/imunologia , Células Th2/imunologia , Células Th2/patologiaRESUMO
Mouse models of allergic asthma are increasingly used to study the immunopathology of this complex disorder. The degree and type of airway inflammation is often studied by determination of differential cell counts on cytospins of bronchoalveolar lavage fluid (BALF) cells stained with May-Grünwald Giemsa, in which the separation of eosinophils (eos) from neutrophils (neutro) and of monocytes (mono) from activated T cells can be quite problematic. In this study, we compared differential cell counts based on morphological criteria on May-Grünwald Giemsa stained cytospins with a newly developed flow cytometric method. BAL fluid cells were identified based on forward and side scatter characteristics (FSC and SSC), autofluorescence of macrophages, and simultaneous one-step staining with antibodies for T cells (CD3-Cy-Chrome), B cells (B220-Cy-Chrome), eosinophils (CCR3-PE), and dendritic cells (DCs) (MHCII-FITC, CD11c-APC). The validity of this flow cytometric determination was tested by morphological analysis of flow-sorted cellular subsets. In an animal model of ovalbumin-induced asthma, this new method correlated very well with the differential counts based on cytospins. Flow cytometric determination of the cellular composition of BAL fluid in mouse models of asthma is a rapid and easy method that can replace differential cell counts based on morphology.
Assuntos
Asma , Líquido da Lavagem Broncoalveolar/citologia , Citometria de Fluxo/métodos , Contagem de Leucócitos/métodos , Animais , Asma/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Leucócitos/classificação , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Dendritic cells are key initiators and regulators of the immune response. Dendritic cell commitment and function require orchestrated regulation of transcription. Gata1 is a transcription factor expressed in several hematopoietic lineages. However, Gata1 function has not been explored in the monocytic or dendritic cell compartment. Here, we show that Gata1 is expressed in myeloid and plasmacytoid dendritic cells and that Gata1 ablation affects the survival of dendritic cells. Furthermore, lipopolysaccharide (LPS) stimulation of dendritic cells prompts Gata1 up-regulation, which is accompanied by increased levels of BclX and Ifng. Our findings show that Gata1 is a transcriptional regulator of dendritic cell differentiation and suggest that Gata1 is involved in the dendritic cell and macrophage lineage separation.
Assuntos
Diferenciação Celular , Linhagem da Célula , Sobrevivência Celular , Células Dendríticas/citologia , Fator de Transcrição GATA1/fisiologia , Animais , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Fator de Transcrição GATA1/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Integrases/metabolismo , Interferon gama/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células Mieloides/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteína bcl-X/metabolismoRESUMO
RATIONALE: Asthma is associated with increased expression of a typical array of genes involved in immune and inflammatory responses, including those encoding the prototypic Th2 cytokines interleukin (IL) 4, IL-5, and IL-13. Most of these genes contain binding sites for activator protein-1 (AP-1) within their promoter and are therefore believed to depend on AP-1 for their expression, suggesting that this transcription factor could be of particular importance in asthma pathophysiology. OBJECTIVE: To clarify the role of AP-1 in the effector phase of pulmonary allergy. METHODS: Ovalbumin (OVA)-sensitized mice were intratracheally given decoy oligodeoxyribonucleotides (ODNs) specifically directed to AP-1 or scrambled control ODNs before challenge with aerosolized OVA. Twenty-four hours after the last OVA challenge, airway hyperresponsiveness was measured and allergic airway inflammation was evaluated quantitatively. AP-1 decoys were localized using flow cytometry and immunohistochemistry. AP-1 activity in the lung was assessed using electrophoretic mobility shift assay. MEASUREMENTS AND MAIN RESULTS: Intratracheally delivered AP-1 decoys efficiently targeted airway immune cells, thus precluding AP-1 activation on OVA challenge. Decoy-mediated local inhibition of AP-1 resulted in significant attenuation of all the pathophysiologic features of experimental asthma-namely, eosinophilic airway inflammation, airway hyperresponsiveness, mucous cell hyperplasia, production of allergen-specific immunoglobulins, and synthesis of IL-4, IL-5, and IL-13. Scrambled control ODNs had no detectable effects. CONCLUSIONS: Our results reveal a key role for AP-1 in the effector phase of pulmonary allergy and indicate that specific AP-1 inhibition in the airways may have therapeutic value in the control of established asthma.
Assuntos
Asma/fisiopatologia , Hipersensibilidade/fisiopatologia , Oligodesoxirribonucleotídeos/farmacologia , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Animais , Asma/etiologia , Asma/imunologia , Asma/patologia , Hiper-Reatividade Brônquica/etiologia , Hiper-Reatividade Brônquica/fisiopatologia , Sobrevivência Celular/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Feminino , Hipersensibilidade/complicações , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/patologia , Sistema Imunitário/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Traqueia/imunologia , Traqueia/patologia , Fator de Transcrição AP-1/antagonistas & inibidoresRESUMO
Asthma is characterized by infiltration of the airway wall with eosinophils. Although eosinophils are considered to be effector cells, recent studies have reported their ability to activate primed Th2 cells. In this study, we investigated whether eosinophils are capable of presenting Ag to unprimed T cells in draining lymph nodes (DLN) of the lung and compared this capacity with professional dendritic cells (DC). During development of eosinophilic airway inflammation in OVA-sensitized and challenged mice, CCR3(+) eosinophils accumulated in the DLN. To study their function, eosinophils were isolated from the bronchoalveolar lavage fluid of mice by sorting on CCR3(+)B220(-)CD3(-)CD11c(dim) low autofluorescent cells, avoiding contamination with other APCs, and were intratracheally injected into mice that previously received CFSE-labeled OVA TCR-transgenic T cells. Eosinophils did not induce divisions of T cells in the DLN, whereas DC induced on average 3.7 divisions in 45.7% of T cells. To circumvent the need for Ag processing or migration in vivo, eosinophils were pulsed with OVA peptide and were still not able to induce T cell priming in vitro, whereas DC induced vigorous proliferation. This lack of Ag-presenting ability was explained by the very weak expression of MHC class II on fresh eosinophils, despite expression of the costimulatory molecules CD80 and ICAM-1. This investigation does not support any role for airway eosinophils as APCs to naive T cells, despite their migration to the DLN at times of allergen exposure. DC are clearly superior in activating T cells in the DLN of the lung.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Movimento Celular/imunologia , Eosinófilos/imunologia , Pulmão/citologia , Pulmão/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Agregação Celular/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/transplante , Eosinófilos/metabolismo , Eosinófilos/transplante , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Injeções Intravenosas , Interfase/imunologia , Intubação Intratraqueal , Mediastino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos T/genética , Subpopulações de Linfócitos T/metabolismoRESUMO
BACKGROUND: Airway dendritic cells (DCs) are crucial for the generation of TH2 cells from naive T cells during sensitization and for reactivation of primed TH2 cells on allergen challenge in mouse models of asthma. It is unknown whether CD80/CD86 costimulation is necessary during both phases of the response because primed T cells rely less on costimulatory molecules compared with naive T cells. OBJECTIVE: We sought to study the contribution of CD80/CD86 costimulatory molecules on DCs during sensitization or challenge in a mouse model of asthma. METHODS: Naive BALB/c mice received an intratracheal injection of ovalbumin (OVA)-pulsed DCs obtained from the bone marrow of wild-type (WT) or CD80/CD86-/- mice and were subsequently challenged with OVA aerosol to address the role of costimulation during sensitization. OVA-sensitized mice received OVA-pulsed WT or CD80/CD86-/- DCs without OVA aerosol to address the role of costimulation during challenge. RESULTS: WT DCs induced the proliferation and effector TH2 differentiation of naive OVA-specific T cells, whereas CD80/CD86-/- DCs induced only proliferation. Not surprisingly, WT DCs but not CD80/CD86-/- DCs induced sensitization to OVA in naive mice. In contrast, in OVA-sensitized mice intratracheal injection of CD80/CD86-/- OVA-pulsed DCs led to eosinophilic airway inflammation, goblet cell hyperplasia, and effector TH2 cytokine production that was not different from that seen after injection with WT OVA-DCs, even when the inducible costimulator ICOS was blocked or cytotoxic T lymphocyte-associated antigen 4 immunoglobulin was given. CONCLUSION: CD80/CD86 costimulation on DCs is only necessary during priming of naive T cells into TH2 cells but not during restimulation of previously primed TH2 cells in the challenge phase.
Assuntos
Antígenos CD/imunologia , Asma/imunologia , Antígeno B7-1/imunologia , Células Dendríticas/imunologia , Glicoproteínas de Membrana/imunologia , Células Th2/imunologia , Animais , Antígeno B7-2 , Feminino , Pulmão , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos AnimaisRESUMO
Nitric oxide (NO) possesses antiinflammatory effects, which may be exerted via its ability to inhibit the transcription factor, NF-kappaB. A commonly proposed mode of action for inhibition of NF-kappaBbyNO involves interference with NF-kappaB binding to DNA. Because activation of inhibitory kappaB kinase (IKK), the prerequisite enzyme complex necessary to induce NF-kappaB, is subject to redox regulation, we assessed whether IKK could present a more proximal target for NO to inhibit NF-kappaB activation. We demonstrate here that S-nitrosothiols (SNO) caused a dose-dependent inhibition of the enzymatic activity of IKK, in lung epithelial cells and in Jurkat T cells, which was associated with S-nitrosylation of the IKK complex. Using biotin derivatization of SNO, we revealed that IKKbeta, the catalytic subunit required for NF-kappaB activation, was a direct target for S-nitrosylation. A mutant version of IKKbeta containing a Cys-179-to-Ala mutation was refractory to inhibition by SNO or to increases in S-nitrosylation, in contrast to wild-type IKKbeta, demonstrating that Cys-179 is the main target for attack by SNO. Importantly, inhibition of NO synthase activity in Jurkat T cells resulted in activation of IKK, in association with its denitrosylation. Moreover, NO synthase inhibition enhanced the ability of tumor necrosis factor alpha to activate IKK, illustrating the importance of endogenous NO in regulating the extent of NF-kappaB activation by cytokines. Collectively, our findings demonstrate that IKKbeta is an important target for the redox regulation of NF-kappaB by endogenous or exogenous NO, providing an additional mechanism for its antiinflammatory properties.