Pollen-induced airway inflammation, hyper-responsiveness and apoptosis in a murine model of allergy

BACKGROUND: Previous studies indicate that murine models are useful tools for studying the allergic diseases, including certain aspects of bronchial asthma such as cellular tissue inflammation and pulmonary function. OBJECTIVE: To develop an experimental model of allergic lung inflammation based on a relevant human allergen, olive pollen, and to establish the immunological, cellular and functional airway features of the allergic response in this model. METHODS: Induction of systemic allergic response was achieved by the subcutaneous administration of Olea europaea extract in BALB/c mice. Olea-specific Igs (IgG1, IgG2a and IgE) and cytokines from splenocyte cultures IL-4, IL-5 IL-10, IL-13 and IFN-gamma were measured. Allergic airway response was generated by transnasal instillation of the allergens. Airway responsiveness was monitored by non-invasive methacholine inhalation challenge. Lungs were paraffin embedded and histologically analysed. Apoptosis was studied by the TUNEL technique in the lung tissue and through cell cycle analysis by flow cytometry in splenocytes. RESULTS: Our results demonstrate that Olea-sensitized mice develop a specific allergic antibody (IgG1 and IgE) and cytokine (IL-4, IL-5, IL-10 and IL-13) response. After transnasal Olea instillation, they show inflammatory infiltration of lung tissue, mucus secretion and non-specific hyper-responsiveness in the airway. Concomitantly, differences in the rate of apoptosis are observed in the lung cells as well as a significant reduction of spontaneous apoptosis in the splenocytes of allergic mice. CONCLUSION: We present a novel animal model of olive pollen-allergic disease. This model presents traits associated with human allergic asthma and could be an interesting tool in the study of underlying molecular mechanisms and in exploring the therapeutic approaches to this disease.     

Suppression of antigen-specific T- and B-cell responses by intranasal or oral administration of recombinant Bet v 1, the major birch pollen allergen, in a murine model of type I allergy

BACKGROUND: Mucosal (nasal or oral) administration of soluble protein antigens induces a state of antigen-specific immunologic hyporesponsiveness. Several studies have shown that induction of mucosal tolerance can prevent the onset or reduce the severity of certain TH1 -mediated experimental autoimmune diseases. Only a few studies describe similar results for type I allergies, which are caused by excessive TH2 cell activities. OBJECTIVE: We sought to investigate whether mucosal tolerance induction would also be efficient in preventing an allergic type I immune response. METHODS: A murine model of inhalative type I allergy, leading to sensitization to birch pollen and its major allergen Bet v 1 in BALB/c mice, was used. Recombinant Bet v 1 was nasally or orally applied in low doses before sensitization. At the time of death, immediate-type skin tests were performed. Blood was taken, and serum was used for measurement of allergen-specific antibodies. Spleen cell cultures were performed to determine cytokine production (IL-4, IL-5, IL-10, and IFN-gamma), as well as levels of TGF-beta mRNA. RESULTS: Both nasal and oral administration of minimal doses of recombinant Bet v 1 before aerosol sensitization with birch pollen suppressed the allergen-specific antibody production of all isotypes. Consequently, the in vivo type I skin test responses to the allergen were negative in the tolerized, in contrast to the sensitized, group. Moreover, allergen-specific lymphoproliferative responses and cytokine production in vitro (ie, IFN-gamma, IL-4, IL-5, and IL-10) were markedly reduced. In contrast, expression of TGF-beta mRNA was markedly increased in spleen cells from nasally tolerized animals, indicating regulatory mechanisms for tolerance induction. CONCLUSION: We conclude from the present study that nasal, as well as oral, administration of recombinant allergen is an effective way to prevent allergen-specific T- and B-cell responses in a TH2 model.     

Lactoferrin decreases pollen antigen-induced allergic airway inflammation in a murine model of asthma

Pollen grains contain reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and in contact with mucosal surfaces generate superoxide anion (O2*-). In the presence of iron, O2*- may be converted to more reactive oxygen radicals, such as to H2O2 and/or *OH, which may augment antigen-induced airway inflammation. The aim of the study was to examine the impact of lactoferrin (LF), an iron-binding protein, on ragweed (Ambrosia artemisiifolia) pollen extract (RWE)-induced cellular oxidative stress levels in cultured bronchial epithelial cells and accumulation of inflammatory and mucin-producing cells in airways in a mouse model of allergic airway inflammation. Results show that LF lowered RWE-induced increase in cellular reactive oxygen species (ROS) levels in bronchial epithelial cells. Most importantly, LF significantly decreased accumulation of eosinophils into airways and subepithelium of intranasally challenged, sensitized mice. LF also prevented development of mucin-producing cells. Amb a 1, the major allergenic ragweed pollen antigen lacking NADPH oxidase activity, induced low-grade airway inflammation. When administered along with glucose oxidase (G-ox), a superoxide-generating enzyme, Amb a 1 induced robust airway inflammation, which was significantly lowered by LF. Surprisingly, LF decreased also inflammation caused by Amb a 1 alone. Iron-saturated hololactoferrin had only a marginal effect on RWE-induced cellular ROS levels and RWE- or Amb a 1 plus G-ox-induced inflammation. We postulate that free iron in the airways chemically reduces O2*- to more reactive species which augment antigen-induced inflammation in a mouse model of asthma. Our results suggest the utility of LF in human allergic inflammatory disorders.     

Allergenic contribution of the IgE-reactive domains of the 1,3-beta-glucanase Ole e 9: diagnostic value in olive pollen allergy.

BACKGROUND: Designing of methods for an accurate diagnosis is a main goal of allergy research. Olive pollen allergy is currently diagnosed using commercially available pollen extracts that do not allow identification of the molecules that elicit the disease. OBJECTIVE: To analyze the suitability of using the N- and C-terminal domains (NtD and CtD, respectively) of the 1,3-beta-glucanase Ole e 9, a major allergen from olive pollen, for in vitro diagnosis. METHODS: Serum samples from 55 olive-allergic patients were assayed using enzyme-linked immunosorbent assay to study hypersensitive patients with IgE reactivity to Ole e 9. The specific IgEs to NtD and CtD, obtained by recombinant technology, were determined by means of immunoblotting, enzyme-linked immunosorbent assay, and inhibition assays. RESULTS: Thirty-one of 33 serum samples from Ole e 9-allergic patients were IgE reactive to recombinant NtD (rNtD) (n = 26 [79%]), recombinant CtD (rCtD) (n = 22 [67%]), or both (n = 17 [52%]). Nine patients (27%) were exclusively reactive to rNtD and 5 (15%) to rCtD. Inhibition assays of IgE binding to Ole e 9 with a mixture of both domains abolished 90% of the binding, whereas 44% and 45% were abolished when rNtD and rCtD were used, respectively. CONCLUSIONS: Because sensitization to NtD or CtD of Ole e 9 could be correlated to vegetable food-latex-pollen cross-reactivity processes or to the exacerbation and persistence of asthma, respectively, these molecules could be used in vitro as markers of disease to classify patients and to design a patient-tailored immunotherapy approach.     

Intranasal treatment with a recombinant hypoallergenic derivative of the major birch pollen allergen Bet v 1 prevents allergic sensitization and airway inflammation in mice

BACKGROUND: The major birch pollen allergen Bet v 1 represents one of the most prevalent environmental allergens responsible for allergic airway inflammation. OBJECTIVE: In the present study we sought to compare the complete recombinant Bet v 1 allergen molecule with genetically produced hypoallergenic fragments of Bet v 1 regarding mucosal tolerance induction in a mouse model of allergic asthma. METHODS: BALB/c mice were intranasally treated with recombinant Bet v 1 or with two recombinant Bet v 1 fragments (F I: aa 1-74; F II: aa 75-160) prior to aerosol sensitization with birch pollen and Bet v 1. RESULTS: Intranasal application of F II, containing the major T cell epitope, led to significant reduction of IgE/IgG1 antibody responses, in vitro cytokine production (IL-5, IFN-gamma, IL-10) and negative immediate cutaneous hypersensitivity reactions comparable to the pretreatment with the complete rBet v 1 allergen. Moreover, airway inflammation (eosinophilia, IL-5) was inhibited by the pretreatment with either the complete Bet v 1 or F II. However, for prevention of airway hyperresponsiveness the complete molecule was required. The mechanisms leading to immunosuppression seemed to differ in their dependence on the conformation of the molecules, since tolerance induced with the complete Bet v 1, but not with F II, was transferable with spleen cells and associated with increased TGF-beta mRNA levels. CONCLUSION: We conclude that mucosal tolerance induction with recombinant allergens and genetically engineered hypoallergenic derivatives thereof could provide a convenient and safe intervention strategy against type I allergy.     

Mucosal tolerance as therapy of type I allergy: intranasal application of recombinant Bet v 1, the major birch pollen allergen, leads to the suppression of allergic immune responses and airway inflammation in sensitized mice

BACKGROUND: Several studies have demonstrated that mucosal administration of soluble antigens can prevent the onset or reduce the severity of certain autoimmune diseases or allergies. Few studies exist showing the efficacy of mucosal tolerance for therapy of such diseases. OBJECTIVE: The aim of the present study was to modulate an allergic immune response by intranasal antigen administration in an already sensitized organism. METHODS: A murine model of allergic asthma to birch pollen (BP) and its major allergen Bet v 1 was utilized. Sensitized mice were intranasally treated with recombinant (r)Bet v 1 in different concentrations and at different intervals. On the day the mice were killed, blood and bronchoalveolar lavage fluids were taken and immediate type I skin tests were performed. T cell proliferation and cytokine production (interleukin (IL)-5, interferon (IFN)-gamma) were measured in spleen and lung cell cultures. RESULTS: Mucosal treatment with rBet v 1 (3 x 50 microg in 4 day intervals) led to a reduction of type I skin reactions, suppressed immunoglobulin (Ig)G1/IgE antibody levels and markedly decreased IL-5 and IFN-gamma production in vitro in spleen and lung cell cultures. Moreover, lung inflammation (i.e. eosinophilia and IL-5 levels in bronchoalveolar lavage fluids) was significantly suppressed by the treatment. CONCLUSION: Our results demonstrate that intranasal treatment with rBet v 1 reduced systemic allergic immune responses as well as airway inflammation in BP-sensitized mice. We therefore suggest that mucosal tolerance induction with recombinant allergens could be a promising concept for the therapy of allergic diseases.     

Histamine type 1 receptor deficiency reduces airway inflammation in a murine asthma model

BACKGROUND: Histamine plays an important role in immediate and late immune responses. The histamine type 1 (H1) receptor is expressed on several immune cell populations, but its role in a murine model of asthma remains unclear. The present study evaluated the role of histamine H1 receptors in airway allergic inflammation by comparing the development of bronchial asthma in histamine H1 receptor gene knockout (H1RKO) and wild-type mice. METHODS: H1RKO and wild-type mice were sensitized by intraperitoneal injection of ovalbumin (OVA) or saline, and then challenged with aerosolized OVA or saline. Ventilatory timing in response to inhaled methacholine was measured, and samples of blood, bronchoalveolar lavage, and lung tissues were taken 24 h after the last OVA challenge. RESULTS: OVA-treatedwild-type mice showed significantly increased airway eosinophilic infiltration, and airway response to methacholine compared to OVA-treated H1RKO mice. The serum level of immunoglobulin E and levels of interleukin (IL)-4, IL-5, IL-13, and TGF-beta1 in bronchoalveolar lavage fluid were lower in OVA-treated H1RKO mice than in OVA-treated wild-type mice, but there was no significant difference in interferon-gamma expression. Overall, deletion of histamine H1 receptors reduced allergic responses in a murine model of bronchial asthma. CONCLUSION: Histamine plays an important role via H1 receptors in the development of T helper type 2 responses to enhance airway inflammation.     

Intranasal immunization with a dominant T-cell epitope peptide of a major allergen of olive pollen prevents mice from sensitization to the whole allergen

Mucosal tolerance induction with vaccines based on peptides representing T-cell epitopes of allergens is a promising way for treating allergic diseases. Ole e 1 is the main allergen of olive pollen, which is an important cause of allergy in Mediterranean countries. The aim of this study was to evaluate the ability of the peptide T109-K130 containing a dominant T-cell epitope of Ole e 1, to modulate the allergen-specific immune response in a prophylactic mouse model. Mice were intranasally treated with the peptide 1 week prior to sensitization with Ole e 1. Blood, lungs and spleens were collected and analysed for immune response. Intranasal pretreatment of mice with the peptide led to suppress serum specific IgE, IgG1 and IgG2a antibody levels, and markedly reduced proliferative T-cell response and Th2-cytokine production, but increased IFN-gamma secretion in spleen cell cultures. Increased mRNA IL-10 levels were observed in lungs from pretreated mice. Pathologic alterations of the lung associated with airway inflammation (peribronchial/perivascular infiltrates, eosinophilia and mucus production) were significantly suppressed after pretreatment. Similar results were obtained when mice were sensitized 10 weeks after treatment. Our results demonstrate that intranasal administration of a single T-cell peptide protects mice against subsequent sensitization to the allergen, possibly via IFN-gamma and IL-10. This study emphasizes the usefulness of nasal peptide T-based vaccines against allergy.     

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.     

Suppressive versus stimulatory effects of allergen/cholera toxoid (CTB) conjugates depending on the nature of the allergen in a murine model of type I allergy.

Recent reports have demonstrated that feeding small amounts of antigen conjugated to the B subunit of cholera toxin (CTB) suppress immune responses in experimental models of certain Th1-based autoimmune diseases. We have established a model of aerosol sensitization leading to Th2-mediated allergic immune responses in BALB/c mice. In the present study two different antigens, the dietary antigen ovalbumin (OVA) and the inhalant allergen Bet v 1 (the major birch pollen allergen), chemically coupled to recombinant CTB were tested for their potential to influence Th2-like immune responses. Intranasal administration of OVA-CTB prior to sensitization with OVA led to a significant decrease of antigen-specific IgE antibody levels, but a marked increase of OVA-specific IgG2a antibodies as compared to non-pretreated, sensitized animals. Antigen-specific lympho-proliferative responses in vitro were reduced by 65% in the pretreated group; IL-5 and IL-4, but not IFN-gamma, production were markedly decreased in responder cells of lungs and spleens of nasally pretreated mice. In contrast, mucosal administration of rBet v 1-CTB conjugates prior to sensitization led to an up-regulation of allergen-specific IgE, IgG1 and IgG2a, increased in vitro lympho-proliferative responses as well as augmented production of IL-5, IL-4, IL-10 and IFN-gamma. Intranasal administration prior to sensitization of unconjugated allergens showed also contrasting effects: OVA could not significantly influence antigen-specific antibody or cytokine production, whereas intranasal pretreatment with unconjugated Bet v 1 suppressed allergen-specific immune responses in vivo and in vitro. These results demonstrated that the two antigens--in conjugated as in unconjugated form--had different effects on the Th2 immune responses. We therefore conclude that the tolerogenic or immunogenic properties of CTB--and probably also other antigen-delivery systems--strongly depend on the nature of the coupled antigen-allergen.     

Suppressive versus stimulatory effects of allergen/cholera toxoid (CTB) conjugates depending on the nature of the allergen in a murine model of type I allergy.

Recent reports have demonstrated that feeding small amounts of antigen conjugated to the B subunit of cholera toxin (CTB) suppress immune responses in experimental models of certain T(h)1-based autoimmune diseases. We have established a model of aerosol sensitization leading to T(h)2-mediated allergic immune responses in BALB/c mice. In the present study two different antigens, the dietary antigen ovalbumin (OVA) and the inhalant allergen Bet v 1 (the major birch pollen allergen), chemically coupled to recombinant CTB were tested for their potential to influence T(h)2-like immune responses. Intranasal administration of OVA-CTB prior to sensitization with OVA led to a significant decrease of antigen-specific IgE antibody levels, but a marked increase of OVA-specific IgG2a antibodies as compared to non-pretreated, sensitized animals. Antigen-specific lympho-proliferative responses in vitro were reduced by 65% in the pretreated group; IL-5 and IL-4 production were decreased in responder cells of lungs and spleens of nasally pretreated mice. In contrast, mucosal administration of rBet v 1-CTB conjugates prior to sensitization led to an up-regulation of allergen-specific IgE, IgG1 and IgG2a, increased in vitro lympho-proliferative responses as well as augmented production of IL-5, IL-4, IL-10 and IFN-gamma. Intranasal administration prior to sensitization of unconjugated allergens showed also contrasting effects: OVA could not significantly influence antigen-specific antibody or cytokine production, whereas intranasal pretreatment with unconjugated Bet v 1 suppressed allergen-specific immune responses in vivo and in vitro. These results demonstrated that the two antigens-in conjugated as in unconjugated form-had different effects on the T(h)2 immune responses. We therefore conclude that the tolerogenic or immunogenic properties of CTB-and probably also other antigen-delivery systems-strongly depend on the nature of the coupled antigen-allergen.     

Serological and skin-test diagnosis of birch pollen allergy with recombinant Bet v I, the major birch pollen allergen

Background Type I allergy represents a severe health problem in industrialized countries where up to 20% of the population suffer froin allergic rhinitis, conjunctivitis and allergic asthma bronchiale and in severe cases from anaphylaxis. leading to death.
Objective The aim of this study was to evaluate recombinant Bet v I, the major birch pollen allergen for in vivo and in vitro diagnosis of birch pollen allergy.
Methods A group of 51 birch pollen allergic patients and eight non-allergic control individuals were tested for birch pollen allergy by skin-prick and intradennal testing, comparing commercial birch pollen extracts with recombinant Bet v I. Quantitative and qualitative serological testing was done with natural and recombinant allergens by radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA) and immunoblotting.
Results Recombinant Bet v I allowed accurate in vivo and in vitro diagnosis of tree pollen allergy in 49/51 patients tested. No false positive results were obtained in any in vitro assay system (ELISA. Westernblot) or by skin testing (skin-prick, intradermal test) with recombinant Bet v I.
Conclusion Our results document that recombinant Bet v I produced in bacterial expression systems allows accurate in vitro and in vivo diagnosis of birch pollen allergy in > 95% of birch pollen allergic patients.     

Allergic inflammation induced by a Penicillium chrysogenum conidia-associated allergen extract in a murine model

BACKGROUND: Recent evidence has shown that viable conidia from the fungus Penicillium chrysogenum induce allergic effects in mice. The present study was conducted to determine the specific allergic dose response of C57BL/6 mice to the protease extract, Pen ch, isolated from viable P. chrysogenum conidia. METHODS: Mice were treated with primary intraperitoneal (IP) injections of 10 or 100 microg of Pen ch adsorbed to alum, followed by weekly IP injections of 0.1, 1.0, or 10.0 microg Pen ch with alum for 4 weeks, and with 10.0 microg of Pen ch by intranasal (IN) inoculations the final 2 weeks before killing. RESULTS: Intraperitoneal injections of 10 and 100 microg of Pen ch for 5 weeks followed by 2 weeks of IN instillation of 10 microg induced significant increases of total serum immunoglobulin (Ig)E and IgG(1). Bronchoalveolar lavage cell counts revealed increased numbers of eosinophils and neutrophils. Histopathological examination of lungs detected perivascular inflammation by eosinophils and neutrophils and increased mucous production. CONCLUSIONS: The data presented in this study indicate that sensitization to protease allergens released by viable P. chrysogenum conidia in vivo induce a strong allergic inflammatory response in a murine model, which could have implications for people exposed to high levels of conidia of this organism.     

Rapid activation of nuclear factor-kappaB in airway epithelium in a murine model of allergic airway inflammation

Bronchiolar epithelium is postulated to play a critical role in the orchestration of responses to inhaled allergens, and may contribute to the pathogenesis of asthma. Using a murine model of allergic airway inflammation and hyperresponsiveness, we demonstrate in mice sensitized with ovalbumin (OVA) that following a single challenge with nebulized OVA, a rapid and protracted activation of inhibitor of kappa B kinase (IKK) occurred in lung tissue. IKK activation was followed by nuclear localization of nuclear factor (NF)-kappaB within the bronchiolar epithelium and increased luciferase activity in lungs of mice containing a NF-kappaB-dependent reporter gene. Challenge of sensitized mice with OVA also induced mRNA expression of the chemokines, macrophage inflammatory protein-2 (MIP-2) and eotaxin in lung tissue, which corresponded temporally with the observed influx of neutrophils and eosinophils, respectively, into the airspaces. Using laser capture microdissection and quantitative polymerase chain reaction, we demonstrated that MIP-2 and eotaxin were predominantly expressed in bronchiolar epithelium, in contrast to distal regions of the lungs, which expressed lower or undetectable levels of these mRNAs. These studies strengthen the potential importance of the bronchiolar epithelial cell as a source of production of NF-kappaB-dependent mediators that play a role in asthma.     

An intranasal administration of Lactococcus lactis strains expressing recombinant interleukin‐10 modulates acute allergic airway inflammation in a murine model

Background Around 300 million people world‐wide suffer from asthma, and the prevalence of allergic diseases has increased. Much effort has been used in the study of mechanisms involved in the immune response observed in asthma to intervene for the treatment of this condition. During inflammation in asthma, Th2 cytokines and eosinophils are essential components of the host immune system. Furthermore, for therapeutic interventions against this disease, IL‐10 is an important cytokine because it has a central role in the regulation of inflammatory cascades. Objective To evaluate the immunomodulatory effect of Lactococcus lactis strains expressing recombinant IL‐10 in a mouse model of ovalbumin (OVA)‐induced acute airway inflammation. Methods L. lactis expressing recombinant IL‐10 in a cytoplasmic (LL‐CYT) or secreted form (LL‐SEC) and wild‐type (LL‐WT) were used. IL‐10 production by the recombinant strains was evaluated by ELISA. After an intranasal administration of L. lactis producing recombinant IL‐10 and the induction of acute allergic airway inflammation in mice, blood samples were collected to detect IgE anti‐OVA, and bronchoalveolar lavage (BAL) was harvested for eosinophil count. Additionally, the lungs were collected for the detection of the eosinophil peroxidase (EPO) activity, measurement of cytokines and chemokines and evaluation of pathology. Results Mice that received LL‐CYT and LL‐SEC strains showed a significant decrease in eosinophils numbers, EPO activity, anti‐OVA IgE and IgG1 levels, IL‐4 and CCL3 production and pulmonary inflammation and mucus hypersecretion, compared with the asthmatic group. Only the LL‐CYT/OVA group showed reduced levels of IL‐5, CCL2, CCL5 and CCL11. Conclusion Treatment with L. lactis producing recombinant IL‐10 used in this study (LL‐CYT and LL‐SEC) modulated experimental airway inflammation in the mouse model independently of Treg cells. Additionally, the LL‐CYT strain was more efficient in the suppression of lung inflammation. Cite this as: F. A. V. Marinho, L. G. G. Pacífico, A. Miyoshi, V. Azevedo, Y. Le Loir, V. D. Guimarães, P. Langella, G. D. Cassali, C. T. Fonseca and S. C. Oliveira, Clinical & Experimental Allergy, 2010 (40) 1541–1551.