Chemicals and cell lines
The RBL-2H3 cell and the human basophilic cell line KU812 were purchased from the Type Cell Culture Collection of the Chinese Academy of Science (Wuhan, China). DB, OVA and monoclonal anti-dinitrophenyl IgE antibody (anti-DNP IgE) and 4-nitrophenyl-N-acetyl-β-D-glucosaminide were obtained from Sigma-Aldrich (St. Louis, MOUSA). Dinitrophenyl–human serum albumin (DNP–HSA) was obtained from Biosearch Technologies, Inc. (Novato, CA,USA). The antibody against FcεRIα was purchased from Abcam (Cambridge, England, UK) and Affinity Biosciences (OH.USA), respectively. Cell culture medium (RPMI 1640 medium, 1640) was from Thermo Fisher Scientific (Waltham, Mass) and cell counting kit-8 (CCK-8) was provided by ApeXBio (Houston, USA). The vendors for the other reagents are included with the relevant assays.
Mice
Female BALB/c mice were supplied by the Department of Animal Science, Nanchang University (Nanchang, China; permission number “SYXK (Gan) 2017-00021). In each animal experiment, mice (6 to 7 weeks old and 20.0 ± 2.0g body weight) were randomly divided into different groups (n = 8/group). All mice used in this study were cared for according to the approved guidelines of the Care and Use of Laboratory Animals guidelines (NIH Publication No. 86 − 23, Revised 1996) and all experimental procedures were approved by the Animal Care Review Committee, Nanchang University (NCU2018-0227).
Human sera preparation and ethics statement
Human sera were prepared as previously described [9]. The sera were pooled from 10 milk allergy patients (Table 1). Bovine specific OVA-IgE and total IgE levels were determined (specific IgE level > 1 IU / mL, total IgE level > 100 IU / mL) with anImmunoCAP 100E instrument (Phadia AB, Uppsala, Sweden). Sera from non-allergic individuals (n = 4) were also pooled and used as negative controls. Human sera were collected under the approval of the Nanchang University First Affiliated Hospital Ethic Committee of (certificate no. 14/2010).
Table 1
Clinical data of patients with milk allergies
Patient No. | Sex | Age (years) | Clinical symptoms | Total IgE Level (IU/mL) | Specific IgE Level (IU/mL) |
1 | Female | 4 | Urticaria | > 100 | 6.5 |
2 | Male | 10 | NKa | > 100 | 9.8 |
3 | Male | 5 | Chronic Rhinitis | > 100 | 2.8 |
4 | Female | 12 | NKa | > 100 | 7.5 |
5 | Female | 3 | Asthma, Bronchitis | > 100 | 4.2 |
6 | Female | 2 | Bronchial Asthma | > 100 | 6.9 |
7 | Female | 4 | NKa | > 100 | 11.7 |
8 | Male | 4 | Urticaria | > 100 | 4.5 |
9 | Male | 18 | Chronic Rhinitis | > 100 | 6.4 |
10 | Female | 1 | Bronchial Asthma | > 100 | 7.3 |
aNK, Not Known |
Treatment of mast cell lines
RBL-2H3 and KU812 cells were grown at 37°C in RPMI-1640 supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (Invitrogen). RBL-2H3 cells were sensitized overnight with 500 ng/mL anti-DNP IgE while KU812 cells were sensitized with 1:5 dilutions of patient serum. The following day, the cells were incubated with various concentrations of DB for 4 hours before challenging the cells for 30 minutes with 50 mg/mL DNP–HAS or 50 mg/mL OVA, for RBL-2H3 and KU812 cells, respectively. A control group received no DB treatment while the naive group was only treated with vehicle substances.
Cell viability and cytotoxicity assays
CCK-8 assays were performed according to manufacturer’s instructions. Briefly, cells were cultured in 96-well plates before 24 h treatment with varying concentrations of DB. Then, cell supernatants were removed, followed by incubation with CCK-8 solution for 4 hours before measuring absorbance intensities at 450 nm in a microplate reader (Thermo Fisher Scientific).
Measurement of degranulation
Degranulation of mast cells was determined by measuring the release of the granule markers histamine and β-hex. Released histamine was measured by an enzyme immunoassay, according to manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI, USA). The β-hex activity assays were as previously described [10]. Briefly, cells were stimulated with DNP-HAS or OVA in Tyrode’s buffer for 30 minutes at 37°C. The supernatants were collected, and the unreleased β-hex was quantified by adding 0.5% Triton X-100 solution to the cells. Samples (50 µL) and the substrate solution (50-µL p-nitrophenyl-N-acetyl-β-D-glucosamide) were added to 96-well microtiter plates, respectively before incubation at 37°C for 60 minutes, followed by addition of 200-µL stop solution (0.2-mol/L glycine, pH 10) to each well. After measuring absorbance values at 405 nm, the amount of β-hex or histamine release into media was expressed as the percentage of the total amount of β-hex or histamine originally in the cells [% release = 100× (experimental β-hex or histamine release-spontaneous β-hex or histamine release) ÷ total cellular β-hex or histamine].
Measurement of intracellular Ca2+ levels
A fluorescence-based assay was used to analyze intracellular Ca2+ levels as previously described [10]. After DB treatment, about 1 × 106 cells were pulsed with 5 µM Fluo-3/AM containing 0.05% PluronicF127 for 30 min before addition of DNP–HAS or OVA. Fluorescent intensity measurements were recorded at 488 nm excitation and 526 nm emission wavelengths using a Varioskan Flash microplate reader (ThermoFisher Scientific, Waltham, MA, USA).
Immunofluorescence microscopy and image analysis
RBL-2H3 cells cultured on coverslips while KU812 cells were cytocentrifuged onto silane-coated glass slides, followed by processing for immunofluorescence microscopy, with minor modifications of previous methods [11]. Cells were incubated with the FcεRIα antibody at 4°C overnight using the manufacturer’s recommended dilution before detection with fluorophore-labelled (Alexa Fluor-488 and 594) secondary antibodies (Invitrogen, OR). DAPI (4′, 6-Diamidino-2-phenylindoledihydrochloride; Invitrogen) was used to counterstain nuclei. Digital images were captured with Nikon Eclipse Ci-L light microscope (Tokyo, Japan) and Nikon Digital Sight DS-Fi2 (Tokyo, Japan) and processed and analyzed using Image-Pro Plus 6.0.
Animal protocol
Mice were tested for their susceptibility to OVA-induced allergy using a previously published protocol [12]. Briefly, mice were twice sensitized, 2 weeks apart, by intraperitoneal injection of 50 µg of OVA in the presence of 1 mg of aluminum potassium sulfate adjuvant [alum: AIK(SO4)2-12H2O] (A-7210; Sigma-Aldrich). Two weeks later, mice were subjected to intra-gastric gavage with 250 µl of sterile saline that contained up to 50 mg of OVA every other day from day 28. Mice were treated intragastrically with 5 mg/kg DB in 250µl sterile saline every other day from day 21 to day 42 (Fig. 1).
Clinical symptoms were recorded every 10 min after the last OVA challenge and were used to derive clinical scores using the anaphylactic symptom scoring system developed previously [13]. Briefly, absence of clinical symptoms was given a score of 0; repetitive facial scratching, a score of 1; reduced activity and facial puffiness, a score of 2; periods of motionlessness for 1 min, a score of 3; and no movement to gentle prodding, a score of 4.
Diarrhea was assessed by visually monitoring mice for up to 1 hour following intragastric challenge. Mice were placed in individual cages and monitored for diarrhea symptoms for one hour. The mice were scored according to feces appearance as follows: score 0 = no change; score 1 = well formed; score2 = soft, non-formed; score 3 = one episode of liquid diarrhea; score 4 = at least two episodes of liquiddiarrhea [14]. Finally, mice were euthanized and serum and small intestine were collected. The serum levels of β-hex and histamine were quantified to evaluate mast cell degranulation.
Morphological analysis
Goblet cell mucins stained with periodic acid-Shiff base, Alcian blue, pH 2.5 (PAS/AB) were used to estimate the amount of mucus accumulation as previously described [15]. After sacrifice, intestines were carefully dissected and fixed overnight in Carnoy’s solution to preserve mucus. The jejunum was paraffin embedded and processed for PAS/AB staining. From each animal, 3 sections were imaged by a digital camera using a 20X objective on a Nikon microscope.
Measurement of OVA-specific IgE
Allergic sensitization to OVA was evaluated by measuring the levels of OVA-specific IgE with slight modifications of the previous method [12]. Briefly, 96-well microtiter plates were coated overnight at 4°C with 10µg/ml purified rat anti-mouse IgE Ab (DB Pharmingen) in 50 nM carbonate-bicarbonate buffer (pH 9.6; Sigma-Aldrich).The coated plates were blocked with 1% BSA/PBS for 2 h at room temperature, washed with 0.05% Tween-20 in Tris-saline before subsequent addition of serum samples (four dilutions of 1/10, 1/50, 1/250, and 1/500) and further incubation for 2–3 h at room temperature. The plates were then washed and biotinylated OVA was added (1:1,000 dilution of 2 mg/ml, 25 µl/well), followed by 100µl horseradish peroxidase-conjugated goat anti-mouse IgE antibody (1:6000; SouthernBiotech, USA) and incubation for 1 h at 37°C. Finally, a tetramethylbenzidine substrate (0.1 mg/ml) solution was added before using 2 M H2SO4to stop the color reaction. The optical density was determined at 490nm with a microplate reader (Benchmark 96, Bio-Rad Laboratories, Hercules, CA, USA).
Isolation of intestinal mucosal mast cells
Lamina propria (LP) mononuclear cells were isolated from the mouse small intestine as previously described [16]. In brief, small intestines were cut longitudinally after removing Peyer patches. To isolate epithelial cells, the segments were incubated in 1× HBSS containing 5 mmol/L EDTA, 5% FCS, and 1 mmol/L dithiothreitol for 20 minutes at 37°C under continuous rotation. An LP dissociation kit and a gentle MACS Dissociator (MiltenyiBiotec, BergischGladbach, Germany) were used to digest the remaining tissue pieces, according to the manufacturer's instructions. A discontinuous Percoll density gradient centrifugation was performed and LP cells were collected from the layer between the 40% and 75% Percoll layers.
Western blot analysis
Crude protein extracts were prepared as previously described [17] and protein concentrations determined using the bicinchoninic acid method. The proteins were resolved by 15% SDS-PAGE and subsequently electro-transferred to nitrocellulose membranes before blocking with 2% bovine serum albumin (Sigma–Aldrich) blocking buffer for 1 h at room temperature. Afterwards, the membranes were probed with FcεRIα and β-actin antibodies (Servicebio Technology, Wuhan, China) overnight at 4°C. After washing three times with TBS-T, the membranes were incubated with HRP–conjugated anti-mouse IgG antibody for 1h at room temperature. Immunocomplexes were visualized by enhanced chemiluminescence detection (Bio-Rad, Hercules, USA), followed by densitometric analysis of protein bands using AlphaEase FC software.
RNA isolation and quantitative RT-PCR
Total RNA was prepared from intestinal mast cells using an RNA extraction kit (Trizol, Thermo, USA) according to the manufacturer's protocol. RNA was converted into first-strand cDNA using a real-time reverse transcription (RT-PCR) kit (Cwbiotech, Beijing, China). Quantitative PCR (qPCR) was performed in a Bio-radCFX96 Touch Real-Time PCR Detection System. The primers used were: GAPDH (NM_017008.4)5’-CTGGAGAAACCTGCCAAGTATG-3’ (sense); and 5’-GGTGGAAGAATGGGAGTTGCT-3’ (antisense); rat high affinity IgE receptor, α-subunit (M21622)5’-TGT GTA CTT GAA CGT GAT GCA A-3’(sense) and 5’-TGT CTA AGA CCA C GT CAG CAG-3’ (antisense). Relative mRNA amounts were calculated using GAPDH as the reference gene using the formula: 2−[Ct(Sample) − Ct(Gapdh)].
Statistical analyses
Statistical comparisons between groups were performed using a single factor ANOVA for analyzing differences among multiple groups. Concentrations of DB were independent variables and mast cell mediators the dependent variables. Differences at p < 0.05 were considered statistically significant. All experiments were performed at least three times.