R. Grönneberg

Clinical effects of immunotherapy with genetically modified recombinant birch pollen Bet v 1 derivatives

Background Birch pollen and pollen from related trees of the Fagales order are a major cause of allergic rhinitis, conjunctivitis, and asthma through the spring season in northern and central Europe.

Immune regulation by CD4+CD25+ T cells and interleukin‐10 in birch pollen‐allergic patients and non‐allergic controls

Background CD4+CD25+ regulatory T (Treg) cells and the cytokines IL‐10 or TGF‐β play key roles in the maintenance of T cell homeostasis and tolerance to infectious and non‐infectious antigens such as allergens.

Allergen provocation increases TH2-cytokines and FOXP3 expression in the asthmatic lung.

To cite this article: Thunberg S, Gafvelin G, Nord M, Grönneberg R, Grunewald J, Eklund A, van Hage M. Allergen provocation increases TH2‐cytokines and FOXP3 expression in the asthmatic lung. Allergy 2010; 65: 311–318.

Nasal challenges with recombinant derivatives of the major birch pollen allergen Bet v 1 induce fewer symptoms and lower mediator release than rBet v 1 wild-type in patients with allergic rhinitis

Background Genetic engineering of the major birch pollen allergen (Bet v 1) has led to the generation of recombinant Bet v 1 derivatives with markedly reduced IgE‐binding capacity, but with retained T cell activating ability.

Allergic asthmatics show divergent lipid mediator profiles from healthy controls both at baseline and following birch pollen provocation.

Background Asthma is a respiratory tract disorder characterized by airway hyper-reactivity and chronic inflammation. Allergic asthma is associated with the production of allergen-specific IgE and expansion of allergen-specific T-cell populations. Progression of allergic inflammation is driven by T-helper type 2 (Th2) mediators and is associated with alterations in the levels of lipid mediators. Objectives Responses of the respiratory system to birch allergen provocation in allergic asthmatics were investigated. Eicosanoids and other oxylipins were quantified in the bronchoalveolar lumen to provide a measure of shifts in lipid mediators associated with allergen challenge in allergic asthmatics. Methods Eighty-seven lipid mediators representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened via LC-MS/MS following off-line extraction of bronchoalveolar lavage fluid (BALF). Multivariate statistics using OPLS were employed to interrogate acquired oxylipin data in combination with immunological markers. Results Thirty-two oxylipins were quantified, with baseline asthmatics possessing a different oxylipin profile relative to healthy individuals that became more distinct following allergen provocation. The most prominent differences included 15-LOX-derived ω-3 and ω-6 oxylipins. Shared-and-Unique-Structures (SUS)-plot modeling showed a correlation (R2 = 0.7) between OPLS models for baseline asthmatics (R2Y[cum] = 0.87, Q2[cum] = 0.51) and allergen-provoked asthmatics (R2Y[cum] = 0.95, Q2[cum] = 0.73), with the majority of quantified lipid mediators and cytokines contributing equally to both groups. Unique structures for allergen provocation included leukotrienes (LTB4 and 6-trans-LTB4), CYP-derivatives of linoleic acid (epoxides/diols), and IL-10. Conclusions Differences in asthmatic relative to healthy profiles suggest a role for 15-LOX products of both ω-6 and ω-3 origin in allergic inflammation. Prominent differences at baseline levels indicate that non-symptomatic asthmatics are subject to an underlying inflammatory condition not observed with other traditional mediators. Results suggest that oxylipin profiling may provide a sensitive means of characterizing low-level inflammation and that even individuals with mild disease display distinct phenotypic profiles, which may have clinical ramifications for disease.

Effects of loratadine on anti‐IgE‐induced inflammation, histamine release, and leukocyte recruitment in skin of atopics

The aim of this study was to assess the ability of the H1‐receptor antagonist loratadine to modify anti‐IgE‐induced cutaneous wheal‐and‐flare and late‐phase reactions (WFR and LPR), as well as histamine release and leukocyte accumulation in skin chambers. For this purpose, 10 atopics with allergic rhinitis were entered into a double‐blind crossover study in which they received either placebo or loratadine (20 mg/day orally) for 8 days separated by a 7‐day washout period. Blisters were induced on both forearms on day 7 of each treatment period, and were unroofed on day 8 and covered with plastic skin chambers. Chamber fluids were collected during 7 h after 1‐h incubation with anti‐IgE or control IgG. Intradermal challenge with histamine and anti‐IgE was performed at the same occasion. As compared to placebo treatment, loratadine inhibited the immediate WFRs to anti‐IgE by 35% (wheal) and 65% (flare), respectively (P < 0.01), and corresponding reactions to histamine challenge by 50% and 70% (P<0.001), respectively. Moreover, the initial phase (0‐2 h) of the LPR induced by anti‐IgE was attenuated by up to ∼60% (P < 0.001) during loratadine treatment. Thereafter, no inhibition of the LPR was observed. The magnitude and time course of histamine release into skin chambers was virtually the same after loratadine and placebo treatment, with a peak during 0‐1 h and a progressive decline during the following 2 h. Accumulation of α2‐macroglobulin, reflecting extravasation of large plasma proteins, also peaked during the first hour and was unaffected by loratadine during the 8‐h observation period. Moreover, loratadine did not reduce the anti‐IgE‐induced recruitment of eosinophils or other subtypes of leukocytes. Altogether, loratadine inhibited both the WFRs to histamine and anti‐IgE and the initial phase of the IgE‐mediated LPR. However, loratadine did not express anti‐inflammatory activity with respect to mast‐cell mediator release or leukocyte recruitment. The latter findings are in contrast to the action of loratadine in allergic rhinitis and conjunctivitis, suggesting that the actions of loratadine may be organ specific and that the effects of loratadine cannot always be extrapolated from one tissue to another.

Eotaxin Increases the Expression of CD11b/CD18 and Adhesion Properties in IL5, But Not fMLP-Prestimulated Human Peripheral Blood Eosinophils

A selective recruitment of eosinophils to sites of inflammation is claimed to be controlled by regulation of cytokines, chemokines and adhesion molecules. In animal models, eotaxin has been suggested to be a potent chemokine since it in cooperation with interleukin-5 induce selective chemotaxis and infiltration of eosinophils to lung tissue after an allergen provocation. We have investigated the in vitro effect of eotaxin on human peripheral blood eosinophils with respect to CD11b/CD18 expression and adhesion properties to the matrix protein fibronectin. We did not find any effect of eotaxin per se on CD11b/CD18 expression, neither on eosinophils from healthy subjects nor from patients with asymptomatic pollen related asthma. However, eotaxin significantly upregulated the quantitative level of CD11b/CD18 and increased the adhesion to fibronectin in eosinophils from healthy subjects preincubated in vitro with interleukin-5, but not in eosinophils preincubated with fMLP. Moreover, eosinophils harvested 24 hours after an in vivo allergen inhalation provocation in asthmatics, upregulated CD11b/CD18 after in vitro incubation with eotaxin alone.

Elevated Exhaled Nitric Oxide in Allergen-Provoked Asthma Is Associated with Airway Epithelial iNOS

Background Fractional exhaled nitric oxide is elevated in allergen-provoked asthma. The cellular and molecular source of the elevated fractional exhaled nitric oxide is, however, uncertain. Objective To investigate whether fractional exhaled nitric oxide is associated with increased airway epithelial inducible nitric oxide synthase (iNOS) in allergen-provoked asthma. Methods Fractional exhaled nitric oxide was measured in healthy controls (n = 14) and allergic asthmatics (n = 12), before and after bronchial provocation to birch pollen out of season. Bronchoscopy was performed before and 24 hours after allergen provocation. Bronchial biopsies and brush biopsies were processed for nitric oxide synthase activity staining with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), iNOS immunostaining, or gene expression analysis of iNOS by real-time PCR. NADPH-d and iNOS staining were quantified using automated morphometric analysis. Results Fractional exhaled nitric oxide and expression of iNOS mRNA were significantly higher in un-provoked asthmatics, compared to healthy controls. Allergic asthmatics exhibited a significant elevation of fractional exhaled nitric oxide after allergen provocation, as well as an accumulation of airway eosinophils. Moreover, nitric oxide synthase activity and expression of iNOS was significantly increased in the bronchial epithelium of asthmatics following allergen provocation. Fractional exhaled nitric oxide correlated with eosinophils and iNOS expression. Conclusion Higher fractional exhaled nitric oxide concentration among asthmatics is associated with elevated iNOS mRNA in the bronchial epithelium. Furthermore, our data demonstrates for the first time increased expression and activity of iNOS in the bronchial epithelium after allergen provocation, and thus provide a mechanistic explanation for elevated fractional exhaled nitric oxide in allergen-provoked asthma.

The degree of natural allergen exposure modifies eosinophil activity markers in the circulation of patients with mild asthma

We have previously found that CD9, CD11b, and intracellular ECP (EG2) may be used as activity markers for eosinophils in vitro. The main object of the present study was to determine whether these markers can reflect eosinophil activation in vivo in relation to allergen exposure. For this purpose, six patients with a history of allergic rhinitis and occasional asthma symptoms during the pollen season participated. Blood donors served as controls. Peripheral blood eosinophils were analyzed according to the FOG method and flow cytometry, before and during one birch pollen season with high pollen load (HPL) and one with low pollen load (LPL). The CD9 expression on peripheral eosinophils from the patients was significantly increased both before (P<0.05) and during (P<0.01) HPL season, and CD11b expression solely during HPL season (P = 0.01) as compared to controls. The intracellular expression of the EG2 epitope was increased before (P<0.01) and during (P<0.05) HPL season, and increased significantly (P<0.05) during season as compared to before. No changes were observed before and during LPL season. The proportion of eosinophils was increased both before (P<0.05) and during (P<0.001) the HPL season as compared to controls. The markers CD9, EG2, and, to a lesser extent, CD11b seem to detect activated eosinophils in the circulation, whereas EG2 may also reflect increased antigen exposure during season.

Antiallergic actions of high topical doses of terbutaline in human nasal airways.

It is debatable whether β2‐receptor agonists produce antiallergic effects in human airways. This question has been addressed in the present study by examination of both mast‐cell indices and the physiologic response to allergen challenge in human nasal airways. Twelve asymptomatic patients with seasonal allergic rhinitis were investigated outside the pollen season. Intranasal allergen provocation was carried out with diluent and three increasing doses of allergen. Topical terbutaline sulfate (1.0 mg) was given 5 min prior to each allergen challenge and nasal lavage was carried out 10 min after each challenge. The study design was double‐blind, placebo‐controlled, crossover, and randomized. The allergen challenge‐induced mast‐cell activation and the ensuing physiologic response of the airway tissue were investigated by measuring a mast‐cell‐derived mediator (tryptase) and plasma proteins (albumin and α2 macroglobulin), respectively, in the lavage fluids. Allergen provocation produced dose‐dependent increments of nasal symptoms and lavage fluid levels of tryptase, albumin and α2‐macroglobulin. Both nasal symptoms (p≤0.05) and lavage fluid levels of tryptase (p≤0.05), albumin (p≤0.05), and α2‐macroglobulin (p≤0.01) were reduced by pretreatment with topical terbutaline sulfate. We conclude that high doses of topical terbutaline may produce significant antiallergic effects in human airways by equally reducing both tryptase release and plasma exudation in the acute allergic reaction in human airways. Further studies are now warranted to determine whether microvascular antipermeability effects of (β2‐receptor stimulation contribute to the present observations.