The following reagents were purchased from Biolegend (San Diego, CA, USA): human red blood cell lysis buffer, Brilliant Violet 510TM (BV510)-conjugated donkey anti-rabbit IgG polyclonal antibody, PE/Cy7-conjugated mouse anti-human CD14 monoclonal antibody, BV510-conjugated rat anti-mouse Ly-6G/Ly-6C (Gr-1) monoclonal antibody, BV510-conjugated rat anti-mouse F4/80 monoclonal antibody, PerCP-conjugated rat anti-mouse CD11b monoclonal antibody, human Fc receptor blocking solution, rat anti-mouse CD16/32 antibody, Brefeldin A, Zombie NIRTM Fixable Viability Kit, and Zombie GreenTM Fixable Viability Kit. Mouse IL-18BPd DuoSet ELISA kit, APC-conjugated mouse anti-human IL-18Rα monoclonal antibody, PE-conjugated mouse anti-human IL-18 monoclonal antibody, APC-conjugated rat anti-mouse IL-18Rα monoclonal antibody and their respective isotype controls (catalog #: IC002A, IC002P, IC005A) were supplied by R&D Systems (Minneapolis, MN, USA). Rabbit anti-human IL-18BPa and rabbit IgG isotype control were obtained from Novus Biologicals (Minneapolis, MN, USA). IFNγ was supplied by PeproTech (Rocky Hill, NJ, USA). Trypan blue dye and Ovalbumin (OVA, Grade V) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human IL-18BPa and IL-18 ELISA kits were from ImmunoWay Biotechnology Company (Plano, TX, USA) and ExCell Bio (Shanghai, China), respectively. Cytofix/CytopermTM Fixation/Permeabilization Solution Kit was bought from BD Biosciences (Beldford, MA, USA). Fetal bovine serum (FBS, Hyclone), penicillin-streptomycin antibiotic mixture and RPMI 1640 medium were obtained from Gibco BRL (Grand Island, NY, USA). Alhydrogel® adjuvant was bought from InvivoGen (San Diego, CA, USA). RBC Lysis Buffer (Multi-species), Mouse IL-18 ELISA kit and TRIzol reagent were purchased from Invitrogen (Carlsbad, CA, USA). anti-human CD14 MicroBeads and autoMACS Running Buffer–MACS Separation Buffer, and Lymphoprep™ were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany) and AXIS-SHIELD PoC AS (N-0504 Oslo, Norway), respectively. Dermatophagoides pteronyssinus allergen extract (DPAE) was bought from Greer Laboratories, Inc. (Lenoir, NC, USA). Artemisia sieversiana wild allergen extract (ASWAE) and Platanus pollen allergen extract (PPAE) were purchased from Macro Union Pharmaceutical Co. Ltd (Beijing, China). Allergens for skin prick tests were supplied by ALK-Abelló, Inc. (Denmark). Most of the general-purpose chemicals such as salts and buffer components were of analytical grade.
Subjects and animals
A total of 33 patients with perennial allergic rhinitis (pAR), 9 patients with sAR and 25 healthy control (HC) subjects were recruited in the study. Their general characteristics were summarized in Additional file 1 (Table S1). The diagnosing criteria of pAR and sAR were conformed to the Chinese Society of Allergy Guidelines for Diagnosis and Treatment of Allergic Rhinitis . The informed consent from each volunteer according to the declaration of Helsinki and agreement with the ethical committee of the First Affiliated Hospital of Jinzhou Medical University and with the General Hospital of Shenyang Military Region of PLA was obtained.
Immediately after confirmed diagnosis (acute stage), peripheral blood from each patient with allergic rhinitis was collected. Blood from HC were collected in the outpatient clinic. From each donor, 5 mL was taken into an EDTA containing tube before centrifugation at 450×g for 10 min. The cells were used for flow cytometric analysis, and plasma was collected and frozen at -80°C for analysis of cytokines. For CD14+ monocytes isolation study, 180 mL of peripheral blood was taken from each donor.
Five-week-old female BALB/c mice were obtained and maintained as described previously . The animal experiment procedures were authorized by the Animal Care Committee at Jinzhou Medical University.
Isolation of CD14+ monocytes and allergen challenge test
CD14+ cells were enriched by density gradient centrifugation and positive selection by using magnetic beads on magnetic cell sorting (MACS) according to the manufacturer’s instructions. Final recovery of cells was determined with an improved Neubauer haemocytometer after being stained with trypan blue solution, and recovered cell purities were assessed by flow cytometry with an anti-human CD14 antibody.
To further investigate the direct action of allergen on the expression of IL-18, IL-18BP and IL-18R in monocytes, the isolated primary monocytes at a density of 1×106 per mL were cultured in RPMI 1640 medium containing 3% FBS and 100 U/ml penicillin/streptomycin in a 12-well cell culture plate (Nest, Wuxi, China) in the presence or absence of ASWAE, PPAE, DPAE (all at a concentration of 1.0 μg/mL) or IFNγ (as positive control) at 5 ng/mL for 10, 30 and 60 min, respectively at 37°C in a 5% (v/v) CO2, water-saturated atmosphere. Brefeldin A at 2 μg/mL was added in wells for detecting the intracellular expression of IL-18 and IL-18BP before stimulation. Cells were then harvested and centrifuged at 450×g for 10 min at 4°C. Cell pellets containing approximately 0.5×106 and 1×106 cells were resuspended in PBS for flow cytometric analysis, and in TRIzol reagent for RT-PCR, respectively. Cell culture supernatant was collected and frozen at -80°C for further use.
Flow cytometric analysis of IL-18, IL-18BP and IL-18R in human peripheral blood monocytes and isolated CD14+ monocytes
The procedures for detecting IL-18, IL-18BP and IL-18R expression in human peripheral blood monocytes were mainly adopted from a previous study by Zhang et al . Briefly, whole blood cells were challenged with or without ASWAE, PPAE, or DPAE (all at a concentration of 1.0 μg/mL) for 1 h, and isolated CD14+ monocytes were challenged for 10, 30 and 60 min at 37°C.
For cell surface molecules, whole blood cells were incubated with human Fc receptor blocking solution and a live/dead cell dye (Zombie GreenTM Fixable Viability Kit)  for 15 min, then stained with PE/Cy7 conjugated anti-human CD14 and APC conjugated anti-human IL-18Rα antibodies. Following red blood cell lysis, cells were analyzed with FACS Verse flow cytometer (BD Biosciences, San Jose, CA, USA). An irrelevant isotype- and concentration-matched antibody of anti-human IL-18Rα was used for fluorescence minus one (FMO) control. Dead cells and doublets were discriminated by SSC-A–live/dead cell dye and FSC-H–FSC-A gating strategies. As for magnetic isolated monocytes, cells were processed as above.
For intracellular molecules, whole blood cells were incubated with human Fc receptor blocking solution and a live/dead cell dye, and then stained with PE/Cy7 conjugated anti-human CD14 as described above. After lysing red blood cells, resuspended leukocytes were fixed and permeabilized, and stained with PE conjugated anti-human IL-18 antibody, and anti-human IL-18BP primary antibody followed by the addition of BV510-conjugated donkey anti-rabbit polyclonal antibody. Finally, cells were processed and analyzed as above. As for isolated primary monocytes, cells were processed as above.
Establishment of mouse allergic rhinitis model
OVA-induced allergic rhinitis mouse model was mainly adopted from a previous study by Mo JH et al . Briefly, mice were sensitized on days 0, 7 and 14 by intraperitoneal injection of 25 μg OVA emulsified in 1 mg of alhydrogel. On days 21–27 mice were challenged by intranasal instillation with 500 μg of OVA dissolved in PBS (10 µL/nostril) once daily. For control experiments, healthy mice received vehicle only instead of OVA solution. At 24 h following the last OVA challenge, blood and nasal lavage fluid (NLF) were collected from each mouse. Total cells were determined and collected after centrifugation as described above. The cells were used for flow cytometric analysis, plasma and NLF supernatant was collected and frozen at -80°C until use.
To evaluate allergic symptoms, numbers of sneezing and nasal-rubbing motions during the first 15 min after each OVA challenge were recorded and compared with healthy control mice (HM) by observers blinded to the study. As presented in Additional file 2 (Fig. S1a, b), the numbers of nasal rubbing and sneezing motion in AR mice were substantially higher than that in HM during a 7-day observation period.
Flow cytometric analysis of IL-18R in mouse blood monocytes and NLF macrophages
To detect IL-18R expression in mouse blood monocytes, whole blood cells were incubated with anti-mouse CD16/32 antibodies, and a live/dead cell dye (Zombie NIRTM Fixable Viability Kit)  for 15 min. Each labeled monoclonal antibody including BV510-conjugated anti-mouse Gr-1, PerCP-conjugated anti-mouse CD11b and APC-conjugated anti-mouse IL-18R was added into tubes for 15 min before red blood cells being lysed. Finally, cells were processed as for human blood samples and analyzed by using flow cytometer.
To detect IL-18R expression in NLF macrophages, cells were incubated with anti-mouse CD16/32 antibodies and a live/dead cell dye, followed by incubation with BV510-conjugated anti-mouse F4/80 and APC conjugated anti-mouse IL-18R antibodies, and analyzed as above.
Real-time PCR for IL-18, IL-18BP and IL-18R in isolated CD14+ monocytes
Total RNA was extracted from magnetically sorted blood monocytes as described previously . Briefly, after synthesizing cDNA from total RNA by using RT Master Mix Perfect Real Time, qPCR was performed with SYBR Premix Ex TaqII Kit on the Real-time Thermal Cycler (Thermo Fisher Scientific Oy, Vantaa, Finland). Each reaction contains 12.5 μL of 2×SYBR green Master Mix, 300 nM oligonucleotide primers, and 10 μL of the cDNA. Untreated controls were chosen as the reference samples, and the ΔCt for all experimental samples were subtracted by the ΔCt for the control samples (ΔΔCt). The magnitude change of test gene mRNA was expressed as 2−ΔΔCt. Each measurement of a sample was conducted in duplicate. The forward and reverse primers for human IL-18, IL-18BP and IL-18R were listed in Additional file 3 (Table S2).
Measurement of cytokine levels in plasma and NLF supernatant, and calculation of molar concentration ratio of plasma IL-18BP/IL-18
Levels of total IL-18 (tIL-18) and IL-18BP (tIL-18BP) in plasma or NLF supernatant were determined by using ELISA kit according to the manufacturer’s instructions. Molar concentration of human IL-18 and IL-18BP was calculated using the equation of mass concentration divided by molecular weight, and free IL-18 (fIL-18) and IL-18BP (fIL-18BP) was calculated on the basis of a 1:1 stoichiometry in the complex of IL-18 and IL-18BP with a dissociation constant of 400 pM .
Human bio-plex panel (Bio-Rad Laboratories, California, USA) was employed to detect human plasma levels of IL-1β and TNF-α. The detection ranges for IL-1β and TNF-α were 0.24–3994 pg/mL and 0.57–9270 pg/mL, respectively.
Statistical analyses were performed by using SPSS software (version 21.0, IBM Corporation). Data are displayed as a boxplot, which indicates the median, interquartile range, the largest and smallest values for the number of experiments indicated. Where Kruskal–Wallis analysis indicated significant differences between groups, a pairwise test was used for multiple comparisons between the groups. Where ANOVA indicated significant differences between groups with ANOVA, a Bonferroni method was applied for further comparison. Correlations were determined by using Pearson’s correlation or Spearman rank correlation analysis. For all analyses, P < 0.05 was considered statistically significant.