Immunotherapy extract was prepared as described before (5). An adequate amount of pollens was collected in the Suwon area during the last 4 weeks of September 2003. The pollens were defatted, dried, and sent to Allergopharma Co., Germany for "Depo-Hop J" preparation, which was used for skin prick test and immunotherapy. Standardization process was carried out and presented as protein nitrogen units (PNU). The initial preparation was composed of three serial vials with strengths 1 (25 PNU), 2 (250 PNU), and 3 (2,500 PNU). For the maintenance treatment, vial 3 (2,500 PNU) was used, and was administered monthly for 3 yr. The initial dose was 0.1 mL, and the maximum tolerable dose was decided on an individual basis. The extracts were lyophilized, which was used for ELISA and IgE-immunoblot analysis.

Twenty patients (13 asthma with rhinitis and 7 hay fever) were enrolled. Asthma severity was classified as mild to moderate degrees based on the revised global initiative for asthma guideline (2004) and they had suffered from seasonal aggravation of asthmatic and rhinitis symptoms. All the patients had high serum-specific IgE antibody to Hop J by ELISA, as well as positive [>3+ (allergen to histamine ratio, A/H)] responses to skin prick tests. To exclude allergen exposure effect, Hop J immunotherapy was started in 1 month after the pollen season. Patients' sera were collected three times: before, one year and three years after the immunotherapy. All the patients gave their informed consent, and this study was approved by the ethical commitee of Ajou University Medical Center, Suwon, Korea.

Skin prick tests were performed three times: before, one year and three years after the immunotherapy and the skin reactivity was presented as A/H ratio. Degrees of airway hyperresponsiveness to methacholine were measured out of season before and every year using the method previously described (6).

Serum total IgE level was measured by Immuno-CAP system (Pharmacia, Sweden) according to the manufacturer's instructions. A change of specific IgE level to Hop J was determined by ELISA according to the previously described method (6). Briefly, microtiter plates (Costar, Corning, NY, U.S.A.) were first coated with 100 µL of Hop J pollen extract (1 µg/well) and left at 4℃ overnight. Each well was washed three times with 0.05% Tween-phosphate-buffered saline (PBS-T), and the remaining binding sites were blocked by incubation with 200 µL of 10% fetal bovine serum for 1 hr at room temperature. Each well was washed three times and then incubated for 1 hr at room temperature with 50 µL of either the patients' sera (1:5 dilution) or control sera from 60 patients who showed negative skin prick test responses to common inhalant allergens and the Hop J pollen. After the wells were washed three times with PBS-T, 100 µL of 1:1,000 (vol/vol) biotin-labeled goat anti-human IgE antibody (Sigma Co., St. Louis, MO, U.S.A.) was added to the wells and incubated for 1 hr at room temperature. The wells were then washed three times with PBS-T and incubated with 1:1,000 (vol/vol) streptavidin peroxidase (Sigma Co.) for 30 min before another washing step, which was followed by incubation with 100 µL of 3.3', 5.5'-tetramethylene benzidine in 0.05 M/L phosphate citrate buffer, pH 5.0, and 10 mL of 30% H₂O₂ in a phosphate citrate buffer for 10 min at room temperature. The reaction was stopped by the addition of 100 µL of 2 N sulfuric acid, and the absorbance was read at 450 nm by an automated reader.

Diluted patient serum or negative control serum (1:3 for soluble IgG₁ and 1:100 for soluble IgG₄ in diluent buffer; PBS containing 10% fetal bovine serum; 200 µL) was added to each well coated with Hop J pollen. After incubation for 1 hr at 25℃, the wells were washed three times with PBS-T. After they were washed three times with PBS-T, 100 µL of 1:1,000 (vol/vol) biotin-labeled goat anti-human IgG₁ or IgG₄ antibody (Sigma Co.) were added to the wells and incubated for 1 hr at room temperature. The wells were then washed three times with PSB-T and incubated with 1:1,000 (vol/vol) streptavidin peroxidase (Sigma Co.) for 30 min before another washing step, which was followed by incubation with 100 µL of 3.3', 5.5'-tetramethylene benzidine in 0.05 M phosphate citrate buffer, pH 5.0, and 10 mL of 30% H₂O₂ in a phosphate citrate buffer for 10 min at room temperature. The reaction was stopped by the addition of 100 µL of 2 N sulfuric acid, and the absorbance was read at 450 nm by an automated reader. The antibody titer was expressed as arbitrary unit (A.U.) absorbance values. The positive cut-off value was determined as mean +3 standard deviations (SD) of 60 controls. All the samples were run on the same day.

12% SDS-PAGE and IgG₄-immunoblot analysis were performed under reducing conditions according to methods described previously (6). The Hop J pollen extracts were mixed with sample buffer (Tris-HCl 31 mM/L, 10% glycerol, 1% SDS, 0.0025%, bromophenol blue, 2.5% β-mercaptoethanol, pH 6.8) and heated in boiling water for 5 min. Then, standard marker (4 to 250 kDa; Novex, San Diego, CA, U.S.A.) and the Hop J pollen extracts were loaded to 12% Tris-glycine gel (Novex) for the separation of the antigens. Electrophoresis was performed with a Bio-Rad protein Mini cell for 90 min at 125 V. The gel was fixed and stained with Coomassie brilliant blue. For immunoblotting, the proteins were transferred onto a polyvinylidene difluoride membrane (PVDF) (Millipore Co., Bedford, MA, U.S.A.) in transfer buffer (Tris-base 25 mM/L, glycine 193 mM/L and methanol 20%) with a Bio-Rad transfer apparatus set at 200 mA for 90 min. The blotted PVDF membrane was sliced into 4 mm widths. The pieces of the membrane were blocked by using 5% skim milk in Tris-buffered saline (TBS)-Tween (TBST) for 1 hr to block nonspecific binding. Each membrane was then incubated overnight at 4℃ with the patient or control sera that had been diluted 1:10 v/v with 5% skim milk TBST. After washing, the membranes were incubated with anti-human IgG₄ conjugated with biotin and streptavidine-alkaline phosphastase (1:1,000 diluted, Sigma Co.) for one hour at room temperature. After washing with TBST, the membrane was developed by using BCIP/NBT alkaline phosphatase substrate (Sigma Co.).

To observe the changes of cell marker and cytokines, the levels of IL-10, soluble CD23 (sCD23), IL-12 and TGF-β₁ were measured by ELISA kits (Benger Med Systems, Vienna, Austria) according to the manufacturer's guidance. The changes between years were presented as the ratio between two values.

Wilcoxon signed rank tests using SPSS (Chicago, IL, U.S.A.) version 11.0 were applied to evaluate the statistical differences between two values. A p value of 0.05 or less was regarded as significant.

Fig. 1A shows the changes of skin reactivity to Hop J. It decreased after 1 yr of immunotherapy (p=0.019), however further decrease was not noted from 1 to 3 yr (p=0.86). Airway hyperresponsiveness to methacholine tended to improve at 1 yr (p=0.21) and improved further during next 2 yr (p=0.05) as shown in Fig. 1B.

Fig. 2 shows the changes of serum specific IgE to Hop J pollen extracts and sCD23 levels during 3 yr study period. There were no significant changes in either total (data not shown) or specific IgE antibody levels during the 3-yr of immunotherapy (p>0.05, respectively), however sCD23 level began to decrease during the first year and significantly decreased during next two years. (p=0.003)

Fig. 3 shows the changes of serum specific IgG₁ (Fig. 3A) and IgG₄ (Fig. 3B) antibodies to Hop J pollen extracts during 3 yr immunotherapy. Specific IgG₁ levels increased significantly for first year with statistical significance (p<0.001), and then decreased significantly during next two years (p=0.0039). However, serum specific IgG₄ levels began to increase remarkably during the first year (p<0.0001) and increased further until 3 yr with high statistical significance (p<0.001, Fig. 3B).

Fig. 4 shows the comparison of IgG₄-binding components within the Hop J pollen extracts using the sera of seven individuals showing remarkable increase of serum specific IgG₄ level during the study period. Five components (94, 78, 22, 13, 7 kDa) showing remarkable increase of IgG₄ binding intensity were generated as shown in Fig. 4B.

Fig. 5 shows the changes of serum IL-10 (Fig. 5A) and TGF-β₁ (Fig. 5B) levels during 3 yr immunotherapy. There were no significant changes in serum IL-10 during the first year (p=0.55), but decreased significantly during next two years with statistical significance (p=0.05). TGF-β₁ levels began to decrease during the first year and decreased further during next two years of immunotherapy with statistical significance (p=0.01). There were no significant changes in serum IL-12 levels during 3 yr study period (data not shown).