Long-term characteristics of hazelnut allergy in an adjuvant-free mouse model

Abstract

Background: Clinically it is recognized that tree nut allergies such as hazelnut allergy are not usually outgrown. Specific mechanisms underlying the persistence of such food allergies are incompletely understood. Here we studied the natural history and the long-term immune and clinical characteristics of hazelnut allergy in an adjuvant-free mouse model.

Methods: BALB/c mice were sensitized to hazelnut protein using a transdermal sensitization protocol that does not use adjuvant. After establishing sensitization, exposure to hazelnut was withdrawn for 3, 5 or 8 months. The fate of circulating IgE antibodies was monitored. Subsequently, mice were given booster exposures and examined for memory IgE antibody and spleen cell IL-4 responses. Clinical characteristics and hypothermia responses upon oral allergen challenge were studied.

Results: Upon allergen withdrawal, circulating hazelnut-specific IgE antibody levels began to drop. Nevertheless, IgE responses once established remained at significantly high levels for up to 8 months (the last time point studied) despite withdrawal of allergen exposure. Memory IgE responses to booster exposures were robust after 3, 5 or 8 months of allergen withdrawal. Furthermore, significant clinical reactivity to oral hazelnut challenge, and hypothermia responses were demonstrable at each of these time points. Long-lasting spleen cell memory IL-4 responses to hazelnut were detectable in these mice explaining the mechanism of sustenance of IgE responses and clinical sensitization.

Conclusions: Hazelnut allergy once established persists for long periods, despite withdrawal of allergen exposure, due to long-lasting, memory IgE and IL-4 responses.

Fig. 1

a–c Circulating hazelnut-specific IgE antibodies persist for long periods despite allergen withdrawal of 3–8 months in BALB/c mice. Groups of mice (n = 10/group) were transdermally exposed to hazelnut (HN) protein (0 or 1 mg/mouse) as described in the Methods; TDE = transdermal exposure. Specific IgE antibody levels were measured before exposure and at several time points after exposure and after allergen withdrawal. The data in a, b and c are from 3-, 5- and 8-month allergen withdrawal experiments, respectively.

Fig. 2

a–f Hazelnut elicits an IgE response that is characterized by long-lasting memory in BALB/c mice. Groups of mice (n = 10/group) were transdermally exposed to hazelnut protein (0 or 1 mg/mouse), and then allergen was withdrawn as described in the Methods. Then mice were given two booster transdermal exposures, and their memory IgE levels were measured. a, b Three-month allergen withdrawal experiment. c, d Five-month allergen withdrawal experiment. e, f Eight-month allergen withdrawal experiment. Data shown as means ± SE. Unpaired t test results: * p < 0.05.

Fig. 3

Long-lasting memory IgE response to hazelnut is associated with clinical reactivity and hypothermia response upon oral challenge with hazelnut. Groups of mice (n = 10/group) were transdermally exposed to hazelnut protein (0 or 1 mg/mouse) and then allergen was withdrawn as described in the Methods. Later, mice were orally challenged with hazelnut protein (15 mg/mouse) and examined for clinical signs of systemic anaphylaxis during the next 60 min. a, c, e Clinical symptom scores are shown as a scatter plot with each symbol representing 1 mouse. Unpaired t test results: hazelnut, 1 and 0 mg/mouse, p < 0.05. b, d, f Rectal temperature before and at 30 min after oral challenge. a, b Three-month allergen withdrawal experiment. c, d Five-month allergen withdrawal experiment. e, f Eight-month allergen withdrawal experiment. Data shown as means ± SE. ANOVA test results: bars with different letters are significantly different (p < 0.05).

Fig. 4

Long-lasting allergic responses are associated with memory IL-4 responses. Spleen cells were isolated from BALB/c mice from various groups and cultured with hazelnut protein (0.1, 0.5 mg/ml) or culture medium alone. Cell culture supernatants were harvested on day 3 and analyzed for cytokines using optimized ELISA. a, b Three-month allergen withdrawal experiment. c, d Five-month allergen withdrawal experiment. e, f Eight-month allergen withdrawal experiment. Data shown are averages ± SE of duplicate analyses. Significance was determined by ANOVA. Bars with different letters are significantly different (p < 0.05).