Pectate lyase pollen allergens: sensitization profiles and cross-reactivity pattern

Abstract

Background: Pollen released by allergenic members of the botanically unrelated families of Asteraceae and Cupressaceae represent potent elicitors of respiratory allergies in regions where these plants are present. As main allergen sources the Asteraceae species ragweed and mugwort, as well as the Cupressaceae species, cypress, mountain cedar, and Japanese cedar have been identified. The major allergens of all species belong to the pectate lyase enzyme family. Thus, we thought to investigate cross-reactivity pattern as well as sensitization capacities of pectate lyase pollen allergens in cohorts from distinct geographic regions.

Methods: The clinically relevant pectate lyase pollen allergens Amb a 1, Art v 6, Cup a 1, Jun a 1, and Cry j 1 were purified from aqueous pollen extracts, and patients' sensitization pattern of cohorts from Austria, Canada, Italy, and Japan were determined by IgE ELISA and cross-inhibition experiments. Moreover, we performed microarray experiments and established a mouse model of sensitization.

Results: In ELISA and ELISA inhibition experiments specific sensitization pattern were discovered for each geographic region, which reflected the natural allergen exposure of the patients. We found significant cross-reactivity within Asteraceae and Cupressaceae pectate lyase pollen allergens, which was however limited between the orders. Animal experiments showed that immunization with Asteraceae allergens mainly induced antibodies reactive within the order, the same was observed for the Cupressaceae allergens. Cross-reactivity between orders was minimal. Moreover, Amb a 1, Art v 6, and Cry j 1 showed in general higher immunogenicity.

Conclusion: We could cluster pectate lyase allergens in four categories, Amb a 1, Art v 6, Cup a 1/Jun a 1, and Cry j 1, respectively, at which each category has the potential to sensitize predisposed individuals. The sensitization pattern of different cohorts correlated with pollen exposure, which should be considered for future allergy diagnosis and therapy.

Fig 1. Geographical distribution of plants producing pollen containing clinically relevant allergenic pectate lyases.

Light colored zones represent areas with low to medium distribution, dark colored zone areas with high distribution of the respective species. Ambrosia artemisiifolia (ragweed) is presented in blue, Artemisia vulgaris (mugwort) in violet, Cupressus arizonica / Cupressus sempervirens (cypress) in red, Juniperus ashei (Mountain cedar) in orange, and Cryptomeria japonica (Japanese cedar) in yellow. The 4 clinical centers of the study are depict as cross lines (www.gbif.org; www.pollenlibrary.com; eol.org) [3, 4].

Fig 2. Taxonomy of Cupressaceae and Asteraceae and SDS-PAGE of purified allergenic proteins.

(A) Phylogenetic tree of Cupressaceae and Asteraceae species. (B) Identity matrix of pectate lyase pollen allergens and, in case of Amb a 1, allergen isoforms. (C) SDS-PAGE of allergenic pectate lyases purified from pollen extracts by anion-exchange chromatography and/or size exclusion chromatography. The gel was stained with Coomassie Brilliant Blue R-250. Natural Cry j 1 migrates as visible double band.

Fig 3. Patients´ serum IgE levels determined by ELISA.

IgE binding of sera from Austrian (n = 28), Canadian (n = 40), Italian (n = 30), and Japanese (n = 28) Asteraceae and Cupressaceae pollen allergic patients were tested on natural Amb a 1, Art v 6, Cup a 1, Jun a 1, and Cry j 1. % values for IgE positive sera are given on the left, IgE levels are presented as mean ± SD on the right. Statistical analysis was performed with Repeated Measures ANOVA and Tukey´s Multiple Comparison Test (* P< .05, ** P< .01, *** P< .001).

Fig 4. Inhibition ELISA of allergic patients´ sera.

IgE binding of sera of pectate lyase pollen allergic patients from Austria (n = 6) and Canada (n = 6) were tested with coated Amb a 1 and Art v 6, respectively, sera from Italian (n = 6) and Japanese (n = 6) patients were tested with coated Cup a 1, Jun a 1, and Cry j 1. Prior to the ELISA all sera were pre-incubated with either 1 μg/ well of Amb a 1, Art v 6, Cup a 1, Jun a 1, or Cry j 1. Inhibitions are presented as box plots, median values are indicated as lines, whiskers indicate the 5–95 percentile. Results are presented as % inhibition compared to maximum IgE of untreated sera. Statistical analysis was performed with Repeated Measures ANOVA and Tukey´s Multiple Comparison Test (* P< .05, ** P< .01, *** P< .001).

Fig 5. Sensitization patter of 10,352 allergic patients from Italy.

(A) Immuno solid-phase allergen chip (ISAC) assays were performed to determine sensitization to pectate lyase allergens. (B) Venn diagram of 10,352 pectate lyase positive patients´ sera. (C) IgE values are presented as kU_A/L, the cut off value was set at 0.01 kU_A/L. Results are presented as box plots, medians are indicated as solid lines, 25–75 percentiles are boxed, whiskers are set at 5–95%, statistics were calculated with Repeated Measures ANOVA and Tukey´s Multiple Comparison Test (*** P< .001). (D-F) Correlations of pectate lyase sensitizations are presented as dot blot and in matrix format. Spearman´s rank correlation coefficients were calculated.

Fig 6. Humoral analysis of murine immune sera.

The immunization scheme is presented as timeline, at each time point animals were immunized with 5 μg antigen adsorbed to Alu-Gel-S. Sera were drawn at day 0 and 49, respectively. Levels of allergen-specific IgG1 and IgG2a of mice (n = 5/ group) immunized with either Amb a 1, Art v 6, Cup a 1, Jun a 1, or Cry j 1 at day 49 were analyzed by ELISA. Titers are displayed as bars ± SEM. Statistical analysis was performed with One-way ANOVA and Tukey´s Multiple Comparison Test (* P< .05, ** P< .01, *** P< .001).

Fig 7. Biologically functional IgE of immunized mice was analyzed by mediator release assays.

RBL-2H3 cells were passively sensitized with pooled immune sera (n = 5 animals/ group) withdrawn at day 49, and mediator release was triggered by addition of 0.3 μg/ml allergen. Relative β-hexosaminidase activity was calculated compared to Triton X-100 treated cells. Results are presented as mean of duplicates ± SEM. Statistical analysis was performed with One-way ANOVA and Tukey´s Multiple Comparison Test (* P< .05, ** P< .01, *** P< .001).

Fig 8. Proliferation of re-stimulated splenocytes.

Activation of T cells was observed by the induction of IL-2 secreting cells. Therefore, splenocytes of immunized animals (n = 5 animals/ group) were re-stimulation with each of the five pectate lyase allergens. IL-2 secreting cells were detected by ELISpot. Results are presented as mean ± SD. Statistical analysis was performed with One-way ANOVA and Tukey´s Multiple Comparison Test (* P< .05, ** P< .01, *** P< .001).