From March to May of 2012, a total 14 patients with AR were admitted into this department: five males and nine females, with a mean age of 29.76years. The diagnosis of AR was made according to its diagnostic criteria.1 Skin- prick test results were positive for dust mite; the severity of AR was moderate or severe in all 14 patients. These patients had not been treated with glucocorticoids in the past month and had not received anti-histamine therapy and allergen-specific immunotherapy. Patients with sinusitis, asthma, and intolerance to acetylsalicylic acid were excluded. Additionally, six healthy volunteers without symptoms of AR were recruited as controls: two males and four females, with a mean age of 30.83years. All subjects signed an informed consent prior to their enrollment in the study.

Flow cytometer (FACSCalibur; BD - USA) was used for detection and Cellquest software (BD - USA) was employed for the acquisition of data and subsequent analysis. Microplate reader (ELX-800; Bio-Tek - USA), PMA, ionomycin calcium, BFA (MultiSciences), Human Regulatory T Cell Staining Kit (eBioscience - USA), APC-conjugated human CD8 mAb (BD - USA), PerCP-Cy5.5 conjugated human CD3 mAb, PE-conjugated human IL-17 mAb and corresponding istype control (eBioscience - USA), fixation solution and membrane rupture solution (Invitrogen - USA), anti-human IL-17 ELISA kit (eBioscience - USA), and TGF- β1 ELISA kit (RayBio - USA) were used in the present study.

In the morning, venous blood samples (4mL) were collected from the ulnar vein and anti-coagulated with heparin. Then, 2mL of blood were subjected to centrifugation at 3,000rpm for 15; the serum was collected and stored at -20°C for the detection of IL-17 and TGF-β1. In addition, the remaining 2mL of blood were used for flow cytometry for the detection of Th17 cells and Treg cells.

In brief, 250μL of peripheral blood were mixed with 50μg/L PMA, 2.0μmol/L monensin, and 750 μmoL/L ionomycin calcium, followed by incubation at 37°C in an environment with 50mL/L CO2 for 4h. The cell suspension was transferred into a 1.5ml-PE tube followed by centrifugation at 2,500rpm for 6min. The supernatant was removed, and the cells were washed with PBS twice for FCM. The cells were treated with 10μL of PECy5-CD3Ab and 10μL of FITC-CD8Ab at room temperature, in a dark environment, for 30min.

After washing in PBS twice, the cells were fixed in 300μL of fixation solution in a dark environment at 4°C for 15min. After centrifugation, the supernatant was removed. Following the addition of membrane rupture solution, centrifugation was performed at 3,000rpm and the supernatant was removed. After washing in PBS twice, cells were transferred into two tubes and treated with 20μL of PE-IL-17Ab and 10μL of isotype control PE-IgG1, respectively, in a dark environment at room temperature for 30min. After washing in PBS twice, cells were re-suspended in 0.3mL of PBS and subjected to FCM. CellQuest software was employed for data analysis.

Cells were transferred into sample tube and control tube. Cells in the sample tube were treated with CD4/CD25/ Foxp3, and those in the control group were treated with Foxp3 isotype control (CD4/CD25/mouse IgG). Then, 100μL of anti-coagulated blood was added followed by incubation in a dark environment at room temperature for 20min. Following the addition of 1mL of hemolytic agent, incubation was performed at room temperature in a dark environment for 10min followed by centrifugation at 1,000rpm for 5min. The supernatant was removed and cells were re-suspended in 1mL of PBS followed by centrifugation at 1,000rpm for 5min. The supernatant was then removed, and 0.5mL of Foxp3 fixation solution were added, followed by incubation in a dark environment at room temperature for 20min. Cells were re-suspended in 1mL of PBS, and centrifuged at 1,000rpm for 5min. The supernatant was removed, and 0.5mL of Foxp3 membrane rupture solution was added to each tube, followed by centrifugation at 1,000rpm for 5min. These cells were re-suspended in 0.5mL of Foxp3 membrane rupture solution, followed by incubation in a dark environment at room temperature for 15min. After centrifugation at 1,000rpm for 5min, the supernatant was removed. Then, 10μL of PE Foxp3Ab were added to the sample tube and 10μL of isotype control were added to the control tube, followed by incubation at room temperature in a dark environment for 30min. Cells were re-suspended in 1mL of PBS and centrifuged at 1,000rpm for 5min. The supernatant was removed and the cells were re-suspended in 0.4mL of PBS. FCM was performed and CellQuest software was employed for data analysis.

The detection of serum contents of IL-17 and TGF- β1 was performed according to the manufacturer’s instructions. The lower limit of detection was 0.5pg/mL for IL-17 and 9pg/mL for TGF- β1.

Statistical analysis was performed with SPSS version 16.0. Data with normal distribution were expressed as mean±standard deviation. Student’s t-test for independent variables was performed for comparisons between two groups. A value of p<0.05 was considered statistically significant.

FCM demonstrated that the proportion of Th17 cells in the AR group was markedly higher than that of the control group (p<0.01; Table 1).

In the AR group, the proportion of Treg cells was markedly reduced when compared to the control group (p<0.01; Table 1).

ELISA was performed to detect the serum contents of IL-17 and TGF- β1 in the AR group and control group. Data are shown in Table 2. Results demonstrated that the IL-17 level in the AR group was markedly higher than that of the control group (p<0.01). In the AR group, the TGF- β1 level was dramatically reduced when compared with the control group (p<0.01).