In vivo and T cell cross-reactivity between walnut, cashew and peanut

Abstract

Background: Examination of IgE cross-reactivity among nuts has been limited to in vitro experiments. Cross-reactivity studies of nuts at the T cell level are difficult to interpret because of the inability to determine which cellular responses are from a true sensitization and which are due to cross-reactivity. Using a mouse model in which the sensitizing nuts are controlled may provide novel methods to investigate in vivo and T cell cross-reactivity.

Methods: C3H/HeJ mice were sensitized by intraperitoneal injection of cashew alone (monosensitized mice), or cashew plus walnut, utilizing alum as an adjuvant. Both groups underwent challenges to cashew, walnut and peanut, with subsequent monitoring of anaphylactic reactions. Anaphylactic antibodies were quantified by ELISA, and protein allergens were identified by Western blotting. Cellular responses were studied via splenocyte proliferation assay and measurement of secreted cytokines.

Results: The monosensitized mice reacted to cashew and walnut during challenges, with significantly weaker reactions induced on challenge with peanut. Cross-reactive IgE to walnut and peanut were detected by ELISA, and the cross-reactive allergens were identified as vicilin proteins. In cellular assays, splenocytes from the monosensitized mice proliferated and produced IL-4 and IL-5 in response to cashew, walnut and peanut. The cashew- plus walnut-sensitized mice experienced stronger clinical reactions to walnut, recognized additional walnut allergens and secreted significantly more IL-4 and IL-5 in walnut-stimulated splenocyte assays compared to the monosensitized mice.

Conclusions: Cross-reactivity in vivo was found between cashew and walnut, while cross-reactivity among cashew, walnut and peanut was demonstrated at the T cell level.

Fig. 1

Schedule of sensitization, blood collection and challenges for the CSH mice, CSH + WN mice and naïve mice. During the sensitization phase, mice were injected intraperitoneally with the nut extract (cashew or walnut) or PBS, plus alum as an adjuvant on the days shown. Blood was collected 2 weeks following the last immunization for studies on the immunoglobulin responses. Mice were then challenged intraperitoneally with cashew, walnut and peanut (PN) on the days shown.

Fig. 2

Anaphylactic symptom scores and change in body temperature following challenges. Groups of mice were challenged intraperitoneally with the quantities of nuts shown (PN = peanut). Anaphylactic reactions were assessed by monitoring clinical symptoms and measuring changes in body temperature. a Symptom scores for the two groups. Circles represent individual mice. Bars represent the median outcome for each challenge. b Temperature changes for the two groups. The average is shown with the standard deviation. ^a p < 0.05: Statistically significant difference compared to 1 mg of peanut; ^b p < 0.05: statistically significant difference compared to 3 mg of peanut.

Fig. 3

Nut-specific antibodies in the serum of mice following sensitization. Values shown are averages with standard deviation. a IgE in response to cashew, walnut and peanut in the sensitized mice. IgE levels in naïve mice for each nut were subtracted from those in the sensitized mice and are thus set to zero (not shown). b IgG1 in response to cashew, walnut and peanut in the sensitized mice. IgG1 levels in naïve mice were less than 0.3 µg/ml for each nut and are not shown. * p < 0.01: Statistically significant differences between the two groups of mice for the walnut-specific immunoglobulins. PN = peanut.

Fig. 4

Western blot analysis showing IgG1-binding proteins in the CSH mice and CSH + WN mice. a Coomassie blue-stained SDS-PAGE gel showing the protein profile of peanut (P), cashew (C) and walnut (W) extracts. b Proteins recognized by IgG1 in pooled CSH mice sera for peanut (P), cashew (C) and walnut (W). Inhibition studies were performed with no inhibitor (Ø), walnut or peanut (PN), and demonstrated that the cross-reacting proteins can be inhibited with the appropriate nut extract. The cross-reactive peanut protein was identified as Ara h 1, and the cross-reactive walnut protein was found to be Jug r 2. c Western blot showing the cashew (C) and walnut (W) allergens in the CSH + WN mice. Notice that additional bands are recognized in walnut compared to the CSH mice.

Fig. 5

Splenocyte proliferation assay for CSH mice and CSH + WN mice in response to various food antigens. Triplicate cultures of splenocytes were used to calculate the stimulation indices, using RPMI stimulation as the reference, for an individual mouse in response to cashew, walnut, peanut (PN), macadamia (MAC) or egg. Naïve mice were always normalized to a stimulation index of 1.0, and the other groups were adjusted accordingly. Values shown are averages of at least 3 individual mouse stimulation indices, with the exception of egg stimulation, which was only carried out on 2 mice per group.

Fig. 6

Secreted IL-4 and IL-5 levels from tree-nut-sensitized mice. Splenocytes were stimulated with cashew, walnut, peanut (PN), macadamia (MAC), egg or medium alone (RPMI) and cultured for 96 h. ELISA was used to quantify IL-4 (a) and IL-5 (b). Values shown are averages with standard deviations and represent at least 3 mice per condition, except for egg, which represents only 2 mice. * p < 0.05: Statistically significant difference between the two groups of mice for walnut-stimulated IL-4 and IL-5 secretion.