Allergen-induced Degranulation Research Articles

Effects of Ser47-Point Mutation on Conformation Structure and Allergenicity of the Allergen of Der p 2, a Major House Dust Mite Allergen.

PurposeHypoallergenic recombinant Der p 2 has been produced by various genetic manipulations, but mutation of a naturally polymorphic amino acid residue known to affect IgE binding has not been studied. This study aimed to determine the effect of a point mutation (S47W) of residue 47 of Der p 2 on its structure and immunoglobulin (Ig) E binding. Its ability to induce pro-inflammatory responses and to induce blocking IgG antibody was also determined.MethodsS47 of recombinant Der p 2.0110, one of the predominant variants in Bangkok, was mutated to W (S47W). S47W secreted from Pichia pastoris was examined for secondary structure and for the formation of a hydrophobic cavity by 8-Anilino-1-naphthalenesulfonic acid (ANS) staining. Monoclonal and human IgE-antibody binding was determined by enzyme-linked immunosorbent assay. Allergen-induced degranulation by human epsilon receptor expressed-rat basophil was determined. Stimulation of the pro-inflammatory cytokine interleukin (IL)-8 release from human bronchial epithelial (BEAS2B) cells and inhibition of IgE binding to the wild type allergen by S47W-induced IgG were determined.ResultsS47W reduced secondary structure and failed to bind the hydrophobic ANS ligand as well as a monoclonal antibody known to be dependent on the nature of the side chain of residue 114 in an adjacent loop. It could also not stimulate IL-8 release from BEAS2B cells. IgE from house dust mite (HDM)-allergic Thais bound S47W with 100-fold weaker avidity, whereas IgE of HDM-allergic Australians did not. S47W still induced basophil degranulation, although requiring higher concentrations for some subjects. Anti-S47W antiserum-immunized mice blocked the binding of human IgE to wild type Der p 2.ConclusionsThe mutant S47W had altered structure and reduced ability to stimulate pro-inflammatory responses and to bind IgE, but retained its ability to induce blocking antibodies. It thus represents a hypoallergen produced by a single mutation of a non-solvent-accessible amino acid.

Bet v 1 – a Trojan horse for small ligands boosting allergic sensitization?

Birch pollen allergy represents the main cause of winter and spring pollinosis in the temperate climate zone of the northern hemisphere and sensitization towards Bet v 1, the major birch pollen allergen, affects over 100 million allergic patients. The major birch pollen allergen Bet v 1 has been described as promiscuous acceptor for a wide variety of hydrophobic ligands. In search of intrinsic properties of Bet v 1, which account responsible for the high allergenic potential of the protein, we thought to investigate the effects of ligand-binding on immunogenic as well as allergenic properties. As surrogate ligand of Bet v 1 sodium deoxycholate (DOC) was selected. Recombinant and natural Bet v 1 were characterised physico-chemically as well as immunologically in the presence or absence of DOC, and an animal model of allergic sensitization was established. Moreover, human IgE binding to Bet v 1 was analysed by nuclear magnetic resonance (NMR) spectroscopy. Ligand-binding had an overall stabilizing effect on Bet v 1. This translated in a Th2 skewing of the immune response in a mouse model. Analyses of human IgE binding on Bet v 1 in mediator release assays revealed that ligand-bound allergen-induced degranulation at lower concentrations; however, in basophil activation tests with human basophils ligand-binding did not show this effect. For the first time, human IgE epitopes on Bet v 1 were determined using antibodies isolated from patients' sera. The IgE epitope mapping of Bet v 1 demonstrated the presence of multiple binding regions. Deoxycholate binding stabilizes conformational IgE epitopes on Bet v 1; however, the epitopes themselves remain unaltered. Therefore, we speculate that humans are exposed to both ligand-bound and free Bet v 1 during sensitization, disclosing the ligand-binding cavity of the allergen as key structural element.

Sake Lees Fermented with Lactic Acid Bacteria Prevents Allergic Rhinitis-Like Symptoms and IgE-Mediated Basophil Degranulation

We tested the effect of oral administration of fermented sake lees with lactic acid bacteria (FESLAB) on a murine model of allergic rhinitis upon immunization and nasal sensitization with ovalbumin (OVA). We used Lactobacillus paracasei NPSRIk-4 (isolated from sake lees), and L. brevis NPSRIv-8 (from fermented milk) as starter strains to produce the FESLAB. Oral FESLAB administration resulted in the development of significantly fewer sneezing symptoms than those seen in sham control animals given sterile water. We also found that FESLAB suppressed the allergen-induced degranulation of RBL2H3 rat basophilic leukemia cells.

IL-9 Promotes but Is Not Necessary for Systemic Anaphylaxis

Anaphylaxis represents an extreme form of allergic reaction, consisting of a sensitization phase during which allergen-specific IgE are produced and an acute effector phase triggered by allergen-induced degranulation of mast cells. We studied the role of IL-9, a Th2 cytokine implicated in asthma, in different models of murine anaphylaxis. Using a passive model of systemic anaphylaxis, in which anti-DNP IgE Abs were administered before challenge with DNP-BSA, we found that IL-9-transgenic mice or wild-type mice treated with IL-9 for 5 days were highly sensitive to fatal anaphylaxis. This effect was reproduced in both anaphylaxis-susceptible and -resistant backgrounds (FVB/N or [FVB/N x BALB/c] F(1) mice, respectively) and correlated with increased serum concentrations of mouse mast cell protease-1 level, a protein released upon mast cells degranulation. By contrast, IL-9 did not increase the susceptibility to passive cutaneous anaphylaxis. IL-9 expression also increased the susceptibility to fatal anaphylaxis when mice were sensitized by immunization against OVA before challenge with the same Ag. In this model, serum from sensitized, IL-9-transgenic mice was more potent in transferring susceptibility to OVA challenge into naive mice, indicating that IL-9 also promotes the sensitization stage. Finally, using IL-9R-deficient mice, we found that despite its anaphylaxis-promoting activity, IL-9 is dispensable for development of both passive and active anaphylaxis, at least in the C57BL/6 mouse background. Taken together, the data reported in this study indicate that IL-9 promotes systemic anaphylaxis reactions, acting at both the sensitization and effector stages, but is not absolutely required for this process.

Saturation of Immunoglobulin E (IgE) Binding Sites by Polyclonal IgE Does Not Explain the Protective Effect of Helminth Infections against Atopy

One hypothesis for the decreased rates of atopy observed among helminth-infected individuals is that parasite-induced polyclonal immunoglobulin E (IgE) out-competes allergen-specific IgE for FcepsilonRI binding on basophils and mast cells. In experiments with fresh blood drawn from filaria-infected patients, we found no association between ratios of polyclonal to Brugia malayi antigen (BmAg)-specific IgE (range, 14:1 to 388:1) and basophil responses to BmAg as measured by histamine release. Using serum samples from a filaria-infected patient who also had dust mite (Dermatophagoides pteronyssinus)-specific IgE antibodies from time points with various ratios of polyclonal to D. pteronyssinus-specific IgE (16:1 to 86:1), we demonstrated that increased ratios of polyclonal to D. pteronyssinus-specific IgE did not attenuate basophil sensitization as measured by D. pteronyssinus-specific histamine release. Suppression of histamine release was likely not observed in either of these sets of experiments because polyclonal to antigen-specific IgE ratios were not sufficiently high, as concurrent passive sensitization of basophil experiments required ratios of polyclonal to antigen-specific IgE of greater than 500:1 to suppress basophil histamine release. Further, the intensity of IgE staining in basophil populations from 20 patients with active filaria infections correlated strongly with total serum IgE levels (rho = 0.698; P = 0.0024) with no plateau in intensity of IgE staining, even though some patients had total IgE levels of greater than 10,000 ng/ml. Our data therefore suggest that in helminth infections (and in filarial infections in particular), the ratios of polyclonal to allergen-specific IgE rarely reach those levels necessary to inhibit allergen-specific IgE-FcepsilonRI binding and to suppress allergen-induced degranulation of mast cells and basophils.

Degranulation status of airway tissue eosinophils in mouse models of allergic airway inflammation.

Eosinophil degranulation is a characteristic feature of asthma and allergic rhinitis. However, degranulated eosinophils have not been convincingly demonstrated in the common mouse models of these airway diseases. This study uses eosinophil peroxidase (EPO) histochemistry and transmission electron microscopy (TEM) analysis to assess eosinophil degranulation in the airways of ovalbumin (OVA)-sensitized and challenged BALB/c and C57BL/6 mice. Using TEM we also examined mouse and human blood eosinophils after in vitro incubation with formyl-Met-Leu-Phe (fMLP) or phorbol myristate acetate (PMA). Although OVA exposure induced significant nasal and lung eosinophilia, we did not observe any of the known cellular processes by which eosinophils release their granule products, i.e., eosinophil cytolysis, piecemeal degranulation, and exocytosis. The occurrence of other allergen-induced degranulation events was ruled out because no difference in granule morphology was observed between lung-tissue eosinophils and blood or bone-marrow eosinophils from control animals. Accordingly, there was no detectable extracellular EPO in lung tissues of allergic mice. Similarly, mouse blood eosinophils remained nondegranulated in vitro in the presence of fMLP and PMA, whereas the same treatment of human eosinophils resulted in extensive degranulation. This investigation indicates that OVA-induced airway inflammation in the present mouse strains does not involve significant eosinophil degranulation. It is speculated that this dissimilarity from the human disease may be due to a fundamental difference in the regulation of mouse and human eosinophils.

IL-13 overexpression predisposes to anaphylaxis following antigen sensitization.

Anaphylaxis represents an extreme form of allergic reaction. This acute-phase component of allergy and asthma is triggered by allergen-induced degranulation of mast cells following the cross-linking of cell surface-bound, allergen-specific IgE, resulting in the liberation of inflammatory mediators and the development of bronchoconstriction. We used IL-13 transgenic mice to investigate the role of this Th2 cell-derived cytokine in the onset of allergic disease. Strikingly, IL-13-transgenic mice were highly predisposed to fatal anaphylaxis following Ag sensitization. This response correlated with substantially elevated levels of circulating Ag-specific IgE, mast cell degranulation, and histamine release. Furthermore, allergen exposure also induced phenotypic changes typical of asthma, including pulmonary fibrosis, goblet cell hyperplasia, elevated Th2 cytokines, eosinophilia, and airways occluded by mucus and Charcot-Leyden crystals. Expression of IL-4 was not required for the induction of IgE-mediated responses. These data represent the first characterization of a functional role for IL-13-induced IgE in the generation of immediate hypersensitivity reactions and highlight the importance of IL-13 in the development of the symptoms of atopy. The systemic regulation of this response makes these mice an important resource for studying atopic responses.

Mast cell tryptase release and asthmatic responses to allergen increase with regular use of salbutamol

Background: Increased asthmatic responses to allergen, both early and late, have been demonstrated after regular use of β2-agonists in as few as 7 days. Desensitization of β2-adrenergic receptors on airway mast cells may contribute to this effect by allowing greater release of mast cell mediator on allergen-induced degranulation. Tryptase released from lung mast cells can be measured in serum 1 hour after allergen challenge and serves as a marker of mast cell degranulation. Objective: To examine the effect of regular treatment with salbutamol, a β2-agonist, on mast cell mediator release after allergen challenge and its influence on the early asthmatic response (EAR) and the late allergic response, we measured the EAR, serum tryptase levels, the 7-hour FEV1, and sputum tryptase levels and cell profiles. Methods: We conducted a placebo-controlled, double-blind, randomized cross-over comparison of treatments for 10 days with either a salbutamol metered-dose inhaler (100 μg, 2 puffs 4 times daily) or a matched placebo inhaler with at least a 7-day washout between treatments. Atopic subjects (n = 14) with mild-to-moderate asthma performed same-dose allergen inhalation tests after both treatments 12 to 15 hours after the last dose of study inhaler. Baseline and 7-hour FEV1 and the EAR to allergen were measured by using spirometry; venous blood was drawn at 1 hour for analysis of serum tryptase; and sputum was induced and collected at 1 and 7 hours. Results: Salbutamol treatment resulted in a significantly greater EAR (20% ± 1.6% [SEM] vs 15% ± 2.1%; P = .047); increased 1-hour serum tryptase levels (9.09 ± 1.57 vs 7.52 ± 1.12 μg/L; P = .011); increased proportions of eosinophils in the 7-hour sputum sample (39.1% ± 5.1% vs 28.4% ± 4.4%; P < .05); increased proportion of metachromatic cells in the 7-hour sputum sample (4.4% ± 1.1% vs 2.2% ± 0.6%; P = .032); and lower 7-hour FEV1 (2.77 ± 0.18 vs 2.97 ± 0.20 L; P = .014). Baseline FEV1 was not significantly different after salbutamol treatment compared with values after placebo treatment (2.90 ± 0.20 vs 3.00 ± 0.19 L; P = .11). Conclusion: Regular 10-day treatment with salbutamol increases the allergen-induced release of mediator from airway mast cells, and this is reflected in an increased EAR to allergen. Late-phase responses to allergen were also enhanced, as demonstrated by decreased 7-hour FEV1 and increased eosinophilia and percentage of metachromatic cells in the 7-hour sputum sample. Increased allergen-induced mast cell degranulation could, in part, explain the increased asthmatic responses to allergen after β2-agonist treatment and could contribute to the deterioration of asthma control that is associated with regular use of β2-agonist by potentiating allergic inflammation. (J Allergy Clin Immunol 2000;106:57-64.)

In vitro allergen-induced degranulation of pulmonary mast cells from horses with recurrent airway obstruction (heaves)

Abstract Objective To determine the capacity of pulmonary mast cells (PMC) to degranulate in response to various potential allergens and other secretagogues in horses with recurrent airway obstruction (heaves) and clinically normal horses before and after exposure to moldy hay. Animals 5 horses with heaves and 5 clinically normal horses. Procedures Heaves was characterized as an increased clinical respiratory score and maximum change in transpulmonary pressure of &gt; 20 cm H2O after exposure. Bronchoalveolar lavage was performed during each period. Washed and resuspended cells were exposed for 20 minutes at 37 C with whole reconstituted freeze-dried preparations of Aspergillus fumigatus, Alternaria tenuis, and Ambrosia elation, fungal extracts of Aspergillus fumigatus, Alternaria tenuis, and Micropolyspora faeni; A23187; and compound 48/80. Histamine release (HR) was used as a marker of degranulation. Results Compared with clinically normal horses, HR was significantly greater from PMC from horses with heaves during remission and exacerbation in response to whole preparations and extracts of Aspergillus fumigatus and whole preparations of Alternarla tenuis. Extracts of Alternaria tenuis caused significantly greater HR from PMC from horses with heaves during exacerbation. Histamine was also released from PMC in response to A23187 and to changes in osmolality of the medium, but only as a result of cell lysis by compound 48/80. Conclusions Increased degranulation of PMC after antigenic challenge may contribute to the pathogenesis of heaves in horses. Clinical Relevance Strategies for prevention and treatment that attenuate degranulation of PMC may assist in the clinical management of horses with heaves. (Am J Vet Res 1999;60:841–847)

CME questions to asthma

Background: Increased asthmatic responses to allergen, both early and late, have been demonstrated after regular use of β2-agonists in as few as 7 days. Desensitization of β2-adrenergic receptors on airway mast cells may contribute to this effect by allowing greater release of mast cell mediator on allergen-induced degranulation. Tryptase released from lung mast cells can be measured in serum 1 hour after allergen challenge and serves as a marker of mast cell degranulation. Objective: To examine the effect of regular treatment with salbutamol, a β2-agonist, on mast cell mediator release after allergen challenge and its influence on the early asthmatic response (EAR) and the late allergic response, we measured the EAR, serum tryptase levels, the 7-hour FEV1, and sputum tryptase levels and cell profiles. Methods: We conducted a placebo-controlled, double-blind, randomized cross-over comparison of treatments for 10 days with either a salbutamol metered-dose inhaler (100 μg, 2 puffs 4 times daily) or a matched placebo inhaler with at least a 7-day washout between treatments. Atopic subjects (n = 14) with mild-to-moderate asthma performed same-dose allergen inhalation tests after both treatments 12 to 15 hours after the last dose of study inhaler. Baseline and 7-hour FEV1 and the EAR to allergen were measured by using spirometry; venous blood was drawn at 1 hour for analysis of serum tryptase; and sputum was induced and collected at 1 and 7 hours. Results: Salbutamol treatment resulted in a significantly greater EAR (20% ± 1.6% [SEM] vs 15% ± 2.1%; P = .047); increased 1-hour serum tryptase levels (9.09 ± 1.57 vs 7.52 ± 1.12 μg/L; P = .011); increased proportions of eosinophils in the 7-hour sputum sample (39.1% ± 5.1% vs 28.4% ± 4.4%; P < .05); increased proportion of metachromatic cells in the 7-hour sputum sample (4.4% ± 1.1% vs 2.2% ± 0.6%; P = .032); and lower 7-hour FEV1 (2.77 ± 0.18 vs 2.97 ± 0.20 L; P = .014). Baseline FEV1 was not significantly different after salbutamol treatment compared with values after placebo treatment (2.90 ± 0.20 vs 3.00 ± 0.19 L; P = .11). Conclusion: Regular 10-day treatment with salbutamol increases the allergen-induced release of mediator from airway mast cells, and this is reflected in an increased EAR to allergen. Late-phase responses to allergen were also enhanced, as demonstrated by decreased 7-hour FEV1 and increased eosinophilia and percentage of metachromatic cells in the 7-hour sputum sample. Increased allergen-induced mast cell degranulation could, in part, explain the increased asthmatic responses to allergen after β2-agonist treatment and could contribute to the deterioration of asthma control that is associated with regular use of β2-agonist by potentiating allergic inflammation. (J Allergy Clin Immunol 2000;106:57-64.)

Leflunomide, a novel immunomodulating agent, prevents the development of allergic sensitization in an animal model of allergic asthma.

Leflunomide is a new anti-inflammatory and immunomodulating agent which is showing promise in several immune disorders, especially rheumatoid arthritis. Its activity profile suggests it may be of use in modulating allergic sensitization. To investigate the effectiveness of leflunomide in preventing the development of allergic sensitization. Fifty-three brown Norway rats were sensitized by intraperitoneal injection of ovalbumin and adjuvant (ricin) on day 0. To determine the ability of leflunomide to inhibit primary allergic sensitization six rats were treated with A77 1726, the active metabolite of leflunomide, from day 0 through day 5, six were treated from day 5 through day 10, and nine rats acted as controls. On day 14, ovalbumin-specific serum antibody levels and the magnitude of the early-phase airway response (EAR) after inhalation allergen challenge were assessed. To determine the ability of acute topical treatment with leflunomide to inhibit mast cell degranulation, three groups of five animals received either vehicle, 100 microg A77 1726, or 1000 g A77 1726 60 min prior to aerosol allergen challenge. To determine the effects of leflunomide treatment in vivo on mast cell function in vitro, mast cells were obtained by bronchoalveolar lavage from 17 rats (nine treated with leflunomide and eight controls). Allergen-specific and non-specific degranulation (48/80 induced) were studied. In the leflunomide treated rats both ovalbumin-specific IgE and IgG levels were significantly reduced, and the increases in lung resistance and lung elastance were essentially abolished, compared to those of the control group. Non significant differences were found in any of the parameters between the two leflunomide treated groups. Topical pre-treatment with leflunomide did not prevent the allergen-induced EAR. Treatment with leflunomide in vivo prevented allergen-induced mast cell degranulation in vitro because the mast cells lacked IgE on their surface. Non allergen-specific degranulation was normal and allergen-induced degranulation could be restored by passive sensitization. These data suggests that leflunomide can prevent primary allergic sensitization and prevent allergen-induced EAR by inhibiting production of allergen-specific IgE antibodies. Further studies in atopic conditions are warranted.

Allergen-specific IgG1 and IgG3 through Fc gamma RII induce eosinophil degranulation.

Evidence suggests that eosinophils contribute to inflammation in bronchial asthma by releasing chemical mediators and cytotoxic granule proteins. To investigate the mechanism of eosinophil degranulation in asthma, we established an in vitro model of allergen-induced degranulation. We treated tissue culture plates with short ragweed pollen (SRW) extract and sera from either normal donors or SRW-sensitive patients with asthma. Eosinophils were incubated in the wells and degranulation was assessed by measurement of eosinophil-derived neurotoxin in supernatants. We detected degranulation only when sera from SRW-sensitive patients were reacted with SRW. Anti-IgG and anti-Fc gamma RII mAb, but not anti-IgE or anti-Fc epsilon RII mAb, abolished the degranulation. IgG-depleted serum did not induce degranulation; IgE-depleted serum triggered as much degranulation as untreated serum. Furthermore, serum levels of SRW-specific IgG1 or IgG3 correlated with the amounts of released eosinophil-derived neurotoxin. When eosinophils were cultured in wells coated with purified IgG or IgE, eosinophil degranulation was observed only with IgG. Finally, human IgG1 and IgG3, and less consistently IgG2, but not IgG4, induced degranulation. Thus, sera from patients with SRW-sensitive asthma induce eosinophil degranulation in vitro through antigen-specific IgG1 and IgG3 antibodies. These antibodies may be responsible for degranulation of eosinophils in inflammatory reactions, such as bronchial asthma.

Management of allergic rhinitis: focus on intranasal agents.

The clinical manifestations of allergic rhinitis are the result of an immune-mediated process after exposure of a sensitized individual to airborne allergens. The primary symptomatology includes nasal congestion, rhinorrhea, nasal and conjunctival pruritus, and sneezing. Principles of management include allergen avoidance, palliative therapy, immunotherapy, and pharmacotherapy. Oral decongestants stimulate alpha-adrenergic receptors in the nasal cavity, resulting in vasoconstriction and decreased edema. Oral antihistamines block histamine1 (H1) receptors, and may relieve rhinorrhea, sneezing, and nasal and conjunctival pruritus. Topical decongestants have a local effect on adrenergic receptors in the nasal mucosa, resulting in rapid, marked vasoconstriction. Intranasal corticosteroids inhibit mediator release from mast cells and basophils, and reduce edema of the nasal mucosa. Dexamethasone sodium phosphate, beclomethasone dipropionate, and flunisolide are currently available for intranasal administration. Cromolyn sodium inhibits allergen-induced degranulation and mediator release from sensitized cells, and is useful primarily as a prophylactic agent. Several agents, including the corticosteroids budesonide and flucortin butylester, the mast cell-stabilizing agent nedocromil sodium, the anticholinergic agent ipratropium bromide, and the H1 receptor antagonist levocabastine are being investigated for intranasal use in the management of allergic rhinitis.