We investigated the changes of IgE binding components to major tree and weed pollens (alder, birch, ragweed, and mugwort) (1, 12) in pollinosis patients undergoing allergen-SIT. We enrolled nine pollinosis patients with an increasing tendency of serum sIgE levels or A/H (allergen/histamine wheal) ratio on following skin prick tests (SPTs) to alder, birch, ragweed and mugwort pollens during the pollen-SIT from the Allergy and Clinical Immunology Department of Ajou University Hospital, Suwon, Korea. We analyzed the clinical data (including age, sex, underlying allergic diseases, SPTs, and serum specific IgE level) and investigated the underlying allergic diseases (AR, AC, BA, and AD). The A/H ratio represents the results of SPTs performed with 50 common aeroallergens (Bencard, Bretford, UK). Serum sIgE levels to each pollen were monitored by an ImmunoCAP® system during SIT. Blood sampling was done before SIT (initial sampling, i) and 31.2±25.7 months after maintenance SIT (follow-up sampling, f). We performed an enzyme-linked immunosorbant assay (ELISA) to detect the changes of serum sIgE to alder, birch, ragweed and mugwort pollen extracts. Pollen extracts were prepared the same as described in a previous study method (17); pollen of Alnus incana (alder), Betula pendula (birch), Ambrosia artemisiifolia (ragweed), and Artemisia vulgaris (mugwort) were purchased at Allergon® (ThermoFisher Scientific, Uppsala, Sweden). After the preparation of a crude extraction in a phosphate buffered saline (PBS), a Bradford protein assay measured the protein concentration and a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the protein bands. ELISA was performed in sera sampled from two time-point with each pollen extract in a concentration of 5 µg/mL for 2 hr to detect the serum sIgE to each pollen. Serum sIgE levels were presented as absorbance values and positive cutoff levels were defined as (mean±3*standard deviation) of absorbance values from non-atopic healthy controls. IgE immunoblot analysis was performed with transfer 40 µg/mL pollen extracts to polyvinylidene difluoride (PVDF membrane, Millipore Co., Bedford, MA, USA) and incubation of sera for 12 hr at 4℃. The major allergen of each pollen was defined according to previous reports; Aln g 1 (17 kDa) in alder, Bet v 1 (17 kDa) in birch, Amb a 1 (38 kDa) in ragweed and Art v 1 (24 kDa) in mugwort (17, 18). A densitometry (Bio-Rad Universal Hood II Gel Imager, BIO-RAD Laboratories, Milan, Italy) observed the changes of IgE binding components.

This study enrolled six male and three female pollinosis patients. Their mean age was 38.9±10.6 yr. All nine patients had seasonal AR and AC polysensitized to tree and weed pollens. BA was comorbid in five patients while two patients had AD. Initial sampling (i) was done before SIT and follow-up sampling (f) was done during SIT (31.2±25.7 months after maintenance SIT). All nine patients were treated with pollen-SIT with causative pollens that included the four pollens. Table 1 shows patient characteristics such as SPTs and ImmunoCAP® results. A Wilcoxon signed rank test compared the changes of serum sIgE levels in ELISA. Significant increases of serum sIgE levels to alder, birch, ragweed and mugwort pollen extracts by ELISA were noted during the maintenance of SIT compared to the initial levels presented in Fig. 1 (alder, P=0.001; birch, P=0.006; ragweed, P=0.001; mugwort, P=0.001). IgE immunoblot analysis showed two changing patterns: 1) intensification or a new generation of major allergen and 2) generation of additional IgE binding components (Fig. 2). Fig. 2A shows the changing patterns of alder pollen extracts; two patients (No. 1, 2) showed a new generation of major allergen (17 kDa) and additional IgE binding components (9-175 kDa); however, one patient (No. 3) showed a new generation of additional components (39-125 kDa, Fig. 2A). Fig. 2B showed the changing patterns of birch pollens. Two patients (No. 1, 5) showed new generations of major allergen (17 kDa) and additional components (9-125 kDa) while one patient (No. 4) showed new generation of additional components (11 kDa). Fig. 2C showed changing patterns of ragweed pollens; various changes of major allergen (38 kDa, showed no change in patient No. 6, decreased in No. 7, and intensified in No. 8) and a new generation of additional components ranged from 9-13 kDa. Fig. 2D showed the patterns of mugwort pollens; intensification (No. 9) or new generation (No. 1) of major (24 kDa) and additional components (9-80 kDa) were noted (Fig. 2D).

Serum sIgE levels increased at the first year of SIT and then decreased along with an increase of a blocking antibody, IgG and immunomodulation of B-cell itself or by regulatory T-cells (6, 19). We demonstrated increased sIgE levels to major tree and weed pollens (that included alder, birch, ragweed and mugwort pollens) in a subpopulation of pollinosis patients despite a few years of maintenance SIT with corresponding allergen extracts. ELISA confirmed increased serum sIgE levels and an IgE immunoblot analysis confirmed the intensification of major allergens or a new generation of IgE binding components to each pollen. Recent studies have demonstrated that environmental changes (especially climate change) can increase the allergenic potency of major allergens in pollens such as birch, ragweed and Japanese hop (10, 11, 12, 16). Our previous study demonstrated that environmental changes over the last 10 yr could induce the intensification of major allergenic component of Japanese hop pollen at 10 kDa and a new generation of additional IgE binding components within the pollens. The findings suggest that climate change can increase the allergenic potency of major pollens through intensification or the generation of new IgE binding components within major tree or weed pollens (alder, birch, ragweed and mugwort pollens) that can contribute to the increased prevalence and severity of pollinosis in Korea (10, 11, 12, 13, 14, 15, 20). The present study demonstrates that sIgE levels to offending allergens did not decrease; conversely, the results indicated a paradoxical increase in IgE binding components to alder, birch, ragweed and mugwort pollens after allergen-SIT in a subpopulation of pollinosis patients that could reduce clinical outcomes of SIT. The results suggest that the regular monitoring of serum sIgE levels and symptom changes in patients undergoing SIT with tree and weed pollens is needed to detect and improve clinical outcomes of SIT in patients who may have a paradoxical increase of sIgE.