Asthmalike biphasic airway responses in Brown Norway rats sensitized by dermal exposure to dry trimellitic anhydride powder

BACKGROUND: Trimellitic anhydride (TMA) can induce specific IgE and occupational asthma. The significance of dermal exposure to TMA in immunologic sensitization and on subsequent airway responses is not clearly known. An animal model displaying both an early-phase airway response (EAR) and a late-phase airway response (LAR) after sensitization and subsequent inhalation challenge to a low-molecular-weight chemical has not been previously reported. OBJECTIVE: The present study investigated EAR and LAR after TMA inhalation challenge in Brown Norway rats sensitized by skin exposure to TMA dry powder. METHODS: Twenty milligrams of dry TMA powder was applied to the skin of each clipped rat's dorsum on days 0, 7, 14, and 21 and occluded overnight with surgical tape. Rats were challenged for 10 minutes with 0.2 to 40 mg/m(3) of TMA aerosol after day 35. Enhanced pause (an index of airway resistance) was recorded overnight in a whole-body plethysmography system. Specific IgE and pulmonary eosinophilia were also measured. RESULTS: Concentration-dependent responses to TMA were observed: provocation with 0.2 mg/m(3) produced no response; 1 mg/m(3) induced only EAR; and 5 mg/m(3) and 40 mg/m(3) induced both EAR and LAR. Specific IgE was positive; airway eosinophilic inflammation was observed. CONCLUSION: TMA powder applied to the skin can lead to both immunologic sensitization and subsequent dose-dependent biphasic airway responses after TMA aerosol challenge.     

Validation of a mouse model of chemical-induced asthma using trimellitic anhydride, a respiratory sensitizer, and dinitrochlorobenzene, a dermal sensitizer

BACKGROUND: Occupational asthma can be caused by chemicals. Previously, we established a murine model of immunologically mediated chemical-induced asthma using toluene diisocyanate. OBJECTIVE: We sought to verify this model using trimellitic anhydride (TMA), a respiratory sensitizer, and 1-chloro-2,4-dinitrobenzene (DNCB), a dermal sensitizer. METHODS: BALB/c mice received dermal applications (vehicle or chemical) on days 1 and 7. On day 10, they received an intranasal instillation (vehicle or chemical). Whole-body plethysmography (enhanced pause) was used to monitor changes in ventilatory function and methacholine reactivity. Pulmonary inflammation was assessed by using bronchoalveolar lavage (cells, TNF-alpha levels, and macrophage inflammatory protein 2 levels). Immunologic parameters included total serum IgE levels, lymphocyte distribution in auricular and cervical lymph nodes, and IL-4 and IFN-gamma levels in supernatants of lymph node cells incubated with or without concanavalin A. RESULTS: Mice dermally treated and intranasally challenged with TMA experienced markedly increased enhanced pause immediately after intranasal challenge and increased methacholine reactivity (24 hours later). Mice similarly treated with DNCB did not show any ventilatory changes. Neutrophil influx and increased macrophage inflammatory protein 2 and TNF-alpha levels were found in bronchoalveolar lavage fluid in both TMA- and DNCB-treated mice. The proportion of CD19+ B cells was increased in auricular and cervical lymph nodes of TMA-treated mice. IL-4 and IFN-gamma levels were increased in supernatants of concanavalin A-stimulated auricular and cervical lymph node cells of TMA- or DNCB-treated mice; however, the relative proportions of IL-4 and IFN-gamma levels differed between TMA- and DNCB-treated mice. Serum total IgE levels were increased in TMA-treated mice only. CONCLUSION: Both compounds induce a mixed T(H)1-T(H)2 response, but only TMA induced ventilatory changes. CLINICAL IMPLICATIONS: In the workplace avoiding skin contact with chemical sensitizers might be advised to prevent chemical-induced asthma.     

A model of immunologic lung injury induced by trimellitic anhydride inhalation: antibody response

We studied lung injury induced in Sprague-Dawley rats by trimellitic anhydride (TMA) inhalation. Groups of 40 male and 20 female rats were exposed to TMA by inhalation at target concentrations of 0, 10, 30, 100, and 300 micrograms/m3, 6 hours per day, 5 days per week, for 2 weeks. Rats in each exposure group were sacrificed after 10 exposures or rested for 12 days and either sacrificed or received a 6-hour TMA challenge at their initial exposure levels and sacrificed at 24 hours. At each sacrifice, serum antibody to radiolabeled trimellityl rat serum albumin (RSA-TM) was measured by an ammonium sulfate technique, and lung pathology was determined. After 10 days of exposure, external hemorrhagic lung foci were directly related to the exposure concentration of TMA. Serum antibody binding of RSA-TM correlated with exposure concentration, hemorrhagic lung foci, and lung weight. There was healing of lung lesions 12 days after exposure with a return of lung lesions only 18 hours after the 6-hour inhalation challenge. A correlation between serum antibody to RSA-TM, hemorrhagic foci, and lung weight existed after challenge. This model clarifies two clinical entities observed in exposed workers, the late respiratory systemic and the pulmonary disease-anemia syndromes.     

Atrial natriuretic peptide gene transfer by means of intranasal administration attenuates airway reactivity in a mouse model of allergic sensitization

Atrial natriuretic peptide (ANP) is a bronchodilator; however, the short half-life of ANP in vivo limited its therapeutic utility to treat asthma. The efficacy of intranasally administered plasmid DNA-expressing ANP (pANP; amino acid 99-126; Acc. No. XM131840) on the prevention of allergen-induced airway hyperresponsiveness (AHR) was examined in this study by using a mouse model of asthma. Ovalbumin-sensitized mice were treated with pANP versus control plasmids, and AHR was monitored after ovalbumin challenge for 5 weeks on 10-day intervals starting 4 days after gene transfer. Mice administered pANP demonstrated significantly less AHR for 20 days after treatment. The results demonstrate that pANP gene transfer protects against AHR and might be useful in the treatment of asthma.     

Role of eicosanoids in airflow obstruction and airway plasma exudation induced by trimellitic anhydride-conjugate in guinea-pigs 3 and 8 weeks after sensitization

Abstract. Trimellitic anhydride (TMA) is a low molecular weight chemical which can cause occupational asthma. We studied the role of eicosanoids in airway responses to TMA at different times after sensitization in actively sensitized guinea-pigs. Sensitization was performed by two intradermal injections of free TMA (0.1 ml of 0.3% TMA in corn oil). At 3 and 8 weeks after sensitization, the guinea-pigs were anaesthetized and challenged with intratracheal instillation of 0.5% TMA conjugated to guinea-pig serum albumin (TM A-GPSA; 50 μl). Lung resistance (RL) was measured to assess airflow obstruction, and the tissue content of Evans Blue dye was measured to assess airway plasma exudation. Intratracheal instillation of TMA-GPSA induced a slowly progressing increase in R_L, reaching a peak at approximately 3.5 min after the challenge (6.0 ± 2.0cm H₂O/ml/s in the 3-week group and 3.8 + 0.6 in the 8-week group). Pretreatment before challenge with pyrilamine (anti-histamine: 2 mg/kg. intravenously) slowed the onset of the increase in R_L following challenge with TMA-GPSA, and significantly attenuated the peak response. A combination of pyrilamine and IC1-192, 605 (thromboxane receptor antagonist; 0.5 mg/kg, intravenously) completely abolished the increase in R_Lin both week groups. A combination of pyrilamine and ICI-198, 615 (leukotrieneC₄/D₄/ E₄ receptor antagonist: 0.5 mg/kg, intravenously) did not further attenuate the increase in R_L compared with pretreatment with pyrilamine alone, but the induced Evans Blue dye extravasation was completely inhibited in the 3-week group, whereas a remaining extravasation was observed in the 8-week group. We conclude that the bronchoconstrictor response to TMA-GPSA in actively sensitized guinea-pigs is mediated by histamine and thromboxane A₂ both early and late after sensitization, whereas leukotrienes and histamine partially mediate TMA-induced airway plasma exudation.     

Exposure to endotoxins during sensitization prevents further endotoxin-induced exacerbation of airway inflammation in a mouse model of allergic asthma

BACKGROUND: We have shown previously that lipopolysaccharides (LPS) inhibited airway inflammation in allergen-sensitized and challenged mice when administered during sensitization, while exacerbating the inflammation when given upon challenge. We have here investigated the effect of LPS administered during both sensitization and challenge on airway inflammation, as well as on the profile of the T-helper (Th) response to allergen. METHODS: Mice were sensitized and challenged with ovalbumin (OVA), in the presence or absence of effective doses of LPS, namely 1 mug during sensitization and 1 ng during challenge. Inflammation was assessed by measuring cell counts and cytokine levels in bronchoalveolar lavage fluid (BALF). The profile of the Th response was determined by quantifying OVA-specific IgE and IgG2a in serum and Th1/Th2 cytokines in the culture medium of splenocytes and in BALF. RESULTS: Allergen-induced airway eosinophilia was increased in mice exposed to LPS during challenge only when compared with controls, whereas it was similarly reduced in animals exposed during sensitization only and during both sensitization and challenge. Mice exposed to LPS during sensitization only or during both sensitization and challenge also displayed a decrease in IgE and an increase in IgG2a, suggesting a switch in the immune response toward the Th1 profile. This was confirmed by quantification of Th1/Th2 cytokines in culture medium of splenocytes and in BALF. CONCLUSIONS: Our data demonstrate that exposure to endotoxins during sensitization prevents allergen-induced airway inflammation, as well as its exacerbation triggered by further exposure to endotoxins during challenge, while switching the immune response to allergen from a Th2 to a Th1 profile.     

Changes in asthma-like responses after extended removal from exposure to trimellitic anhydride in the Brown Norway rat model

Background Organic acid anhydride-induced occupational asthma is considered to be IgE-mediated. Airway and skin exposure are the two main routes of sensitization in the work place. Recently we developed an allergic asthmatic Brown Norway rat model sensitized by dermal exposure to trimellitic anhydride (TMA) using an occlusion patch application.
Objectives The objectives of this study were (1) to develop a model of non-occluded dermal exposure leading to allergic sensitization and (2) to examine the effect of extended removal from exposure on persistence of both specific IgE and TMA aerosol-induced airway responses in this model.
Methods TMA powder (4 or 40 mg) was applied, unoccluded, to the skin of rats for 4 h, once/week for 4 weeks. Rats were given a 10-min aerosol challenge to 40 mg/m³ TMA 2 weeks after the last dermal exposure (day 35). Another group was challenged on day 35 and again 18–24 months later. Respiratory enhanced pause (Penh), pulmonary histopathology and inflammation and specific IgE titres were measured.
Results Rats produced dose-dependent specific IgE titres after exposure and developed early-phase (EAR) and late-phase airway responses (LAR) after airway challenge to TMA aerosol as well as airway eosinophilic inflammation. Specific airway responses were still manifested after a second TMA airway challenge given 18–24 months following the initial airway challenge. While persistent, airway inflammation, specific IgE and EAR were significantly attenuated following the second TMA challenge. LAR remained robust at 18–24 months and was not significantly different from the response on day 35.
Conclusions These results demonstrate the persistence of chemical sensitization and further suggest that IgE is not essential for LAR.     

Sphingosine-kinase 1 and 2 contribute to oral sensitization and effector phase in a mouse model of food allergy

Background

Sphingosine-1-phosphate (S1P) influences activation, migration and death of immune cells. Further, S1P was proposed to play a major role in the induction and promotion of allergic diseases. However, to date only limited information is available on the role of S1P in food allergy.

Objective

We aimed to investigate the role of sphingosine-kinase (SphK) 1 and 2, the enzymes responsible for endogenous S1P production, on the induction of food allergy.

Methods and results

Human epithelial colorectal CaCo2 cells stimulated in vitro with S1P revealed a decrease of transepithelial resistance and enhanced transport of FITC labeled OVA. We studied the effect of genetic deletion of the enzymes involved in S1P production on food allergy induction using a mouse model of food allergy based on intragastrically (i.g.) administered ovalbumin (OVA) with concomitant acid-suppression. Wild-type (WT), SphK1^−/− and SphK2^−/− mice immunized with OVA alone i.g. or intraperitoneally (i.p.) were used as negative or positive controls, respectively. SphK1- and SphK2-deficient mice fed with OVA under acid-suppression showed reduced induction of OVA specific IgE and IgG compared to WT mice, but had normal responses when immunized by the intraperitoneal route. Flow cytometric analysis of spleen cells revealed a significantly reduced proportion of CD4⁺ effector T-cells in both SphK deficient animals after oral sensitization. This was accompanied by a reduced accumulation of mast cells in the gastric mucosa in SphK-deficient animals compared to WT mice. Furthermore, mouse mast cell protease-1 (mMCP-1) levels, an IgE-mediated anaphylaxis marker, were reliably elevated in allergic WT animals.

Conclusion

Modulation of the S1P homeostasis by deletion of either SphK1 or SphK2 alters the sensitization and effector phase of food allergy.     

Common vaccine antigens inhibit allergen-induced sensitization and airway hyperresponsiveness in a murine model

BACKGROUND: Co-vaccination with cellular pertussis vaccine down-regulates allergic sensitization to diphtheria and tetanus antigens. Using a murine model, we investigated whether vaccination with diphtheria/tetanus toxoids, administered separately or simultaneously with the whole cell vaccine of Bordetella pertussis, inhibits subsequent allergen-induced immune and inflammatory responses. METHODS: BALB/c-mice were vaccinated intracutaneously with a combination of diphtheria and tetanus toxoids or a combination of diphtheria and tetanus toxoids with a whole cell vaccine of B. pertussis (three times, days -21 to -7) prior to systemic sensitization (days 1-14) and repeated airway challenges (days 28-30) with ovalbumin. RESULTS: Compared with negative controls, systemic sensitization and airway allergen challenges induced high serum levels of allergen-specific IgE, predominant Th2-type cytokine production, airway inflammation and development of in vivo airway hyperreactivity. Vaccination with diphtheria and tetanus toxoids prior to sensitization suppressed IgE formation and development of eosinophilic airway inflammation. Co-vaccination with a whole cell pertussis vaccine inhibited allergen sensitization, airway inflammation and development of in vivo airway hyperreactivity. Prevention was due to an allergen-specific and general shift from a predominant Th2 towards a predominant Th1 immune response. CONCLUSION: Vaccination with diphtheria and tetanus toxoids alone or in combination with whole cell pertussis vaccine prior to allergen sensitization prevented allergen-induced Th2 immune responses. Vaccine antigens may down-regulate allergic responses to a range of common allergens.     

乳胶致小鼠气道炎症的研究

目的：以乳胶为致敏原，建立具有哮喘主要特征的小鼠模型，研究乳胶与气道炎症之间的关系。方法：乳胶腹腔注射与滴鼻诱发BALB／C小鼠气道炎症，在激发后2，4小时行支气管肺泡灌洗液(BALF)细胞计数分类、肺组织病理检查、血清总IgE及乳胶特异性IgE(sIgE)测定并检测IL-5、IFN-γ的表达。结果：乳胶诱发后，实验组小鼠BALF中细胞总数及嗜酸粒细胞百分比升高；肺组织病理切片示支气管上皮增生、脱落，粘液腺分泌现象，支气管痉挛、收缩，炎症细胞浸润；血清总IgE及乳胶sIgE水平升高；BALF及肺组织局部IFN-γ减少，IL-5增高。结论：用乳胶蛋白作为致敏原，采用腹腔致敏与多次滴鼻激发可使BALB/C小鼠发生气道炎症，具有与变应原诱导的人类哮喘迟发反应相一致的主要特征：气道变应性炎症；外周血IgE浓度升高；肺组织中Th2型CK表达占优势。     

Cyclophosphamide augments inflammation by reducing immunosuppression in a mouse model of allergic airway disease

BACKGROUND: Allergic asthma is a TH2 cell-driven immunological disease, characterized by eosinophilic inflammation. The cytotoxic agent cyclophosphamide paradoxically augments several immune responses. OBJECTIVE: We studied the proposal that cyclophosphamide may aggravate airway inflammation in allergic mice, and these features might result from the loss of regulatory T cells. METHODS: BALB/c mice were immunized with ovalbumin on days 0 and 14 and challenged with aerosolized ovalbumin from days 21 to 27. Some mice also received cyclophosphamide on days -2 and 12. RESULTS: In the lungs of cyclophosphamide-treated animals, pronounced worsening of inflammatory features was noted, including increased eosinophil infiltration, epithelial thickness, mucus occlusion, and eosinophil numbers in bronchoalveolar lavage fluid. There was also increased total and ovalbumin-specific serum IgE, increased IL-4 and IL-5 secretion by peritracheal lymph node cells, and reduced lung mRNA expression of IL-10 and TGF-beta in animals treated with cyclophosphamide. The expression of FoxP3, a marker of regulatory T cells, was significantly reduced in lymphoid organs after the second injection of cyclophosphamide, and in the lung tissue after allergen challenge in cyclophosphamide-treated mice. Lung IL-10+CD4+ T cells and cytotoxic T lymphocyte-associated antigen 4+CD4+ T cells were reduced after allergen challenge in cyclophosphamide-treated mice. CONCLUSION: Cyclophosphamide worsened features of allergic pulmonary inflammation in this model, in association with increased production of IgE and TH2 cytokines. The reduced expression of FoxP3 and immunosuppressive cytokines by cyclophosphamide is consistent with the possibility that toxicity to regulatory T cells may contribute to the increased inflammation.     

Prior exposure to immunosuppressive organophosphorus or organochlorine compounds aggravates the TH1- and TH2-type allergy caused by topical sensitization to 2,4-dinitrochlorobenzene and trimellitic anhydride

Immunosuppressive environmental chemicals may increase the potency of allergens and thereby play a role in the development of allergic diseases. This study’s primary objective was to examine the mechanisms behind the relationship between allergic diseases and the immunosuppression induced by some environmental chemicals. We focused on the modulation of allergic potential in vitro and in mice by the organophosphorus pesticide O,O-diethyl-O-4-nitrophenyl-thiophosphate (parathion) and the organochlorine pesticide 1,1,1-trichloro-2,2-bis(4-methoxy-phenyl)ethane (methoxychlor), with respect to the T_H1-type allergen 2,4-dinitrochlorobenzene (DNCB) and the T_H2-type allergen trimellitic anhydride (TMA). Mice (4-week-old) were orally administered parathion or methoxychlor. Four weeks after the final dosing, the mice were sensitized to DNCB or TMA, and T-lymphocyte proliferation measured in their (using a local lymph node assay [LLNA]). In addition, we analyzed T-lymphocytes via surface antigen expression and local cytokine production in auricular lymph nodes after treatment with 0.1% DNCB or 0.3% TMA. The estimated concentration of DNCB and TMA to yield a stimulation index (SI) of cell proliferation of three decreased markedly in parathion- and methoxychlor-pre-treated mice. Pesticide pre-treatment induced marked increases in the number of helper and cytotoxic T-cells, levels of T_H1 and T_H2 cytokines, and gene expression in lymph node cells. According to our results, T_H1- and T_H2-type allergies are aggravated by prior exposure to immunosuppressive environmental chemicals.     

Sequential development of airway hyperresponsiveness and acute airway obstruction in a mouse model of allergic inflammation

BACKGROUND: Mouse models have been established mirroring key features of human bronchial asthma including airway hyperresponsiveness (AHR). Acute airway obstruction in response to an allergen challenge, however, remains to be demonstrated in these models. OBJECTIVE: A mouse model of allergic lung inflammation was employed to analyze the development of specific (allergen-induced) and nonspecific (methacholine-induced) airway obstruction. METHODS: Mice were sensitized to ovalbumin (OVA) and challenged with OVA aerosol twice each week during four weeks. Changes in lung functions were determined by noninvasive head-out body plethysmography. The development of acute airway obstruction after OVA challenge and AHR after methacholine aerosol application were assessed by a decrease in the mid-expiratory flow rate (EF(50)). Results: Two airway challenges were sufficient to induce AHR (5.7 vs. 15 mg/ml methacholine). Further OVA challenges reduced the baseline EF(50) from 1.85 to 1.20 ml/s (4th week) and induced acute airway obstruction. The OVA-induced obstruction was maximal in the 4th week (EF(50) = 0.91 ml/s). CONCLUSION: The development of acute airway obstruction in allergen-sensitized mice was demonstrated by means of head-out body plethysmography. In our model, AHR was observed before the development of airway obstruction.     

Prenatal lipopolysaccharide-exposure prevents allergic sensitization and airway inflammation, but not airway responsiveness in a murine model of experimental asthma

BACKGROUND: Epidemiological evidence underlines the impact of prenatal environmental factors on the development of postnatal allergies. In this regard an inverse correlation between lipopolysaccharide (LPS) exposure and development of childhood allergy has been found. OBJECTIVE: To assess the impact of prenatal LPS exposure on the development of postnatal respiratory allergies in a mouse model of experimental asthma. METHODS: Female BALB/c mice were exposed to LPS before conception and during pregnancy. Several weeks after birth offspring were sensitized to ovalbumin (OVA) followed by aerosol allergen challenges. RESULTS: Prenatal, maternal LPS-exposure enhanced neonatal IFN-gamma, but not IL-4 and IL-2 production. OVA sensitization of prenatally LPS-exposed mice was accompanied by a marked suppression in anti-OVA IgG1 and IgE as well as unchanged IgG2a antibody responses, paralleled by a significant reduction in IL-5 and IL-13 levels following mitogenic stimulation of splenic leucocytes. Assessment of bronchoalveolar lavage fluids following allergen challenges revealed a marked reduction in eosinophils and macrophages in these mice. Surprisingly, development of airway hyper-responsiveness, a hallmark of bronchial asthma, was not affected. CONCLUSION: This study provides first experimental evidence that LPS may already operate in prenatal life in order to modulate the development of allergies in the offspring.     

Time sequence of airway remodeling in a mouse model of chronic asthma: the relation with airway hyperresponsiveness

During the course of establishing an animal model of chronic asthma, we tried to elucidate the time sequence of airway hyperresponsiveness (AHR), airway inflammation, airway remodeling, and associated cytokines. Seven-week-old female BALB/c mice were studied as a chronic asthma model using ovalbumin (OVA). After sensitization, mice were exposed twice weekly to aerosolized OVA, and were divided into three groups depending on the duration of 4 weeks, 8 weeks, and 12 weeks. At each time point, airway responsiveness, inflammatory cells, cytokines in bronchoalveolar lavage fluids (BALF), serum OVA-specific IgE, IgG1, IgG2a, and histological examination were carried out. AHR to methacholine, increased levels of OVA-specific IgG1 and IgG2a, and goblet cell hyperplasia were continuously sustained at each time point of weeks. In contrast, we observed a time-dependent decrease in serum OVA-specific IgE, BALF eosinophils, BALF cytokines such as IL-13, transforming growth factor-beta1, and a time-dependent increase in BALF promatrix metalloproteinase-9 and peribronchial fibrosis. In this OVA-induced chronic asthma model, we observed airway remodelings as well as various cytokines and inflammatory cells being involved in different time-dependent manners. However, increased airway fibrosis did not directly correlate with a further increase in airway hyperresponsiveness.