Contribution of Nasal IgE Production to the Boost of Systemic Allergen-specific IgE Upon Nasal Allergen Contact

IgE-associated allergies are consistently on the rise forming an enormous wellbeing concern and affecting about 25% of the population around the world, with main prevalence in developed nations. Birch pollen is one important allergen driving allergic conjunctivitis and rhinitis. The characteristics and location IgE producing cells in the nose and their contribution to the subsequent increase in allergen-specific IgE levels in the serum have so far poorly been investigated. Understanding these mechanisms is critical for the development of new therapeutic approaches and is thus the aim of the proposed study. Thus, the investigators plan to conduct a two-armed study of a randomized placebo-controlled double-blind nasal allergen provocation where 30 patients will be provoked with birch pollen extract (n=20) or placebo (n=10) out of the birch pollen season (October) and follow them closely for up to 4 months. Therefore, the objective of this study is to investigate the localization and characteristics of IgE producing cells in the nasal mucosa contributing to the allergen-induced boosts of specific serum IgE upon natural and controlled nasal allergen exposure.

The nose represents an important and first site of allergen contact, both because it is the organ most frequently affected by allergic symptoms and also because it plays an important role for the regulation of IgE production and allergic inflammation. The nasal mucosa acts as a barrier against intruding allergens. This barrier function can be perturbed by various factors such as cigarette smoke, rhinovirus infection or interferon-γ. Exposure to these factors may lead to increased allergen penetration. Nasal allergen contact in sensitized subjects leads to allergic nasal inflammation and symptoms. The investigators of this study have previously demonstrated that allergen-specific IgE levels in sensitized individuals are strongly boosted upon nasal allergen contact during the relevant allergen season or in well controlled intranasal allergen challenge models. This allergen-induced rise in IgE levels consequently leads to increased sensitivity of allergic patients in terms of IgE-mediated mast cell and basophil activation as well as IgE-facilitated antigen presentation via the high (FcεRI) or low (CD23) affinity receptor for IgE by antigen presenting cells, to T cells. In this context, the investigators have recently shown that not only allergen-specific IgE levels themselves but also the crosslinking ability of complexes formed between IgE and allergen dictate the extent of CD23-mediated T cell activation.

The observation that nasal allergen contact triggers boosting of systemic allergen-specific IgE levels indicates the presence of IgE+ producing cells locally in the nasal mucosa which can be stimulated upon allergen contact. This hypothesis is supported by the following observations: (A) there is an evidence that IgE-producing B cells and plasma cells are present in the nasal mucosa but nothing has been proven yet. (B) Upon ex vivo challenge of biopsies from nasal mucosa of allergic patients, local synthesis not only of ε germline, but also of ε circle transcripts as products of mature IgE synthesis were observed. However, to which extent this local nasal IgE production contributes to the observed systemic boost of IgE and the underlying kinetics of IgE production are so far unknown and are one important aim of this proposal.

Also, though local IgE production has been observed, the presence of the IgE producing cells themselves in the nasal mucosa has so far not been confirmed. One important reason for this is the difficulty of unambiguously identifying those cells by flow cytometry or fluorescence microscopy. In the past several step by step flow cytometry protocols have been developed with the aim of excluding non-relevant cells prior to gating for IgE+ B cells. However, it needs to be borne in mind that all currently used anti-IgE antibodies recognize not only surface IgE present in the form of the B cell receptor but also bound to CD23. As a consequence, most current studies overestimate the presence of IgE B cell receptor (BCR) bearing B cells. This has only recently been demonstrated in a study by Jimenez-Saiz et al. where the authors found that only around 0.0015% of B cells bear an IgE BCR. Here the authors employed a single cell nested polymerase chain reaction (PCR) approach to validate the nature of the BCR after flow cytometry-based cell sorting for IgE+ B cells using previously reported protocols. The overestimation is most likely due to detection of IgE+ B cells bearing CD23 bound IgE. One option to improve flow cytometric detection could be to exclude CD23 positive cells but this could also lead to exclusion of IgE producing cells expressing CD23. Thus, the only alternative to overcome this obstacle is to use an anti-IgE antibody that can distinguish between receptor bound and membrane anchored IgE. In this respect, an anti-IgE antibody fulfilling these requirements has recently been shown to successfully identify IgE producing cells. Thus, the investigators will employ this novel strategy to identify IgE producing cells both in the nasal mucosa as well as in the blood of allergic subjects.

In this project, the investigators aim to elucidate the sites and characteristics of allergen-specific IgE production in a two-armed study spanning birch pollen allergic subject undergoing controlled nasal birch pollen challenge outside the season (October). To that aim, IgE producing cells in blood will be determined using flow cytometry and IgE producing cells in the nasal mucosa will be revealed in nasal biopsies using confocal microscopy. Understanding the characteristics and locations of IgE producing cells is critical for the development of future new therapeutic approaches.

Eligibility Criteria:
Inclusion Criteria:

* Birch pollen sensitized subjects

  * Moderate to severe allergic rhinitis to birch pollen allergen for at least two seasons according to their medical history
  * Sensitization to Bet v 1 (Bet v 1 specific IgE: 3.5 kU/L or higher as specified by UniCAP-FEIA)
* CD203c or CD63 upregulation upon challenge with Bet v 1 in flow cytometric basophil activation tests (≥20% upregulation of CD63 or CD203c upon Bet v 1 stimulation in comparison with unstimulated controls as measured by flow cytometry)
* Willingness to follow the protocol.
* Written informed consent
* Standard healthcare insurance
* Subjects should be available during the entire study period

Exclusion Criteria:

* Evidence of acute, chronic, malignant or general diseases- assessed by asking the patient (e.g. Did you suffer or are you currently suffering from any malignant or generalised disease? Did you suffer or are you currently suffering from any chronic disease? Did you have any acute disease in the past two weeks?).
* A History of anaphylaxis.
* Utilization of leukotriene modifiers.
* Utilization of long-acting antihistamines.
* Chronic or intermittent use of oral, inhaled, intramuscular, intravenous, and potent topical corticosteroids.
* Nasal polyps, history of chronic sinusitis or considerable deviation of the nasal septum
* Rhinitis secondary to other causes.
* Contra-indications to skin prick testing, for example, skin irritation in the test area and urticaria factitia.
* Cardiovascular diseases or anti-hypertensive therapy and beta-blockers.
* Any disruption of coagulation system through medication or known clotting disorders
* Prophylactic aspirin therapy
* Chronic use of additional medications that would affect assessment and the results of the study (e.g., tricyclic antidepressants that block both H1 and H2 receptors)
* Pregnant or breastfeeding females.
* Actual disability that would influence subject's ability to participate in the study.
* History of mental illness, intellectual deficiency, drug or alcohol abuse
* Active asthma requiring treatment
* Birch Pollen allergen immunotherapy
* Arranged travel outside the study region for a substantial portion of study period.