Regina Selb

CD23 surface density on B cells is associated with IgE levels and determines IgE-facilitated allergen uptake, as well as activation of allergen-specific T cells

Background: Increasing evidence suggests that the low‐affinity receptor for IgE, CD23, plays an important role in controlling the activity of allergen‐specific T cells through IgE‐facilitated allergen presentation. Objective: We sought to determine the number of CD23 molecules on immune cells in allergic patients and to investigate whether the number of CD23 molecules on antigen‐presenting cells is associated with IgE levels and influences allergen uptake and allergen‐specific T‐cell activation. Methods: Numbers of CD23 molecules on immune cells of allergic patients were quantified by using flow cytometry with QuantiBRITE beads and compared with total and allergen‐specific IgE levels, as well as with allergen‐induced immediate skin reactivity. Allergen uptake and allergen‐specific T‐cell activation in relation to CD23 surface density were determined by using flow cytometry in combination with confocal microscopy and T cells transfected with the T‐cell receptor specific for the birch pollen allergen Bet v 1, respectively. Defined IgE‐allergen immune complexes were formed with human monoclonal allergen‐specific IgE and Bet v 1. Results: In allergic patients the vast majority of CD23 molecules were expressed on naive IgD+ B cells. The density of CD23 molecules on B cells but not the number of CD23+ cells correlated with total IgE levels (RS = 0.53, P = .03) and allergen‐induced skin reactions (RS = 0.63, P = .008). Uptake of allergen‐IgE complexes into B cells and activation of allergen‐specific T cells depended on IgE binding to CD23 and were associated with CD23 surface density. Addition of monoclonal IgE to cultured PBMCs significantly (P = .04) increased CD23 expression on B cells. Conclusion: CD23 surface density on B cells of allergic patients is correlated with allergen‐specific IgE levels and determines allergen uptake and subsequent activation of T cells.

Critical and direct involvement of the CD23 stalk region in IgE binding

Background: The low‐affinity receptor for IgE, Fc&egr;RII (CD23), contributes to allergic inflammation through allergen presentation to T cells, regulation of IgE responses, and enhancement of transepithelial allergen migration. Objective: We sought to investigate the interaction between CD23, chimeric monoclonal human IgE, and the corresponding birch pollen allergen Bet v 1 at a molecular level. Methods: We expressed 4 CD23 variants. One variant comprised the full extracellular portion of CD23, including the stalk and head domain; 1 variant was identical with the first, except for an amino acid exchange in the stalk region abolishing the N‐linked glycosylation site; and 2 variants represented the head domain, 1 complete and 1 truncated. The 4 CD23 variants were purified as monomeric and structurally folded proteins, as demonstrated by gel filtration and circular dichroism. By using a human IgE mAb, the corresponding allergen Bet v 1, and a panel of antibodies specific for peptides spanning the CD23 surface, both binding and inhibition assays and negative stain electron microscopy were performed. Results: A hitherto unknown IgE‐binding site was mapped on the stalk region of CD23, and the non–N‐glycosylated monomeric version of CD23 was superior in IgE binding compared with glycosylated CD23. Furthermore, we demonstrated that a therapeutic anti‐IgE antibody, omalizumab, which inhibits IgE binding to Fc&egr;RI, also inhibited IgE binding to CD23. Conclusion: Our results provide a new model for the CD23‐IgE interaction. We show that the stalk region of CD23 is crucially involved in IgE binding and that the interaction can be blocked by the therapeutic anti‐IgE antibody omalizumab.

Extracorporeal IgE Immunoadsorption in Allergic Asthma: Safety and Efficacy

Background Prevention of IgE-binding to cellular IgE-receptors by anti-IgE (Omalizumab) is clinically effective in allergic asthma, but limited by IgE threshold-levels. To overcome this limitation, we developed a single-use IgE immunoadsorber column (IgEnio). IgEnio is based on a recombinant, IgE-specific antibody fragment and can be used for the specific extracorporeal desorption of IgE. Objective To study safety and efficacy of IgEnio regarding the selective depletion of IgE in a randomized, open-label, controlled pilot trial in patients with allergic asthma and to investigate if IgEnio can bind IgE-Omalizumab immune complexes. Methods Fifteen subjects were enrolled and randomly assigned to the treatment group (n = 10) or to the control group (n = 5). Immunoadsorption was done by veno-venous approach, processing the twofold calculated plasma volume during each treatment. A minimum average IgE-depletion of 50% after the last cycle in the intention-to-treat population was defined as primary endpoint. Safety of the treatment was studied as secondary endpoint. In addition, possible changes in allergen-specific sensitivity were investigated, as well as clinical effects by peak flow measurement and symptom-recording. The depletion of IgE-Omalizumab immune complexes was studied in vitro. The study was registered at clinicaltrials.gov (NCT02096237) and conducted from December 2013 to July 2014. Results IgE immunoadsorption with IgEnio selectively depleted 86.2% (± 5.1% SD) of IgE until the end of the last cycle (p < 0.0001). Removal of pollen allergen-specific IgE was associated with a reduction of allergen-specific basophil-sensitivity and prevented increases of allergen-specific skin-sensitivity and clinical symptoms during pollen seasons. IgEnio also depleted IgE-Omalizumab immune complexes in vitro. The therapy under investigation was safe and well-tolerated. During a total of 81 aphereses, 2 severe adverse events (SAE) were recorded, one of which, an episode of acute dyspnea, possibly was related to the treatment and resolved after administration of antihistamines and corticosteroids. Conclusions This pilot study indicates that IgE immunoadsorption with IgEnio may be used to treat patients with pollen-induced allergic asthma. Furthermore, the treatment could render allergic patients with highly elevated IgE-levels eligible for the administration of Omalizumab and facilitate the desorption of IgE-Omalizumab complexes. This study was funded by Fresenius Medical Care Deutschland GmbH, Bad Homburg, Germany.

An assay that may predict the development of IgG enhancing allergen-specific IgE binding during birch immunotherapy

It has been shown that birch pollen immunotherapy can induce IgG antibodies which enhance IgE binding to Bet v 1. We aimed to develop a serological assay to predict the development of antibodies which enhance IgE binding to Bet v 1 during immunotherapy.

Intranasal administration of allergen increases specific IgE whereas intranasal Omalizumab does not increase serum IgE levels – a pilot study

Administration of the therapeutic anti‐IgE antibody omalizumab to patients induces strong increases in IgE antibody levels.

Betamethasone prevents human rhinovirus- and cigarette smoke- induced loss of respiratory epithelial barrier function

The respiratory epithelium is a barrier against pathogens and allergens and a target for therapy in respiratory allergy, asthma and chronic obstructive pulmonary disease (COPD). We investigated barrier-damaging factors and protective factors by real-time measurement of respiratory cell barrier integrity. Barrier integrity to cigarette smoke extract (CSE), house dust mite (HDM) extract, interferon-γ (IFN-γ) or human rhinovirus (HRV) infection alone or in combination was assessed. Corticosteroids, lipopolysaccharide (LPS), and nasal mucus proteins were tested for their ability to prevent loss of barrier integrity. Real-time impedance-based measurement revealed different patterns of CSE-, HDM-, IFN-γ- and HRV-induced damage. When per se non-damaging concentrations of harmful factors were combined, a synergetic effect was observed only for CSE and HDM. Betamethasone prevented the damaging effect of HRV and CSE, but not damage caused by HDM or IFN-γ. Real-time impedance-based measurement of respiratory epithelial barrier function is useful to study factors, which are harmful or protective. The identification of a synergetic damaging effect of CSE and HDM as well as the finding that Betamethasone protects against HRV- and CSE-induced damage may be important for asthma and COPD.

Characterisation of recombinant CD23 in the trimeric complex with IgE and allergen

Results The characterisation of CD23 via circular dichroism (CD) spectra and gel filtration showed folded, monomeric proteins. Binding of isolated monomeric human monoclonal IgE and of isolated polyclonal serum IgE as well as of both forms of IgE in complex with birch pollen allergen Bet v 1 to recombinant CD23 was demonstrated by ELISA and by surface plasmon resonance analysis. Next, we performed negative stain electron microscopy of the three molecules alone (i.e., CD23, monoclonal human IgE, Bet v 1) and after complex formation. After addition of Bet v 1 allergen (17 kDa) to monoclonal IgE (190 kDa) we could observe an extension of one or both Fab arms of the antibody. Interestingly, further addition of recombinant CD23 molecules (35 kDa) to the IgE-allergen complex resulted in thickening of the antibody’s Fc structure, possibly because CD23 lies in the same plane as the IgE molecule in these pictures. Conclusions In summary, we report the in vitro formation of a trimolecular complex consisting of recombinant CD23, a monoclonal human allergen-specific IgE and the corresponding Bet v 1 allergen and take a first step towards the visualization of this complex using negative stain electron microscopy. Furthermore, the in vitro trimolecular interaction model may be useful for the screening of drugs and compounds for their potential to inhibit the IgE CD23 interaction with the goal to develop new therapeutic strategies for allergy.

Establishment of a cell culture model based on primary epithelial cells to investigate damage and repair of respiratory epithelial cells

Background The nasal epithelium represents the first barrier against entry of airborne particles into the respiratory system and therefore protects against allergens, pollutants and pathogens. We have previously used the respiratory epithelial cell line 16HBE14oto define factors which can impair the barrier function of the respiratory mucosa. However, cultures of primary epithelial cells obtained from human nasal biopsies should resemble the natural situation in the nose better. Here we investigated primary human nasal epithelial cells regarding growth characteristics and sensitivity to damage by interferon-gamma and other factors.

Trimolecular complex between major birch pollen allergen, Bet v 1, monoclonal allergen-specific human IgE and recombinant CD23

CD23 the low affinity receptor for IgE plays an important role in allergic disease because it facilitates allergen presentation to T cells. CD23 is a 45 kDa trans-membrane protein consisting of an alpha-helical stalk and a head domain, which is mainly expressed on B cells. In order to analyze the complex formation between allergen, allergenspecific IgE and CD23 on the molecular level, we expressed four different forms of CD23 in baculovirusinfected SF9 insect cells. Construct A consists of the extracellular part of the molecule, the second construct (B) differs from construct A by a single amino acid exchange in the stalk region to abolish the N-linked glycosylation of CD23. Constructs C and D were smaller molecules consisting mainly of the head domain of the protein. Furthermore we expressed human monoclonal IgE specific for the major birch pollen allergen Bet v 1 in a hybridoma cell line and the Bet v 1 allergen in Escherichia coli. Using circular dichroism analysis we found that all molecules were expressed as folded proteins and gel filtration revealed that the CD23 constructs were monomeric. In ELISA we demonstrated that each of the four CD23 constructs binds monomeric IgE and allergen-IgE complexes in a similar way. Concerning the different CD23 constructs, we observed weaker binding signals when measuring the smaller CD23 molecules. Interestingly, construct B, which was devoid of the N-linked glycosylation site, displayed enhanced IgE binding compared to the glycosylated protein. In conclusion, our results reveal a comparable interaction between isolated IgE as well as IgE-allergen immune complexes with CD23 and provide defined molecules for the detailed structural characterization of the allergenIgE-CD23 interaction. Supported by grants 4605, 4613 and in part by 4604 and P23350-B11 of the Austrian Science Fund (FWF).

Characterisation and culture of primary human nasal epithelial cells and the influence of Interferon-gamma

Background The nasal epithelium with its tight junctions (TJs) represents an important barrier against the penetration of exogenous substances (e.g. allergens, pollutants and pathogens). Damage of the epithelium allows higher amounts of inhaled allergens and pollutants to penetrate the mucosa. Our laboratory has previously used a bronchial epithelial cell line to investigate epithelial damage by various factors [cigarette smoke (Gangl et al., 2009) and cytokines (Reisinger et al., 2005)]. Cultures of primary epithelial cells obtained from nasal epithelium should be superior in resembling the natural situation in the nose and allow a comparison of epithelia from non-allergic and allergic patients. Our aim was to establish primary human nasal epithelial cell culture in a transwell system and to investigate the epithelial barrier function and the properties of the nasal epithelium. Additionally we compared different epithelial cell culture systems to investigate damage and repair in primary human nasal epithelial cells.