RESUMO
Both 2,4-toluene diisocyanate (TDI) and 4,4-methylene diphenyl diisocyanate (MDI) can cause occupational asthma. In this study, we optimized our mouse model of chemical-induced asthma in the C57Bl/6 mice strain using the model agent TDI. Furthermore, we validated MDI in this mouse model and investigated whether cross-reactivity between TDI and MDI is present. On days 1 and 8, C57Bl/6 mice were dermally treated (20 µl/ear) with 3 % MDI, 2 % TDI or the vehicle acetone olive oil (AOO) (3:2). On day 15, they received a single oropharyngeal challenge with 0.04 % MDI, 0.01 % TDI or the vehicle AOO (4:1). One day later, airway hyperreactivity (AHR) and pulmonary inflammation in the bronchoalveolar lavage (BAL) were assessed. Furthermore, total serum IgE levels, lymphocyte subpopulations in auricular lymph nodes and cytokine levels in supernatants of lymphocytes were measured. Both dermal sensitization with TDI or MDI resulted in increased total serum IgE levels along with T and B cell proliferation in the auricular lymph nodes. The auricular lymphocytes showed an increased release of both Th2 and Th1 cytokines. Mice sensitized and challenged with either TDI or MDI showed AHR, along with a predominant neutrophil lung inflammation. Mice sensitized with MDI and challenged with TDI or the other way around showed no AHR, nor BAL inflammation. Both TDI and MDI are able to induce an asthma-like response in this mouse model. However, cross-reactivity between both diisocyanates remained absent.
Assuntos
Poluentes Ocupacionais do Ar/toxicidade , Asma/induzido quimicamente , Isocianatos/toxicidade , Tolueno 2,4-Di-Isocianato/toxicidade , Poluentes Ocupacionais do Ar/imunologia , Animais , Asma/sangue , Asma/imunologia , Reações Cruzadas/efeitos dos fármacos , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Imunoglobulina E/sangue , Isocianatos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Equilíbrio Th1-Th2/efeitos dos fármacos , Tolueno 2,4-Di-Isocianato/imunologiaRESUMO
BACKGROUND: Effective treatments for fibrotic diseases such as idiopathic pulmonary fibrosis are largely lacking. Transforming growth factor beta (TGFß) plays a central role in the pathophysiology of fibrosis. We hypothesized that bone morphogenetic proteins (BMP), another family within the TGFß superfamily of growth factors, modulate fibrogenesis driven by TGFß. We therefore studied the role of endogenous BMP signaling in bleomycin induced lung fibrosis. METHODS: Lung fibrosis was induced in wild-type or noggin haploinsufficient (Nog +/LacZ ) mice by intratracheal instillation of bleomycin, or phosphate buffered saline as a control. Invasive pulmonary function tests were performed using the flexiVent® SCIREQ system. The mice were sacrificed and lung tissue was collected for analysis using histopathology, collagen quantification, immunohistochemistry and gene expression analysis. RESULTS: Nog +/LacZ mice are a known model of increased BMP signaling and were partially protected from bleomycin-induced lung fibrosis with reduced Ashcroft score, reduced collagen content and preservation of pulmonary compliance. In bleomycin-induced lung fibrosis, TGFß and BMP signaling followed an inverse course, with dynamic activation of TGFß signaling and repression of BMP signaling activity. CONCLUSIONS: Upon bleomycin exposure, active BMP signaling is decreased. Derepression of BMP signaling in Nog +/LacZ mice protects against bleomycin-induced pulmonary fibrosis. Modulating the balance between BMP and TGFß, in particular increasing endogenous BMP signals, may therefore be a therapeutic target in fibrotic lung disease.
Assuntos
Bleomicina , Proteínas Morfogenéticas Ósseas/metabolismo , Pulmão/metabolismo , Fibrose Pulmonar/prevenção & controle , Animais , Apoptose , Proteínas de Transporte/genética , Proliferação de Células , Colágeno/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Haploinsuficiência , Pulmão/patologia , Pulmão/fisiopatologia , Complacência Pulmonar , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/fisiopatologia , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
RATIONALE: Airway hyperreactivity (AHR) is a key feature of bronchial asthma, and inhalation of irritants may facilitate development of nonallergic AHR. Swimmers exposed to hypochlorite (ClO(-))-containing water show a higher risk of developing AHR. We developed a mouse model in which instillation of ClO(-) before ovalbumin (OVA) induces AHR without bronchial inflammatory cells. OBJECTIVES: To investigate the mechanisms of ClO(-)-OVA-induced nonallergic AHR. METHODS: The involvement of the transient receptor potential ankyrin (TRPA)1 channel was checked in vivo by the use of TRPA1(-/-) mice and in vitro by Ca(2+) imaging experiments. The role of substance P (SP) was investigated by pretreating animals with the receptor antagonist RP67580, by replacing ClO(-) with SP in vivo, and by immunofluorescent staining of large airways of exposed mice. The role of mast cells was evaluated by exposing mast cell-deficient Kit(Wh)/Kit(Wsh) mice to ClO(-)-OVA with or without mast cell reconstitution. MEASUREMENTS AND MAIN RESULTS: ClO(-)-OVA did not induce AHR in TRPA1(-/-) mice, and ClO(-) generates a Ca(2+) influx in TRPA1-transfected cells. Pretreatment with RP67580 reduces ClO(-)-OVA-induced AHR, although no increased SP expression was shown in the airways. SP-OVA exposure resulted in the same AHR as induced by ClO(-)-OVA. Kit(Wsh)/Kit(Wsh) mice did not develop AHR in response to ClO(-)-OVA unless they were reconstituted with bone marrow-derived mast cells. CONCLUSIONS: Induction of AHR by exposure to ClO(-)-OVA depends on a neuroimmune interaction that involves TRPA1-dependent stimulation of sensory neurons and mast cell activation.
Assuntos
Hiper-Reatividade Brônquica/fisiopatologia , Ácido Hipocloroso/efeitos adversos , Irritantes/efeitos adversos , Mastócitos/imunologia , Canais de Potencial de Receptor Transitório/imunologia , Animais , Hiper-Reatividade Brônquica/etiologia , Células Cultivadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neuroimunomodulação , Nociceptores/imunologia , Ovalbumina/efeitos adversos , Substância P/metabolismo , Canal de Cátion TRPA1RESUMO
Some reactive chemicals, such as diisocyanates, are capable of initiating an allergic response, which can lead to occupational asthma after a latency period. Clinical symptoms such as cough, wheezing, and dyspnea occur only late, making it difficult to intervene at an early stage. So far, most studies using proteomics in lung research have focused on comparisons of healthy versus diseased subjects. Here, using 2D-DIGE, we explored proteome changes in the local draining lymph nodes and serum of mice dermally sensitized once or twice with toluene-2,4-diisocyanate (TDI) before asthma is induced. In the lymph nodes, we found 38 and 58 differentially expressed proteins after one and two treatments, respectively, between TDI-treated and vehicle-treated mice. In serum, seven and 16 differentially expressed proteins were detected after one and two treatments, respectively. We identified 80-85% of the differentially expressed proteins by MS. Among them, lymphocyte-specific protein-1, coronin 1a, and hemopexin were verified by Western blotting or ELISA in an independent group of mice. This study revealed alterations in the proteomes early during sensitization in a mouse model before the onset of chemical-induced asthma. If validated in humans, these changes could lead to earlier diagnosis of TDI-exposed workers.
Assuntos
Asma/induzido quimicamente , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/imunologia , Linfonodos/imunologia , Proteoma/análise , Proteoma/imunologia , Animais , Asma/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais , Tolueno 2,4-Di-Isocianato , Eletroforese em Gel Diferencial BidimensionalRESUMO
Placental growth factor (PlGF) and its receptor vascular endothelial growth factor receptor 1 (VEGFR1) play an important role in pathological conditions related to angiogenesis, vascular leakage, and inflammation. This study investigated their contributions to inflammation and the formation of edema in allergic asthma. The expression of PlGF and VEGFR1 was measured in induced sputum of patients with asthma (n = 11) and healthy subjects (n = 11), and in bronchial biopsies of house dust mite (HDM)-allergic patients stimulated with HDM allergens. The effects of the endonasal administration of human PlGF-2 and PlGF deficiency on inflammation and edema were evaluated in a murine model of allergic asthma. The migration of human neutrophils in response to hPlGF-2 was tested in vitro. The expression of PlGF and VEGFR1 was significantly higher in the sputum of patients with asthma, and in Der p 1-induced PlGF in biopsies from HDM-allergic patients. PlGF was increased in the bronchi of ovalbumin (OVA)-challenged mice compared with control mice (65 ± 17 pg/mg versus 18 ± 1 pg/mg, respectively; P < 0.01), and VEGFR1 was expressed in bronchial epithelium, endothelium (control mice), and inflammatory cells (OVA-challenged mice). The endonasal instillation of hPlGF-2 in wild-type, OVA-challenged mice led to an increase in bronchial neutrophils, lung tissue wet/dry ratio, and IL-17. PlGF-deficient mice showed lower numbers of BAL-infiltrating neutrophils, a reduced lung wet/dry ratio, and lower production of IL-17, macrophage inflammatory protein-2, and granulocyte chemotactic protein-2/LPS-induced chemokine compared with wild-type, OVA-challenged mice. hPlGF-2 induced the migration of human neutrophils in vitro in a VEGFR1-dependent way. PlGF and its receptor VEGFR1 are up-regulated in allergic asthma and play a proinflammatory role by inducing tissue edema, and increasing tissue neutrophilia and the production of IL-17.
Assuntos
Asma/imunologia , Brônquios/imunologia , Edema/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Proteínas da Gravidez/fisiologia , Animais , Feminino , Humanos , Masculino , Camundongos , Fator de Crescimento Placentário , Reação em Cadeia da Polimerase em Tempo RealRESUMO
We determined the ability of a model nanoparticle (NP) (titanium dioxide, TiO(2)) to modulate sensitization induced by a known potent dermal sensitizer (dinitrochlorobenzene) using a variant of the local lymph node assay called lymph node proliferation assay.BALB/c mice received sub-cutaneous injections of vehicle (2.5 mM sodium citrate), TiO(2) NPs (0.004, 0.04 or 0.4 mg/ml) or pigment particles (0.04 mg/ml) both stabilized in sodium citrate buffer at the base of each ear (2x50µl), before receiving dermal applications (on both ears) of 2,4-Dinitrochlorobenzene (DNCB) (2x25µl of 0.1%) or its vehicle (acetone olive oil - AOO (4:1)) on days 0, 1 and 2. On day 5, the stimulation index (SI) was calculated as a ratio of (3)HTdR incorporation in lymphocytes from DNBC-treated mice and AOO-treated controls. In a second experiment the EC(3)-value for DNCB (0 to 0.1%) was assessed in the absence or presence of 0.04 mg/ml TiO(2). In a third experiment, the lymphocyte subpopulations and the cytokine secretion profile were analyzed after TiO(2) (0.04 mg/ml) and DNCB (0.1%) treatment. Injection of NPs in AOO-treated control mice did not have any effect on lymph node (LN) proliferation. DNCB sensitization resulted in LN proliferation, which was further increased by injection of TiO(2) NPs before DNCB sensitization. The EC(3) of DNCB, with prior injection of vehicle control was 0.041%, while injection with TiO(2) decreased the EC(3) of DNCB to 0.015%. TiO(2) NPs pre-treatment did not alter the lymphocyte subpopulations, but significantly increased the level of IL-4 and decreased IL-10 production in DNCB treated animals.In conclusion, our study demonstrates that administration of nano-TiO(2) increases the dermal sensitization potency of DNCB, by augmenting a Th(2) response, showing the immunomodulatory abilities of NPs.
Assuntos
Fatores Imunológicos/administração & dosagem , Linfonodos/efeitos dos fármacos , Nanopartículas Metálicas/administração & dosagem , Titânio/administração & dosagem , Administração Cutânea , Animais , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Dinitroclorobenzeno/administração & dosagem , Dinitroclorobenzeno/farmacologia , Orelha Externa/efeitos dos fármacos , Injeções Subcutâneas , Irritantes/administração & dosagem , Irritantes/farmacologia , Linfonodos/imunologia , Linfonodos/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Pulmonary function analysis is an important tool in the evaluation of mouse respiratory disease models, but much controversy still exists on the validity of some tests. Most commonly used pulmonary function variables of humans are not routinely applied in mice, and the question of which pulmonary function is optimal for the monitoring of a particular disease model remains largely unanswered. Our study aimed to delineate the potential and restrictions of existing pulmonary function techniques in different respiratory disease models, and to determine some common variables between humans and mice. A noninvasive (unrestrained plethysmography) and two invasive pulmonary function devices (forced maneuvers system from Buxco Research Systems [Wilmington, NC] and forced oscillation technique from SCIREQ [Montreal, PQ, Canada]) were evaluated in well-established models of asthma (protein and chemical induced): a model of elastase-induced pulmonary emphysema, and a model of bleomycin-induced pulmonary fibrosis. In contrast to noninvasive tests, both invasive techniques were efficacious for the quantification of parenchymal disease via changes in functional residual capacity, total lung capacity, vital capacity, and compliance of the respiratory system. Airflow obstruction and airflow limitation at baseline were only present in emphysema, but could be significantly induced after methacholine challenge in mice with asthma, which correlated best with an increase of respiratory resistance. Invasive pulmonary functions allow distinction between respiratory diseases in mice by clinically relevant variables, and should become standard in the functional evaluation of pathological disease models.
Assuntos
Asma/metabolismo , Pneumopatias/patologia , Pulmão/patologia , Enfisema Pulmonar/metabolismo , Animais , Testes de Provocação Brônquica , Modelos Animais de Doenças , Fibrose , Humanos , Pulmão/metabolismo , Pneumopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oscilometria , Pletismografia , Enfisema Pulmonar/fisiopatologia , Mecânica RespiratóriaRESUMO
Occupational asthma is the principal cause of work-related respiratory disease in the industrial world. Toluene-2,4-diisocyanate (TDI) is one of the most common respiratory sensitizers leading to occupational asthma. Using a mouse model of chemical-induced asthma, we explored proteome changes in multiple compartments of mice sensitized and challenged with TDI or acetone-olive oil (AOO; vehicle). Airway reactivity to methacholine and a bronchoalveolar lavage (BAL) cell count was assessed in treated and control mice, 1 day after challenge. Subsequently, two-dimensional differential gel electrophoresis (2D-DIGE) was performed on auricular lymph nodes, BAL, and serum comparing TDI-treated and vehicle-treated control mice. The differentially expressed proteins were identified by mass spectrometry and pathway analysis was performed. TDI-treated mice exhibit increased airway reactivity (2.6-fold increase) and a neutrophilic inflammation in the BAL fluid, compared to control mice. 2D-DIGE showed 53, 210, and 40 differentially expressed proteins in the auricular lymph nodes, BAL, and serum of TDI-treated versus vehicle-treated mice, respectively. Several of the identified proteins could be linked with inflammation, neutrophil chemotaxis, and/or oxidative stress. Physiologic and immunologic readouts of the asthmatic phenotype, such as inflammation, were confirmed in three compartments by several of the differentially expressed proteins via 2D-DIGE and computerized pathway analysis.
Assuntos
Asma/induzido quimicamente , Proteoma/análise , Tolueno 2,4-Di-Isocianato/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar/química , Modelos Animais de Doenças , Inflamação , Espectrometria de Massas , Cloreto de Metacolina , Camundongos , Neutrófilos , Estresse OxidativoRESUMO
BACKGROUND: Persulfate salts are the main cause of occupational asthma (OA) in hairdressers. The aim of this study was to verify whether ammonium persulfate ((NH(4))(2)S(2)O(8), AP) is capable of triggering an asthma-like response in mice. METHODS: BALB/c mice were dermally treated on days 1 and 8, with dimethylsulfoxide (DMSO), 1% AP or 5% AP (20 microl/ear). On day 15, the auricular lymph nodes were removed and an in vitro lymphocyte proliferation test (LPT) was performed. AP was tested for its ability to elicit an asthmatic response using a locally developed mouse model of chemical-induced asthma. On days 1 and 8, BALB/c mice received 20 microl AP (5%) or DMSO on each ear. On day 15, they received an intranasal instillation of AP (1%) or saline. Afterwards, ventilatory, inflammatory and immunological parameters were assessed. RESULTS: The LPT showed that in vitro stimulation of lymphocytes with AP leads to specific proliferation of lymphocytes from AP-sensitised mice. In vivo, AP induced, in AP-sensitised mice only, an 'early' ventilatory response (increased Penh (enhanced pause)) immediately after challenge, and airway hyper-reactivity to methacholine 22 h later. Pulmonary inflammation was mainly characterised by neutrophils (10-15%). AP-sensitised mice showed an increase in total number of T helper (Th) and B lymphocytes together with an increased in vitro secretion of interleukin-4 (IL-4), IL-10 and IL-13 and an increase in total serum immunoglobulin E. CONCLUSIONS: In a mouse model, it was confirmed that dermal sensitisation to AP can lead to asthma-like responses after a single administration via the airway.
Assuntos
Sulfato de Amônio/toxicidade , Asma/induzido quimicamente , Animais , Asma/imunologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica/induzido quimicamente , Broncoconstritores , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Imunoglobulina E/sangue , Linfonodos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Cloreto de Metacolina , Camundongos , Camundongos Endogâmicos BALB CRESUMO
To improve our mouse model of chemical-induced asthma we compared a single with a multiple intranasal challenge protocol. BALB/c mice received toluene diisocyanate (TDI) or vehicle on each ear (days 1 and 8) with the first challenge by intranasal instillation given on day 15. In a "long" protocol, the mice received 1 to 6 intranasal instillations, with 1-week interval. In a "short" protocol, the mice received 6 intranasal challenges over a period of 10 days. The "early" ventilatory response and methacholine reactivity were measured. Broncho-alveolar-lavage (BAL), total serum immunoglobulins and draining lymph nodes were analyzed. After 1, 2 or 3 TDI challenges, a significant increase in airway reactivity, total cell count and neutrophils (15-20%) was found in TDI-treated mice. This response diminished with increasing numbers of challenges in both models. The percentage CD4(+) and CD8(+) cells decreased and the percentage CD19(+) cells increased in the lymph nodes, but these returned to control values with multiple challenges. IL-4 secretion increased in cervical lymph node cells in vitro. Total serum IgE levels were persistently increased in TDI-treated mice. Although humoral signs of allergy remain increased after multiple challenges, diminishing ventilatory and inflammatory responses are indicative of the induction of tolerance.
Assuntos
Formação de Anticorpos/efeitos dos fármacos , Asma/induzido quimicamente , Asma/imunologia , Tolerância Imunológica/efeitos dos fármacos , Inflamação/patologia , Mecânica Respiratória/efeitos dos fármacos , Administração Intranasal , Animais , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/patologia , Líquido da Lavagem Broncoalveolar/citologia , Broncoconstritores , Citocinas/biossíntese , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Pulmão/patologia , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , Cloreto de Metacolina , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: To assess the importance of the route of challenge in an existing mouse model of chemical-induced asthma, we replaced intranasal instillation by oropharyngeal aspiration. To our knowledge, oropharyngeal aspiration as a challenge route has not yet been investigated in a mouse model of chemical-induced asthma. METHODS: On days 1 and 8, mice were dermally sensitized with toluene diisocyanate (TDI) (0.3%) [or vehicle (acetone/olive oil)] and on day 15 they received a single challenge, via oropharyngeal aspiration, with TDI (0.01%) or vehicle. One day after challenge, airway reactivity to methacholine was measured by a forced oscillation technique (FlexiVent) and total and differential cell counts, as well as levels of KC, IL-5, IL-17 and TNF-alpha, were assessed in the bronchoalveolar lavage (BAL) fluid. Lymphocytes from the auricular and mediastinal lymph nodes were cultured to determine the concanavaline A-induced secretion of IL-2, IL-4, IL-10, IL-13, IL-17 and IFN-gamma. Total serum IgE was measured. RESULTS: In TDI-sensitized mice, a significant increase in airway reactivity was found after a single oropharyngeal challenge with TDI. BAL neutrophils and eosinophils were increased 7- and 5-fold, respectively. An upregulation of Th1 (IFN-gamma), Th2 (IL-4, IL-10, IL-13) and Th17 (IL-17) cytokines was found in the auricular lymph nodes, in the mediastinal lymph nodes only IL-4 was upregulated. The total serum IgE level in TDI-sensitized mice was significantly increased when compared to control mice. CONCLUSION: We conclude that challenging mice via oropharyngeal aspiration mimics the characteristics of human asthma well, without the possible drawbacks of other techniques.
Assuntos
Asma/induzido quimicamente , Hiper-Reatividade Brônquica/induzido quimicamente , Modelos Animais de Doenças , Aspiração Respiratória , Tolueno 2,4-Di-Isocianato/administração & dosagem , Administração Intranasal , Animais , Asma/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Broncoconstritores , Citocinas/imunologia , Eosinófilos/imunologia , Imunoglobulina E/sangue , Masculino , Cloreto de Metacolina , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Tolueno 2,4-Di-Isocianato/imunologia , Tolueno 2,4-Di-Isocianato/toxicidade , Regulação para Cima/imunologiaRESUMO
BACKGROUND: Persulfate salts have been associated with both allergic contact dermatitis and bronchial asthma. Because there is currently no experimental data available on the sensitizing properties of persulfate salts (ammonium, sodium, and potassium persulfates), we determined their dermal sensitizing capacity, using the murine local lymph node assay (LLNA). MATERIAL AND METHODS: For three consecutive days, BALB/c mice were dermally treated with ammonium, sodium, or potassium persulfate or with the vehicle alone (dimethyl sulfoxide) on each ear (2 x 25 microl). On D6, mice were injected intravenously with [(3)H]-methyl thymidine. The draining auricular lymph nodes were removed, and the incorporation of [(3)H]-methyl thymidine was compared with that of vehicle-treated control mice. A stimulation index (SI) relative to the vehicle-treated control value was derived. The sensitizing potency of the chemicals tested was determined by estimating the concentration of chemical required to induce a SI of 3 (EC3). RESULTS: All three chemicals provoked positive responses in the LLNA, with dose-dependent increases in proliferation. Maximal SIs recorded were 6.8 +/- 1.8, 6.5 +/- 1.2, and 5 +/- 1.0 at 5% for ammonium, sodium or potassium persulfate, respectively. The EC3 values were 1.9%, 0.9%, and 2.4% for ammonium, sodium, and potassium persulfates, respectively. CONCLUSIONS: All three persulfate salts need to be considered strong-to-moderate sensitizers according to the murine LLNA.
Assuntos
Dermatite Alérgica de Contato/diagnóstico , Dermatite Alérgica de Contato/etiologia , Tinturas para Cabelo/toxicidade , Sulfatos/toxicidade , Alérgenos/toxicidade , Sulfato de Amônio/toxicidade , Animais , Ensaio Local de Linfonodo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Potássio/toxicidade , Testes de Irritação da Pele/métodosRESUMO
BACKGROUND: Years after removal from exposure, workers with occupational asthma still show respiratory symptoms and airway hyperresponsiveness on re-exposure to the offending agent. OBJECTIVE: We investigated the persistence of the respiratory responsiveness to toluene diisocyanate (TDI) in a mouse model. METHODS: BALB/C mice received dermal applications of TDI on days 1 and 8, and a single intranasal instillation of TDI on day 10, 15, 20, 25, 30, 40, 50, 60, or 90. After instillation, early (1 hour) changes in ventilatory function and methacholine responsiveness (22 hours) were assessed. Cell counts and macrophage inflammatory protein 2 were measured in bronchoalveolar lavage. Total serum IgE, IgG(1), and IgG(2a) were quantified. Lymphocyte subpopulations were assessed in auricular and cervical lymph nodes, and release of IL-4 and IFN-gamma by these lymph node cells was measured. RESULTS: Toluene diisocyanate-treated mice showed immediate ventilatory changes, increased methacholine reactivity, and an influx of neutrophils and macrophage inflammatory protein 2 in bronchoalveolar lavage as long as 50 days after initial treatment. These mice also showed a relative increase in CD19(+) cells and a decrease in CD4(+) and CD8(+) cells in auricular lymph nodes. Increased release of IL-4 and IFN-gamma in auricular lymph node cells was observed only until 20 days after sensitization. Total serum IgE, IgG(1), and IgG(2a) remained significantly elevated in TDI-sensitized mice until 90 days after dermal sensitization. CONCLUSION: Ventilatory and lung inflammatory responses decrease with increasing delay between sensitization and challenge, despite persistent humoral signs of sensitization.
Assuntos
Imunização , Hipersensibilidade Respiratória/fisiopatologia , Pele/imunologia , Tolueno 2,4-Di-Isocianato/imunologia , Administração Intranasal , Animais , Peso Corporal , Líquido da Lavagem Broncoalveolar/química , Broncoconstritores , Quimiocina CXCL2/metabolismo , Orelha Externa , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Interferon gama/metabolismo , Interleucina-4/metabolismo , Linfonodos/metabolismo , Linfonodos/patologia , Subpopulações de Linfócitos/patologia , Masculino , Cloreto de Metacolina , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/patologia , Respiração , Hipersensibilidade Respiratória/complicações , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Fatores de Tempo , Tolueno 2,4-Di-Isocianato/administração & dosagemRESUMO
Lipopolysaccharides (LPS), the major components of the wall of gram-negative bacteria, trigger powerful defensive responses in the airways via mechanisms thought to rely solely on the Toll-like receptor 4 (TLR4) immune pathway. Here we show that airway epithelial cells display an increase in intracellular Ca2+ concentration within seconds of LPS application. This response occurs in a TLR4-independent manner, via activation of the transient receptor potential vanilloid 4 cation channel (TRPV4). We found that TRPV4 mediates immediate LPS-induced increases in ciliary beat frequency and the production of bactericidal nitric oxide. Upon LPS challenge TRPV4-deficient mice display exacerbated ventilatory changes and recruitment of polymorphonuclear leukocytes into the airways. We conclude that LPS-induced activation of TRPV4 triggers signaling mechanisms that operate faster and independently from the canonical TLR4 immune pathway, leading to immediate protective responses such as direct antimicrobial action, increase in airway clearance, and the regulation of the inflammatory innate immune reaction.
Assuntos
Sinalização do Cálcio , Células Epiteliais/imunologia , Lipopolissacarídeos/imunologia , Mucosa Respiratória/imunologia , Canais de Cátion TRPV/metabolismo , Animais , Cílios/fisiologia , Escherichia coli , Células HEK293 , Humanos , Imunidade Inata , Camundongos Knockout , Óxido Nítrico/metabolismo , Técnicas de Patch-Clamp , Cultura Primária de CélulasRESUMO
INTRODUCTION AND AIM: The role of B-lymphocytes in chemical-induced asthma is largely unknown. Recent work demonstrated that transferring B lymphocytes from toluene diisocyanate (TDI)-sensitized mice into naïve mice, B cell KO mice and SCID mice, triggered an asthma-like response in these mice after a subsequent TDI-challenge. We applied two-dimensional difference gel electrophoresis (2D-DIGE) to describe the "sensitized signature" of B lymphocytes comparing TDI-sensitized mice with control mice. RESULTS: Sixteen proteins were identified that were significantly up- or down-regulated in B lymphocytes of sensitized mice. Particularly differences in the expression of cyclophilin A, cofilin 1 and zinc finger containing CCHC domain protein 11 could be correlated to the function of B lymphocytes as initiators of T lymphocyte independent asthma-like responses. CONCLUSION: This study revealed important alterations in the proteome of sensitized B cells in a mouse model of chemical-induced asthma, which will have an important impact on the B cell function.
Assuntos
Asma/metabolismo , Linfócitos B/metabolismo , Proteoma/metabolismo , Animais , Asma/induzido quimicamente , Asma/imunologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , Tolueno 2,4-Di-IsocianatoRESUMO
BACKGROUND: The pathogenesis of pulmonary fibrosis remains poorly understood. The Wnt signaling pathway regulates fibrogenesis in different organs. Here, we studied the role of two extracellular Wnt antagonists, secreted frizzled-related protein-1 (SFRP1) and frizzled-related protein (FRZB) on lung fibrosis in vitro and in vivo. For this purpose, we used an alveolar epithelial cell line and a lung fibroblast cell line, and the bleomycin-induced lung fibrosis model, respectively. RESULTS: During the course of bleomycin-induced lung fibrosis, Sfrp1 and Frzb expression are upregulated. Expression of Sfrp1 appears much higher than that of Frzb. In vitro, recombinant SFRP1, but not FRZB, counteracts the transforming growth factor ß1 (TGFß1)-induced upregulation of type I collagen expression both in pulmonary epithelial cells and fibroblasts. Both SFRP1 and FRZB inhibit the TGFß1-induced increase of active ß-catenin, but do not influence the TGFß1-induced phosphorylation levels of SMAD3, positioning Wnt signaling activity downstream of the active TGFß signal in lung fibroblasts, but not in alveolar epithelial cells. In vivo, Sfrp1 (-/-) and Frzb (-/-) mice showed identical responses to bleomycin in the lung compared to wild-type controls. CONCLUSIONS: Although SFRP1 counteracts the effect of TGFß1 in pulmonary cells in vitro; loss of neither SFRP1 nor FRZB alters fibrotic outcomes in the lungs in vivo. The lack of in vivo effect in the absence of specific SFRPs suggests functional redundancy within this family of Wnt antagonists.
RESUMO
T-lymphocytes and B-lymphocytes are key players in allergic asthma, with B-lymphocytes producing antigen-specific immunoglobulins E (IgE). We used a mouse model of chemical-induced asthma and transferred B-lymphocytes from sensitized animals into naïve wild type mice, B-lymphocyte knock-out (B-KO) mice or severe combined immunodeficiency (SCID) mice. On days 1 and 8, BALB/c mice were dermally sensitized with 0.3% toluene diisocyanate (TDI) (20 µl/ear). On day 15, mice were euthanized and the auricular lymph nodes isolated. B-lymphocytes (CD19(+)) were separated from the whole cell suspension and 175,000 cells were injected in the tail vein of naïve wild type, B-KO or SCID mice. Three days later, the mice received a single oropharyngeal challenge with 0.01% TDI (20 µl) or vehicle (acetone/olive oil (AOO)) (controls). Airway reactivity to methacholine and total and differential cell counts in the bronchoalveolar lavage (BAL) fluid were measured 24 hours after challenge. B-lymphocytes of AOO or TDI-sensitized mice were characterized for the expression of surface markers and production of cytokines. We found that transfer of B-cells obtained from mice dermally sensitized to toluene diisocyanate (TDI) into naïve wild type mice, B-KO mice or SCID mice led, within three days, to an acute asthma-like phenotype after an airway challenge with TDI. This response was specific and independent of IgE. These B-lymphocytes showed antigen presenting capacities (CD80/CD86 and CD40) and consisted of B effector (Be)2- (IL-4) and Be1-lymphocytes (IFN-γ). The transferred B-lymphocytes were visualized near large airways, 24 hours after TDI challenge. Thus, B-lymphocytes can provoke an asthmatic response without the action of T-lymphocytes and without major involvement of IgE.
Assuntos
Asma/induzido quimicamente , Asma/imunologia , Subpopulações de Linfócitos B/imunologia , Animais , Asma/genética , Asma/patologia , Subpopulações de Linfócitos B/patologia , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Broncoconstritores/farmacologia , Antígenos CD40/genética , Antígenos CD40/imunologia , Modelos Animais de Doenças , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Masculino , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , Tolueno 2,4-Di-Isocianato/toxicidadeRESUMO
Diisocyanates are an important cause of chemical-induced occupational asthma. This type of immunologically mediated asthma is often characterized by a predominant granulocytic inflammation in the airways, rather than an infiltration by lymphocytes. We sought to determine the contribution of granulocytes in the outcome of chemical-induced asthma using general and specific leukocyte depletion strategies in an established mouse model of isocyanate asthma. On days 1 and 8, BALB/c mice received dermal applications with toluene-2,4-diisocyanate (TDI) or vehicle (acetone olive oil), followed by two ip injections of cyclophosphamide (CP, days 11 and 13), or one iv injection of antigranulocyte receptor 1 (aGR1, day 13) monoclonal antibody (mAb), or two ip injections of Ly6G-specific mAb (1A8, days 13 and 14). On day 15, the mice were challenged (oropharyngeal administration) with TDI or vehicle. The next day, we assessed methacholine airway hyperreactivity (AHR); bronchoalveolar lavage differential cell count; histopathology and total serum IgE; and auricular lymphocyte subpopulations and release of interleukin (IL)-2, IL-4, IL-10, IL-13, and gamma interferon by these lymphocytes. CP depleted all leukocyte types and completely prevented AHR and airway inflammation. aGR1 depleted granulocytes and CD8(+) lymphocytes, which resulted in a partial prevention in AHR but no decrease in airway inflammation. Depletion of Ly6G-positive granulocytes, i.e., both neutrophils and eosinophils, prevented AHR and lung epithelial damage and significantly reduced airway inflammation. Injection of aGR1 or 1A8 led to significantly changed cytokine release patterns in TDI-treated mice. Granulocytes, both neutrophils and eosinophils, are key cellular players in this model of chemical-induced asthma.
Assuntos
Asma/induzido quimicamente , Ciclofosfamida/toxicidade , Eosinófilos/imunologia , Neutrófilos/imunologia , Tolueno 2,4-Di-Isocianato/toxicidade , Animais , Asma/imunologia , Peso Corporal , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Epidemiological studies indicate that elderly persons are particularly susceptible to the cardiovascular health complications of air pollution, but pathophysiological mechanisms behind the increased susceptibility remain unclear. Therefore, we investigated how continuous traffic-related air pollution exposure affects haemostasis parameters in young and old mice. Young (10 weeks) and old (20 months) mice were placed in an urban roadside tunnel or in a clean environment for 25 or 26 days and markers of inflammation and endothelial cells or blood platelet activation were measured, respectively. Plasma microvesicles and pro/anticoagulant factors were analysed, and thrombin generation analysis was performed. Despite elevated macrophage carbon load, tunnel mice showed no overt pulmonary or systemic inflammation, yet manifested reduced pulmonary thrombomudulin expression and elevated endothelial von Willebrand factor (VWF) expression in lung capillaries. In young mice, soluble P-selectin (sP-sel) increased with exposure and correlated with soluble E-selectin and VWF. Baseline plasma factor VIII (FVIII), sP-sel and VWF were higher in old mice, but did not pronouncedly increase further with exposure. Traffic-related air pollution markedly raised red blood cell and blood platelet numbers in young and old mice and procoagulant blood platelet-derived microvesicle numbers in old animals. Changes in coagulation factors and thrombin generation were mild or absent. Hence, continuous traffic-related air pollution did not trigger overt lung inflammation, yet modified pulmonary endothelial cell function and enhanced platelet activity. In old mice, subchronic exposure to polluted air raised platelet numbers, VWF, sP-sel and microvesicles to the highest values presently recorded, collectively substantiating a further elevation of thrombogenicity, already high at old age.
Assuntos
Poluição do Ar/efeitos adversos , Trombose/etiologia , Envelhecimento/sangue , Animais , Biomarcadores/sangue , Contagem de Células Sanguíneas , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Fator VIII/metabolismo , Hemostasia , Humanos , Interleucina-6/metabolismo , Pulmão/irrigação sanguínea , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Veículos Automotores , Selectina-P/metabolismo , Material Particulado/efeitos adversos , Contagem de Plaquetas , Fatores de Risco , Trombose/sangue , Saúde da População Urbana , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: We optimized an adoptive transfer protocol in our mouse model of TDI-induced asthma in order to investigate the mechanisms of this type of occupational asthma. METHODS: On days 1 and 8, BALB/c mice were dermally sensitized with 0.3% TDI or vehicle (acetone/olive oil), and on day 15, they were sacrificed and a cell suspension was made from auricular lymph nodes. First, 0.1 x 106, 0.5 x 106, 1 x 106 or 5 x 106 cells were injected intravenously into naïve mice and three days later these mice received an oropharyngeal challenge with 0.01% TDI or vehicle. Second, mice were challenged with 0.01% TDI 1, 3, 5 or 7 days after transferring 0.5 x 106 cells. The following endpoints were measured one day after challenge: methacholine reactivity; differential cell counts in bronchoalveolar lavage (BAL) and total serum IgE. RESULTS: Naïve mice receiving 0.5 x 106, 1 x 106 or 5 x 106 cells showed significant increases in airway reactivity one day after TDI challenge; BAL neutrophils were increased after transferring 0.5 x 106 and 1 x 106 cells. A TDI challenge 3 days after transferring 0.5 x 106 cells gave a 3-fold increase in airway resistance and a pronounced airway inflammation, whereas challenging at other time points gave no differences. CONCLUSION: We were able to passively sensitize naïve mice using lymph node cells from TDI-sensitized mice, resulting in an asthma-like response after an airway challenge. In comparison to other adoptive transfer protocols we used substantially lower number of cells to obtain the desired response.