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Keywords:

  • Bet v 1 homologue;
  • hornbeam;
  • oak;
  • tree pollen allergy

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background:  Birch pollen allergy is one of the most common causes of spring pollinosis often associated with hypersensitivity reactions to pollen of other Fagales species. Yet, only the major disease eliciting allergens of alder and hazel have been fully characterized. Therefore, the aim of this study was to perform cloning, expression and immunologic characterization of the Bet v 1 homologues from oak (Que a 1) and hornbeam (Car b 1).

Methods:  The isoform pattern of Car b 1 and Que a 1 was analyzed by proteomics using 2D gel electrophoresis and LC ESI-QTOF MS. Isoallergens showing high IgE-binding were cloned and expressed in Escherichia coli. IgE-binding activity of the recombinant proteins was determined by enzyme-linked immunosorbent assay (ELISA) and basophil mediator release assays using serum samples from patients mainly exposed either to oak and hornbeam or to birch pollen. Cross-reactivity of the allergens was further investigated at the T-cell level.

Results:  Dominant isoforms of Car b 1 and Que a 1, identified by mass spectrometry, showed different IgE-binding properties when testing Fagales pollen-allergic patients living in birch-free areas as compared to birch-sensitized individuals.

Conclusion:  Tree pollen-allergic patients who are primarily exposed to Fagales pollen other than birch reacted stronger with rCar b 1 and rQue a 1 than with rBet v 1, as determined by inhibition ELISA and basophil mediator release assays. Thus, rCar b 1 and rQue a 1 allergens should be considered for improving molecule-based diagnosis and therapy of tree pollen allergies manifesting in birch-free areas.

In the temperate climate zones of the northern hemisphere, spring pollinosis is mainly caused by pollen from birch or from closely related trees, all belonging to the order Fagales. Following the identification and cloning of the major birch pollen allergen Bet v 1 (1), several homologous pollen allergens have been identified from related species i.e. alder Aln g 1, hornbeam Car b 1, chestnut Cas s 1, hazel Cor a 1, and oak Que a 1 (2–5). The high clinical relevance of Bet v 1 is reflected by the fact that over 95% of birch pollen-allergic patients were tested positive with this allergen and 60% reacted exclusively with Bet v 1.

A skin prick test study including 173 Bet v 1-allergic patients found that approximately 98% of the individuals showed a positive wheal and flare reaction to hornbeam, 93% to alder and hazel, and 75% to oak and beech pollen extract (4). This high level of immunologic cross-reactivity of Bet v 1 with its homologues is per se not surprising and has been known for many years (6, 7). Still, for some Bet v 1 homologues, limited or no immunological data are available resulting in difficulties to estimate the clinical relevance of such allergens. To date, routine diagnosis and therapy of Fagales pollen-allergic patients are mostly based on the use of Bet v 1 or birch pollen extract only (8) without taking into account the differences in the geographic distribution of the various Fagales species (4). It is possible that depending on the exposure patterns of patients, the inclusion of other Bet v 1 family members in allergy diagnosis and therapy might increase the efficacy of the treatment, particularly for those individuals living in birch-free areas (9). For instance in the Mediterranean area, oak and hornbeam are among the dominating Fagales species, whereas birch trees are virtually absent. Nevertheless, little is known about the major allergens from these trees. Here, we report the molecular cloning and characterization of Bet v 1 homologues from hornbeam and oak pollen. To identify the dominating allergen isoforms, a proteomic approach was used. Allergen isoforms that were abundant in aqueous pollen extracts and showed high patients’ IgE-binding were identified by two dimensional (2D) gel electrophoresis in combination with mass spectrometry. Selected isoallergens were produced as recombinant proteins in Escherichia coli and characterized physicochemically and immunologically. Sera from pollen-allergic patients living in birch-free areas (southern Italy) showed different IgE-binding activity towards Car b 1 and Que a 1 when compared to sera from patients living in birch-dominated areas (Austria). These results demonstrate the clinical relevance of hornbeam and oak pollen allergens and highlight their importance as candidates for improving diagnosis and therapy of Fagales pollen allergies (10).

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients and sera

Fagales pollen-allergic patients from Austria (n = 10) and Italy (n = 11) were selected on the basis of typical case history, positive in vivo skin prick test and in vitro IgE detection (ImmunoCAP system; Phadia AB, Uppsala, Sweden). The patients recruited from Austria included six female and four male persons, aged between 17 and 48 years. The average specific IgE level to birch pollen extract was 63.56 kUA/l. Three patients had rhino-conjunctivitis and asthma, five rhino-conjunctivitis, one suffered from conjunctivitis, and one from rhinitis. The patients recruited from Italy included five female and six male persons, aged between 19 and 45 years. The average specific IgE level to birch pollen extract was 17.37 kUA/l, to hornbeam pollen extract 20.67 kUA/l, and to oak pollen extract 10.24 kUA/l. Six patients had rhino-conjunctivitis and asthma, and five suffered from rhino-conjunctivitis. Case histories of patients whose serum samples have been used for ELISA and basophil mediator release assays are listed Table 1. Experiments with blood samples from pollen-allergic patients were approved by the Ethic Committee of the Medical University and General Hospital of Vienna (no. EK028/2006) and by the ethic committee of IDI-IRCCS (project IDI-CACeS SeraBank, no. 106-CE-2005). Informed written consent was obtained from all subjects included in the study.

Table 1.   Patients’ sera
CodeCountrySexTotalsIgEsIgEsIgE 
IgEBirchHazelOakSymptoms
  1. sIgE, specific IgE; R, rhinitis; C, conjunctivitis; RC, rhino-conjunctivitis; n.d., not determined.

  2. Total IgE was determined by PRIST. sIgE values are given in kUA/l (Phadia ImmunoCAP®).

P1ItalyFemale741918.79.7RC
P2ItalyMale10918.1327RC, asthma
P3ItalyMale25831.236.523.9RC
P4ItalyMale13283.799.535.5RC, asthma
P5ItalyMale5103.77.15.6RC, asthma
P6AustriaMale226581.7n.d.n.d.RC, asthma
P7AustriaFemale18354.2n.d.n.d.C
P8AustriaMale46972.6n.d.n.d.RC
P9AustriaMale11168n.d.n.d.RC
P10AustriaFemale484>100n.d.n.d.R

Pollen extracts

Extract of hornbeam (Carpinus betulus) pollen (Batch 027303701; Allergon AB, Ängelholm, Sweden) was prepared by incubation of pollen in distilled H2O (0.18 g pollen/ml) for 30 min at room temperature followed by centrifugation at 4°C for 10 min at 14000 g. The centrifugation step was repeated with the collected supernatant. Oak (Quercus alba) pollen (Batch 030803201; Allergon AB) extract was prepared by incubation in 250 mM ammonium bicarbonate buffer pH 7.9 (0.18 g pollen/ml) for 2 h at room temperature, followed by centrifugation at 4°C for 10 min at 14000 g. The centrifugation step was repeated with the collected supernatant.

Cloning of Car b 1 and Que a 1

Total RNA was isolated from hornbeam and oak pollen (Allergon AB). Briefly, 1 mg pollen was ground and homogenized in 20 ml 4.2 M of guanidine thiocyanate, 50 mM of BES pH 7.2, 4 mM of EDTA. After high-speed centrifugation (15 000 g, 15 min), the supernatant was used for total RNA isolation using the TRIzol LS Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Reverse transcription of allergen genes was performed using the SuperScript III RTS One-Step RT-PCR Kit (Invitrogen). Car b 1 was amplified with the primer pair Car_Fwd and Car_Rev. Que a 1 was cloned in a two-step procedure: first, the cDNA was generated using the following primers: Que_Fwd and oligo_dT. The gene was cloned into a pGEM T Easy vector (Promega, Madison, WI, USA), sequenced and a gene specific reverse primer was designed. Finally, Que a 1 was amplified from the Que a 1::pGEM T Easy plasmid using the primers Que_Fwd and Que_Rev. Full-length Car b 1 and Que a 1 were cloned into the pET28b vector (EMD Biosciences Inc., San Diego, CA, USA) using the NcoI and EcoRI restriction sites. Several clones of each allergen were sequenced. One isoform from each pollen allergen was cloned into the pHis-Parallel2 vector using the NdeI and XhoI sites to allow allergen production as 6×His tagged fusion proteins (11). Therefore, the genes were amplified with either the primers Car_6×His_Fwd and Car_6×His_Rev in case of Car b 1 or Que_6×His_Fwd and Que_6×His_Rev for Que a 1. All primers are listed in Table 2.

Table 2.   Primer list
PrimerSequence
  1. (Y, pyrimidine; R, purine; M = A + C); restriction sites are underlined.

Car_Fwd5′-GGCATGCCATGGGTGTTTTYAAYTAYGARGCMGAAACC-3′
Car_Rev5′-CGCGAATTCTTAGTTGTATTCAGCAGTGTGTGCC-3′
Que_Fwd5′-CATGCCATGGGTGTTTTYAC -3′
Oligo_dt5′-CCGCTCGAGTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′
Que_Rev5′-CGCGAATTCTTACTTGTAGAGATCAGGGTGTGCC-3′
Car_6×His_Fwd5′-GGC-CCATATGGGTGTTTTTAATTATGAGG-3′
Car_6×His_Rev5′-TCGGCTCGAGGTTGTATTCAGCAGTGTGTGC-3′
Que_6×His_Fwd5′-GGCCCATATGGGTGTTTTCACTTATGAATC-3′
Que_6×His_Rev5′-TCGGCTCGAGCTTGTAGAGATCAGGGTGTGC-3′

Gel electrophoresis and mass spectrometry

Proteins were precipitated from aqueous pollen exudates (ProteoExtract Protein Precipitation Kit; Calbiochem, San Diego, CA, USA) and separated by 2D gel electrophoresis. Isoelectric focusing was performed using an immobilized gradient from pH 4 to 7 on 17 cm IPG strips (ReadyStrip IPG Strips, Protean IEF system; Bio-Rad, Hercules, CA, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the Protean II XL cell (Bio-Rad). Spots were excised from Coomassie-stained 2D gels and in-gel digested (ProteoExtract All-In-One Trypsin Digestion Kit; Calbiochem). Proteolytic digests were separated by reversed phase capillary HPLC (Nanoease Symmetry 300TM trap column and 0.075× 15 mm Nanoease Atlantis dC18TM separating column) on CapLC (Micromass-Waters, Milford, MA, USA) and directly infused into a Global Ultima Q-Tof instrument (Waters, Manchester, UK) with electrospray ionization. Data were acquired in the Data Directed Analysis (DDA) mode. Survey and fragment spectra were analyzed using the software plgs version 2.2.5 (Waters) with automatic and manual data verification. For sequence identification, combined SwissProt/Trembl database and a custom-made database containing Bet v 1 isoforms and homologous sequences were used.

Expression and purification of recombinant Car b 1 and Que a 1

Expression constructs, either based on the pET28b or pHis-Parallel2 vectors, were transformed into E. coli BL21 (DE3) pLysS cells (Stratagene, La Jolla, CA, USA), grown at 37°C to an OD600 of 0.8 in LB medium supplemented with either 25 mg/l kanamycin or 100 mg/l ampicillin. Cultures were cooled to 16°C and protein expression was induced by addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After incubation for 18 h, cells were harvested by low-speed centrifugation and resuspended in appropriate buffer. Recombinant Car b 1 and Que a 1 were purified as 6×His-tagged fusion proteins from soluble bacterial lysates by immobilized metal affinity chromatography (11). An additional purification step on a DEAE Sepharose Fast Flow column (GE Healthcare Bio-Sciences, Little Chalfont, UK) was used for rCar b 1. Recombinant proteins were dialyzed against 20 mM Tris–HCl buffer, pH 8 and stored at −20°C.

SDS-PAGE and immunoblots

Escherichia coli lysates, pollen extracts, and purified proteins were analyzed by denaturing SDS-PAGE using 15% gels. Proteins were visualized by Coomassie Brilliant Blue R-250 staining (Bio-Rad, Hercules, CA, USA). For immunoblot analysis, proteins were separated by SDS-PAGE and electroblotted onto nitrocellulose membrane (Whatman, Brentford, UK). Sera from atopic individuals were diluted 1 : 10 in 50 mM of sodium phosphate buffer, pH 7.4, 0.5% (v/v) Tween-20, 5% (w/v) bovine serum albumin, 0.05% (w/v) sodium azide. Bound IgE antibodies were detected using 125I-rabbit anti-human IgE (IBL, Hamburg, Germany) on a phosphorimager screen (Fujifilm BAS-1800 II, Fujifilm Europe, Düssseldorf, Germany). For 2D blots, a serum pool of 11 patients showing a positive skin prick test reaction to both hornbeam and oak pollen extracts as well as a positive ImmunoCAP (Phadia) to Bet v 1 were used.

Physicochemical characterization of recombinant allergens

Circular dichroism spectra of allergens were recorded in 5 mM of sodium phosphate pH 7.4 with a JASCO J-810 spectropolarimeter (Jasco, Tokyo, Japan) fitted with a Neslab RTE-111M temperature control system (Thermo Fischer Scientific Inc., Waltham, MA, USA). Data are baseline-corrected and presented as mean residue molar ellipticity [Θ]MRW at a given wavelength. Mass spectra of intact proteins were acquired using electrospray ionization coupled to a Quadrupole Time-of-Flight Mass Spectrometry (Waters).

Enzyme-linked immunosorbent assay

For chimeric ELISA, plates were coated with 2 μg/ml BIP-1 antibody in 50 μl TBS/well overnight at 4°C (12). Plates were washed, blocked, and incubated with 50 μl purified allergens (c = 2 μg/ml) or allergen extracts (c = 30 μg/ml) in 50 μl TBS/well overnight at 4°C. After further wash, plates were incubated with patients’ sera (dilution 1 : 10) overnight at 4°C. Bound IgE was detected with alkaline phosphatase-conjugated monoclonal anti-human IgE antibodies (BD Biosciences, Franklin Lakes, NJ, USA). For inhibition ELISA, plates were coated with 30 μg/ml pollen extracts in 50 μl TBS/well overnight at 4°C. Patients’ sera (dilutions 1 : 5 or 1 : 10) were preincubated with allergen titrations overnight at 4°C, transferred to the antigen-coated ELISA plates, and incubated at 4°C overnight. Bound IgE was detected with alkaline phosphatase-conjugated monoclonal anti-human IgE antibodies (BD Biosciences).

Rat basophilic leukemia (RBL) cell mediator release

Rat basophilic degranulation assays were performed as previously described (13). Briefly, RBL-2H3 cells transfected with human high-affinity IgE receptor (FcɛR1) were passively sensitized with serum IgE from tree pollen-allergic patients. Degranulation was triggered by addition of serial dilutions of the respective antigens. β-hexosaminidase in the supernatant was measured by enzyme reaction with the fluorogenic substrate 4-methyl umbelliferyl-N-acetyl-β-d-glucosamide and expressed as % of total release from Triton X100-treated cells.

T-cell proliferation assays

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll–Hypaque density gradient centrifugation (GE Healthcare Bio-Sciences) and proliferation assays were performed as described elsewhere (14). The stimulation index was calculated as a ratio between counts per minute obtained in cultures with T cells plus autologous antigen-presenting cells (APCs) plus stimulus and counts per minute obtained in cultures containing T cells and APCs.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Cloning of Car b 1 and Que a 1 isoallergens

Car b 1 and Que a 1 were cloned by RT-PCR using mRNA isolated from hornbeam and oak pollen, respectively. Five full-length isoforms of Car b 1 [Car b 1.0109 (EU283857), Car b 1.0110 (EU283858), Car b 1.0111 (EU283859), Car b 1.0112 (EU283860), Car b 1.0113 (EU283861)] and three isoforms of Que a 1 [Que a 1.0201 (EU283862), Que a 1.0301 (EU283863), Que a 1.0401 (EU283864)] were identified after DNA sequence analysis of eight individual clones derived from each pollen source (Fig. 1). The Database of the I.U.I.S. Allergen Nomenclature Sub-committee (http://www.allergen.org) lists 15 isoforms of Car b 1. Among these Car b 1 isoforms, 10 correspond to unique protein sequences, whereas five show only differences at the DNA level. All deduced amino acid sequences of Car b 1 isoforms reported here differ from the isoform sequences in the IUIS database. In case of Que a 1, only the N-terminal sequence of the protein has been published (2, 15).

image

Figure 1.  Deduced amino acid and sequence alignment of the isoallergens Bet v 1.0101, Car b 1.0109, and Que a 1.0301. Protein sequence identities are as follows: Bet v 1.0101 × Car b 1.0109: 74%, Bet v 1.0101 × Que a 1.0301: 58%. Multiple sequence alignment and calculations of identity scores were performed with ClustalW/EMBL-EBI, dashes indicate identical amino acids.

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Following DNA sequence analysis, Car b 1 as well as Que a 1 isoforms were expressed in E. coli and human IgE-binding was analyzed by dot blot using sera from birch pollen-allergic individuals (data not shown). The dot blot analysis revealed that all Car b 1 and Que a 1 isoforms were recognized by patients’ serum IgE. Previously, three Car b 1 isoforms (Car b 1.0101, Car b 1.0102, Car b 1.0201) have been produced as recombinant proteins in E. coli and patients’ IgE-binding was demonstrated by immunoelectrophoresis using crude bacterial lysates (3). Very recently, Moverare et al. (15) could demonstrate human serum IgE binding to purified natural Que a 1.

2D gel electrophoresis and mass spectrometry of IgE-binding isoforms in pollen extracts

The isoform composition of Car b 1 and Que a 1 in aqueous extracts of hornbeam and oak pollen was evaluated by 2D gel electrophoresis. After 2D separation, the protein spots were digested with trypsin and analyzed by mass spectrometry. In hornbeam pollen extracts, 15 IgE positive spots in the molecular weight region of 17 kDa were observed. The assignment of IgE-positive spots to the respective isoforms depends on the identification of ‘diagnostic peptides’, which are peptides unique for one particular isoform. Certain Car b 1 isoforms differ in only one amino acid. Consequently, isoforms showing amino acid sequence identities of above 98% could not be separated by 2D gel electrophoresis because of similar molecular weights and similar or even identical isoelectric points (e.g. Car b 1.0109, Car b 1.0110, Car b 1.0112, Car b 1.0113). Therefore, several excised spots showed a peptide pattern that could not be unequivocally assigned to a single Car b 1 isoform. Additionally, post-translational modifications often influence the pI of proteins thus resulting in multiple spots of diversely modified, differently charged variants of the same protein isoform. In case of Car b 1, deamidation of Asn47 leads to an acidic pI shift, which was observed for Car b 1.0109, Car b 1.0110, Car b 1.0112 (see spots 1, 5, and 9 in Fig. 2A), and Car b 1.0107 (see spots 3 and 7 in Fig. 2A). Deamidation of Asn47 is commonly observed within the Bet v 1-related pollen allergen family and does neither influence IgE-binding activity (16) nor the antigenicity of the proteins. The isoforms Car b 1.0109, Car b 1.0110, and Car b 1.0112 reported here were identified as the most dominant spots on the 2D gel and showed high IgE-binding in immunoblots. On the basis of these results, we selected isoform Car b 1.0109 for further investigations (Fig. 2A,B). Interestingly, four spots (spots 2, 4, 6, and 8) corresponding to truncated versions of Car b 1 isoforms were identified, which appeared at a lower molecular weight on the 2D gel. For all four spots, the N-terminal sequence was confirmed. As no tryptic peptide corresponding to the C-terminal part was found, we assumed that these spots represent C-terminal degradations of the Car b 1 isoforms. These C-terminally degraded Car b 1 isoforms showed a markedly reduced or even no IgE-binding capacity as compared to the intact protein (e.g. spots 1 and 2). The sequence coverage for Car b 1 averaged out at 74.6%.

image

Figure 2.  Hornbeam (A, B) and oak (D, E) pollen extracts were separated by 2D gel electrophoresis. Gels were either coomassie-stained (A, D) or electroblotted onto nitrocellulose membranes and IgE-binding spots detected using 125I-rabbit anti-human IgE (B, E). Encircled spots were analyzed by MS and assigned to respective isoforms in hornbeam (C) and oak (E). The isoform assignment was based on ‘diagnostic peptides’ identified for the different spots. (+/+) indicates that both N- and C-terminal peptides, (+/−) that only the N-terminal peptide and (−/−) that neither of the two peptides could be identified by MS.

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Only three isoforms of Que a 1 were identified by cDNA cloning. After 2D gel electrophoresis, four distinct IgE-binding spots were identified at a molecular weight of 17 kDa. Two of the spots could be assigned to Que a 1.0201 and two to Que a 1.0301. These spot doublets resulted from a pI shift caused by deamidation of Asn47 in the Que a 1 sequence. In contrast to Car b 1, shorter versions of Que a 1 as a result of C-terminal degradation were not observed. Sequence coverage of Que a 1 isoforms was approximately 69.5% (Fig. 2C,D).

Isoform Que a 1.0301 corresponded to approximately 50% of total pollen Que a 1. Additionally, Que a 1.0301 showed strong serum IgE-binding and thus, was selected for more detailed analysis.

Recombinant production and characterization of Car b 1 and Que a 1 isoallergens

The isoforms Car b 1.0109 and Que a 1.0301 were produced as recombinant proteins in E. coli and purified to homogeneity (Fig. 3A). The primary sequence of both recombinant allergens was confirmed by mass spectrometry. The molecular masses of the proteins were measured as follows (theoretical mass values given in parenthesis): rCar b 1.0109 18 248.6 (18 248.6), rQue a 1.0301 18 412 (18 411.7). Circular dichroism spectroscopy showed that rCar b 1.0109 and rQue a 1.0301 isoforms are folded proteins containing both alpha helices and beta sheets, similar to rBet v 1.0101 (also know as Bet v 1a) (Fig. 3B).

image

Figure 3.  SDS-PAGE analysis of purified rCar b 1.0901 and rQue a 1.0301 (5 μg/lane). Proteins were visualized by coomassie staining (A). Circular dichroism analysis of rCar b 1.0901, rQue a 1.0301, and rBet v 1.0101 isoallergens. Data are presented as mean residue molar ellipticity [Θ]MRW at a given wavelength and baseline corrected (B). Comparison of human IgE binding to natural or recombinant Car b 1 or Que a 1 by chimeric ELISA. Presented data are baseline subtracted. r-values were calculated with Pearson’s correlation (Car b 1: = 0.946, < 0.01, n = 10; Que a 1: = 0.94, < 0.01, n = 10) (C).

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IgE-binding properties and allergenic activity of rCar b 1.0901 and rQue a 1.0301 isoallergens

Patients’ serum IgE binding of recombinant Car b 1.0901 and Que a 1.0301 was compared to natural hornbeam and oak pollen allergens by chimeric ELISA using the monoclonal antibody BIP-1 as capture antibody (12). IgE binding to natural and recombinant Car b 1 as well as Que a 1 was found to correlate significantly (Pearson’s correlation for Car b 1: = 0.946, < 0.01, n = 10; Pearson’s correlation for Que a 1: = 0.94, < 0.01, n = 10), denoting that both recombinant isoallergens behave similar as their natural counterparts (Fig. 3C). For inhibition ELISA, pollen extracts from hornbeam or oak were coated on ELISA plates, patients’ sera were preincubated with either rCar b 1.0901, rQue a 1.0301, or rBet v 1.0101, and bound IgE antibodies were detected (Fig. 4A). Serum samples used in these experiments originated from Fagales pollen-allergic patients living in birch-free areas or in a birch-dominated area. As birch trees are almost absent in Rome and its surrounding areas, this leads to the assumption that there is virtually no exposure to birch pollen for people living in this area, which is dominated by oak trees, followed by alder, hornbeam, and hazel (4). Thus, the selected Italian cohort should rather be sensitized by Fagales species other than birch, whereas the second group of allergic individuals recruited in Austria is predominantly exposed to birch pollen. However, this does not exclude a possible cross-sensitization of Italian patients with birch pollen and vice versa. Nevertheless for Italian patients, less rCar b 1.0901 (2.6 times) and rQue a 1.0301 (2.8 times) were necessary to obtain 50% inhibition of serum IgE-binding as compared to rBet v 1.0101. In contrast, lower levels of rBet v 1.0101 compared to rCar b 1.0901 or rQue a 1.0301 were necessary to achieve 50% inhibition of Austrian patients’ IgE-binding towards the respective pollen extracts. Yet, at high concentrations all three antigens could inhibit to almost 100% the IgE binding to coated pollen extracts (Fig. 4B).

image

Figure 4.  IgE binding properties of rCar b 1.0109 and rQue a 1.0301 were tested by inhibition ELISA (A). Protein concentrations (pg/ml) required to inhibit 50% of serum IgE-binding to coated pollen extracts are indicated. P1 to P4 represent sera from Fagales pollen-allergic patients living in Italy, P5–P8 sera from Fagales pollen-allergic patients living in Austria. Mean values for Italian patients are labeled with mean (I), mean values for Austrian patients with mean (A). ELISA inhibition curves are shown for a representative patient from Italy and Austria, respectively (B). Rat basophil leukemia cells transfected with the human FcɛRI were passively sensitized with human serum IgE. Recombinant Fagales pollen allergens were used to induce mediator release (C). P1–P4 represent sera from Fagales pollen-allergic patients living in Italy, P5–P8 sera from Fagales pollen-allergic patients living in Austria. Mean values for Italian patients are labeled with mean (I), mean values for Austrian patients with mean (A). Protein concentrations required to induce 50% of maximum basophil mediator release are given in pg/ml. N.d. indicates that no β-hexosaminidase release could be detected.

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Basophil mediator release assays

Rat basophils loaded with sera from Italian patients reacted best with rCar b 1.0901. Two- and threefold higher levels of rBet v 1.0101 and rQue a 1.0301, respectively, were necessary to obtain half-maximal levels of mediator release (Table 1). The half maximal mediator release per patient was defined by the dose-dependent mediator release curve of rBet v 1.0101. For basophils loaded with serum IgE from Austrian patients, rBet v 1.0101 was the best stimulus. Eightfold and 28-fold higher concentrations of rCar b 1.0901 and rQue a 1.0301, respectively, were necessary to achieve half maximal mediator release. Interestingly, for both Italian and Austrian patients the maximum mediator release obtained with rBet v 1.0101 and rCar b 1.0901 was nearly identical, whereas rQue a 1.0301 reached only 60% of the levels obtained with rBet v 1.0101 and rCar b 1.0901. Thus, Que a 1 seems to display a lower allergenic activity when compared to Bet v 1 or Car b 1 allergens.

Human T-cell recognition

Proliferative responses of human PBMCs from Austrian pollen-allergic donors were analyzed upon stimulation with 3 μg/ml of rBet v 1.0101, rCar b 1.0901, or rQue a 1.0301. All allergens triggered T-cell proliferation to a similar extent. These results demonstrate strong cross-reactivity of oak, hornbeam, and birch pollen allergens at the T-cell level (Fig. 5).

image

Figure 5.  Human T-cell recognition. PBMCs were stimulated with 3 μg/ml of the respective antigens. Stimulation indices were calculated as a ratio between counts per minute obtained in cultures with T cells plus autologous APCs plus stimulus and counts per minute obtained in cultures containing T cells and APCs. Symbols represent individual patients, bars means.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Bet v 1 homologues found in the pollen of Fagales trees have been identified as major allergens causing spring pollinosis in sensitized individuals. Several of these allergens have been extensively characterized (e.g. Bet v 1, Cor a 1, Aln g 1) and some of them were shown to exist in pollen as complex mixtures of isoforms displaying distinct immunologic properties (17–19). Bet v 1.0101, the isoform showing the highest patients’ IgE-binding activity, is also the most abundant (more than 50% of total Bet v 1) isoform in birch pollen (18). One could speculate that the predominance of Bet v 1.0101 in birch pollen leads to higher levels of high-affinity IgE antibodies directed against this particular protein as compared to other isoforms expressed at lower levels.

In this study, we undertook a detailed molecular analysis of isoforms of hornbeam Car b 1 and oak Que a 1, both species belonging to the Fagales order. A combination of cDNA cloning and mass spectrometry-based proteomics allowed us to identify Car b 1 and Que a 1 isoallergens, which are both abundant in the pollen and show strong IgE antibody-binding activity. Therefore, these isoforms were considered to be relevant for allergic individuals. The isoforms were produced as recombinant proteins and their IgE-binding properties were extensively analyzed.

Because of the fact that in birch-free areas, such as parts of North America and Mediterranean countries, exposure and sensitization to oak, hornbeam, hazel, or alder pollen is much more common than to birch (20, 21), patients from geographically distinct areas were recruited for this study. Assuming that the levels of allergen exposure influence the sensitization patterns, individuals living in central Italy are expected to be sensitized by Fagales trees other than birch, whereas individuals living in Austria should be primarily sensitized to birch pollen. Though it seems unlikely, this does not exclude a cross-sensitization of Italian patients with birch pollen and vice versa. Nevertheless, we found that Italian patients reacted stronger with rCar b 1.0109 and rQue a 1.0301 than with rBet v 1.0101, as determined by inhibition ELISA and basophil mediator release assays, which again supports the concept of sensitization of patients by the dominating trees in the particular area. In contrast, Austrian patients showed stronger IgE reactivity towards rBet v 1.0101 than to rCar b 1.0109 (eight-times lower) and rQue a 1.0301 (28-times lower). To summarize, our results demonstrate that Car b 1 and Que a 1 are relevant allergens, especially for those patients living in birch-free areas. Therefore, one should consider Car b 1 and Que a 1 as candidates to improve diagnosis and allergen-specific immunotherapy of Fagales tree pollen allergy to be used in certain geographic areas. Still further studies on larger cohorts of Fagales allergic patients from selected and well characterized geographic areas should be undertaken with an extended panel of Bet v 1 homologous molecules to define their pattern of IgE recognition and their biological activities by both basophil activation and T-cell proliferation assays.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We thank Dr Christof Ebner (Allergieambulatorium Reumannplatz, Vienna, Austria) and Dr Thomas Hawranek (Department of Dermatology, Paracelsus Private Medical University, Salzburg, Austria) for serum samples. Further, we would like to thank the Christian Doppler Research Association, the Oesterreichische Nationalbank (Project no. 12533) and the Italian Ministry of Health, Ricerca Corrente 2006-7, for financial support of this project.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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