RESUMEN
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.
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Señalización del Calcio , Células Epiteliales/inmunología , Lipopolisacáridos/inmunología , Mucosa Respiratoria/inmunología , Canales Catiónicos TRPV/metabolismo , Animales , Cilios/fisiología , Escherichia coli , Células HEK293 , Humanos , Inmunidad Innata , Ratones Noqueados , Óxido Nítrico/metabolismo , Técnicas de Placa-Clamp , Cultivo Primario de CélulasRESUMEN
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.
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Contaminantes Ocupacionales del Aire/toxicidad , Asma/inducido químicamente , Isocianatos/toxicidad , 2,4-Diisocianato de Tolueno/toxicidad , Contaminantes Ocupacionales del Aire/inmunología , Animales , Asma/sangre , Asma/inmunología , Reacciones Cruzadas/efectos de los fármacos , Reacciones Cruzadas/inmunología , Modelos Animales de Enfermedad , Inmunoglobulina E/sangre , Isocianatos/inmunología , Masculino , Ratones Endogámicos C57BL , Balance Th1 - Th2/efectos de los fármacos , 2,4-Diisocianato de Tolueno/inmunologíaRESUMEN
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.
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Asma/metabolismo , Linfocitos B/metabolismo , Proteoma/metabolismo , Animales , Asma/inducido químicamente , Asma/inmunología , Masculino , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , 2,4-Diisocianato de ToluenoRESUMEN
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.
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Bleomicina , Proteínas Morfogenéticas Óseas/metabolismo , Pulmón/metabolismo , Fibrosis Pulmonar/prevención & control , Animales , Apoptosis , Proteínas Portadoras/genética , Proliferación Celular , Colágeno/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Haploinsuficiencia , Pulmón/patología , Pulmón/fisiopatología , Rendimiento Pulmonar , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/fisiopatología , Transducción de Señal , Proteínas Smad Reguladas por Receptores/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Asma/inducido químicamente , Asma/inmunología , Subgrupos de Linfocitos B/inmunología , Animales , Asma/genética , Asma/patología , Subgrupos de Linfocitos B/patología , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Antígeno B7-2/genética , Antígeno B7-2/inmunología , Broncoconstrictores/farmacología , Antígenos CD40/genética , Antígenos CD40/inmunología , Modelos Animales de Enfermedad , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Masculino , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , 2,4-Diisocianato de Tolueno/toxicidadRESUMEN
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.
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Hiperreactividad Bronquial/fisiopatología , Ácido Hipocloroso/efectos adversos , Irritantes/efectos adversos , Mastocitos/inmunología , Canales de Potencial de Receptor Transitorio/inmunología , Animales , Hiperreactividad Bronquial/etiología , Células Cultivadas , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neuroinmunomodulación , Nociceptores/inmunología , Ovalbúmina/efectos adversos , Sustancia P/metabolismo , Canal Catiónico TRPA1RESUMEN
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.
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Asma/inducido químicamente , Ciclofosfamida/toxicidad , Eosinófilos/inmunología , Neutrófilos/inmunología , 2,4-Diisocianato de Tolueno/toxicidad , Animales , Asma/inmunología , Peso Corporal , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
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.
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Asma/inducido químicamente , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/inmunología , Ganglios Linfáticos/inmunología , Proteoma/análisis , Proteoma/inmunología , Animales , Asma/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Transducción de Señal , 2,4-Diisocianato de Tolueno , Electroforesis Bidimensional Diferencial en GelRESUMEN
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.
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Contaminación del Aire/efectos adversos , Trombosis/etiología , Envejecimiento/sangre , Animales , Biomarcadores/sangre , Recuento de Células Sanguíneas , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Factor VIII/metabolismo , Hemostasis , Humanos , Interleucina-6/metabolismo , Pulmón/irrigación sanguínea , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Vehículos a Motor , Selectina-P/metabolismo , Material Particulado/efectos adversos , Recuento de Plaquetas , Factores de Riesgo , Trombosis/sangre , Salud Urbana , Factor de von Willebrand/metabolismoRESUMEN
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.
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Factores Inmunológicos/administración & dosificación , Ganglios Linfáticos/efectos de los fármacos , Nanopartículas del Metal/administración & dosificación , Titanio/administración & dosificación , Administración Cutánea , Animales , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Dinitroclorobenceno/administración & dosificación , Dinitroclorobenceno/farmacología , Oído Externo/efectos de los fármacos , Inyecciones Subcutáneas , Irritantes/administración & dosificación , Irritantes/farmacología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Linfocitos/patología , Ratones , Ratones Endogámicos BALB CRESUMEN
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.
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Asma/inmunología , Bronquios/inmunología , Edema/inmunología , Inflamación/inmunología , Neutrófilos/inmunología , Proteínas Gestacionales/fisiología , Animales , Femenino , Humanos , Masculino , Ratones , Factor de Crecimiento Placentario , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Some epidemiologic studies have indicated that attendance to chlorinated swimming pools is associated with airway hyperreactivity (AHR), allergies and asthma. AIM: To investigate the effects of sodium hypochlorite (NaClO), the main pool disinfectant, on allergic sensitization and airway inflammation in mice. METHODS: In a first series of experiments, mice were sensitized to ovalbumin (OVA), followed by OVA aerosols with or without prior nasal instillation of NaClO (3ppm active chlorine). In a second series, naïve mice received 1-7 nasal instillations of OVA, 10min after instillations of NaClO or water. After 1, 3, 5 and 7 exposures airway reactivity to methacholine, cellular inflammation in bronchoalveolar lavage (BAL), serum OVA-specific IgEs and lung Th2 cytokines were measured. RESULTS: In the first mouse model, airway allergy parameters were not significantly altered upon NaClO administration. However in the second model, NaClO exposure prior to OVA did induce AHR, already after 1 combined application. Combined NaClO+OVA exposure did not lead to an influx of inflammatory cells in BAL fluid or production of anti-OVA IgEs. No AHR developed when OVA was heat-denatured, pre-chlorinated, or replaced by bovine serum albumin or lipopolysaccharide. CONCLUSION: Nasal instillation of NaClO prior to OVA induces AHR without allergic sensitization. This response is OVA-specific.
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Hiperreactividad Bronquial/etiología , Hipersensibilidad/etiología , Ácido Hipocloroso/toxicidad , Ovalbúmina/inmunología , Animales , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
Invasive lung function measurements are useful tools to describe respiratory disease models in mice but only result in one time-point measurements because of tracheostomy. We explored if intubation may overcome the need for tracheostomy thereby allowing invasive lung function monitoring of individual mice over time. Repeated invasive lung function measurements with Scireq(©) - FlexiVent or Buxco(©) - Forced Pulmonary Maneuvers(®) were performed three times in BALB/c mice with intervals of 10 days. Each lung function assessment following intubation was compared with a similar measurement in age-matched tracheostomized mice, the golden standard in lung function measurements. Tracheostomy and intubation gave similar results for resistance, elastance and compliance of the whole respiratory system as assessed by Flexivent. Likewise, Forced Pulmonary Maneuvers used to measure lung volumes such as total lung capacity, functional residual capacity, forced expiratory volume in 0.1 s and forced vital capacity, resulted in identical outcomes for both airway approaches. No interaction was found between the procedures for any of the pulmonary function variables. The observed changes over time were rather related to animal growth than to repetitive intubation. Eighty percent of the animals survived three consecutive intubations, which were hampered by transient breathing difficulties, weight loss and neutrophilic bronchoalveolar lavage immediately postextubation. Repetitive invasive lung function measurements by intubation are feasible and reproducible in healthy mice and results are comparable to the standard method. This may open new perspectives for longitudinal research in animal models of respiratory diseases.
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Estudios de Factibilidad , Intubación/métodos , Pruebas de Función Respiratoria/métodos , Traqueostomía/métodos , Animales , Lavado Broncoalveolar/métodos , Modelos Animales de Enfermedad , Volumen Espiratorio Forzado , Capacidad Residual Funcional , Pulmón/fisiopatología , Masculino , Ratones , Ratones Endogámicos BALB C , Capacidad VitalRESUMEN
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.
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Traslado Adoptivo/métodos , Asma/inmunología , Modelos Animales de Enfermedad , Linfocitos/inmunología , 2,4-Diisocianato de Tolueno/toxicidad , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Broncoconstrictores/farmacología , Inmunoglobulina E/sangre , Masculino , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Factores de TiempoRESUMEN
BACKGROUND: The development of occupational asthma is the result of interactions between environmental factors and individual susceptibility. We assessed how our model of chemical-induced asthma is influenced by using different mouse strains. METHODOLOGY/PRINCIPAL FINDINGS: On days 1 and 8, male mice of 7 different strains (BALB/c, BP/2, A/J, C57Bl/6, DBA/2, CBA and AKR) were dermally treated with toluene-2,4-diisocyanate (TDI) (0.3%) or vehicle (acetone/olive oil, AOO, 2:3) on each ear (20 microl). On day 15, they received an oropharyngeal instillation of TDI (0.01%) or AOO (1:4). Airway reactivity to methacholine, total and differential cell counts in bronchoalveolar lavage (BAL) and total serum IgE and IgG(2a) levels were measured. Lymphocyte subpopulations in auricular lymph nodes and in vitro release of cytokines by ConA stimulated lymphocytes were assessed. In TDI-sensitized and challenged mice, airway hyper-reactivity was only observed in BALB/c, BP/2, A/J and AKR mice; airway inflammation was most pronounced in BALB/c mice; numbers of T-helper (CD4(+)), T-activated (CD4(+)CD25(+)), T-cytotoxic (CD8(+)) and B- lymphocytes (CD19(+)) were increased in the auricular lymph nodes of BALB/c, BP/2, A/J and CBA mice; elevated concentrations of IL-4, IL-10, IL-13 and IFN-gamma were detected in supernatant of lymphocytes from BALB/c, BP/2, A/J, C57Bl/6 and CBA mice cultured with concanavaline A, along with an increase in total serum IgE. CONCLUSION: The used mouse strain has considerable and variable impacts on different aspects of the asthma phenotype. The human phenotypical characteristics of chemically-induced occupational asthma were best reproduced in Th2-biased mice and in particular in BALB/c mice.
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Asma/inmunología , Modelos Animales de Enfermedad , Ratones , Animales , Asma/inducido químicamente , Asma/genética , Citocinas , Humanos , Masculino , Ratones/genética , Ratones/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Ratones Endogámicos , Linfocitos T/inmunologíaRESUMEN
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.
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Asma/inducido químicamente , Proteoma/análisis , 2,4-Diisocianato de Tolueno/efectos adversos , Animales , Líquido del Lavado Bronquioalveolar/química , Modelos Animales de Enfermedad , Inflamación , Espectrometría de Masas , Cloruro de Metacolina , Ratones , Neutrófilos , Estrés OxidativoRESUMEN
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.
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Sulfato de Amonio/toxicidad , Asma/inducido químicamente , Animales , Asma/inmunología , Asma/fisiopatología , Hiperreactividad Bronquial/inducido químicamente , Broncoconstrictores , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inmunoglobulina E/sangre , Ganglios Linfáticos/inmunología , Activación de Linfocitos/efectos de los fármacos , Masculino , Cloruro de Metacolina , Ratones , Ratones Endogámicos BALB CRESUMEN
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.
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Asma/metabolismo , Enfermedades Pulmonares/patología , Pulmón/patología , Enfisema Pulmonar/metabolismo , Animales , Pruebas de Provocación Bronquial , Modelos Animales de Enfermedad , Fibrosis , Humanos , Pulmón/metabolismo , Enfermedades Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Oscilometría , Pletismografía , Enfisema Pulmonar/fisiopatología , Mecánica RespiratoriaRESUMEN
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.