6-8 week old weight- and sex-matched 129SvEvBrd mice (Lexicon Genetics, The Woodlands, Texas, USA ) and BALB/c (originally provided by The Jackson Laboratory, Bar Harbor, Maine, USA) mice were maintained in a barrier facility, and animals were handled under IACUC-approved protocols from Cincinnati Children's Hospital Medical Center.
Passive anaphylaxis model
IgE-mediated anaphylaxis was induced by intravenous (i.v.) injection of rat IgG2a anti-mouse IgE mAb (rat IgG2a, clone: EM-95) (10 μg/200 μl saline) and IgG mediated anaphylaxis was induced by intravenous injection of rat IgG2b anti-mouse FcγRII/III mAb (rat IgG2b, clone: 2.4G2) (500 μg/200 μl saline) as we have previously described . Rectal temperature was monitored for 60 minutes with a rectal probe (Physitemp Model BAT-12) as previously described . Concentration of anti-mouse IgE and anti- mouse FcγRII/III mAb used were determined in preliminary dose-response experiments and also as we have previously demonstrated [19, 20].
Experimental oral antigen-induced active anaphylaxis
6-8 week old mice were sensitized subcutaneously with 50 μg of ovalbumin [OVA] (Sigma-Aldrich, St. Louis, MO) in the presence of 2 mg of aluminum potassium sulfate adjuvant [alum: AIK(SO4)2-12H2O] (Sigma-Aldrich, St.Louis, USA) in sterile saline. Two weeks later, mice were deprived of food for 4 hours and received intragastric (i.g) challenge of ovalbumin [OVA] (50 mg/250 μl saline) employing i.g. feeding needles (Fisher Scientific Co., Pittsburgh, Pennsylvania, USA). Rectal temperatures and diarrhea were monitored at 30 and 60 minutes as we have previously described .
Effect of anti-histamine treatment
To assess the effect of anti-histamine treatment on oral OVA-induced active anaphylaxis, 30 minutes prior to the OVA challenge, mice were i.p injected with H1 and H2 receptor antagonists 0.2 mg of Triprolidine and 0.2 mg of Cimetidine in 200 μl saline and subsequently received OVA via oral gavage, after which rectal temperature was monitored at 30 minutes and 60 minutes time points as we have previously described .
Histamine biphosphate monohydrate (Sigma-Aldrich, St.Louis, USA) (25 μg/ml saline per mouse) was i.v injected and body temperature was monitored by rectal thermometry every 10 minutes for 60 minutes as we have previously described .
Mast cell quantification
Ear, tongue and jejunum (7 - 10 cm distal to the stomach) were collected and fixed in 10% formalin and processed by standard histological techniques. Longitudinal sections (5 μm) were stained for mast cells with chloroacetate esterase (CAE) activity, as described previously . At least four random sections per mouse were analyzed. Quantification of stained cells was performed by counting the number of chloroacetate-positive cells in 5 fields for tongue, 10 fields for ear and 20 fields for intestine (magnification 400 X).
Bone marrow-derived cells (BMMCs) isolated from 6-8 week old mice were maintained in complete medium consisting of RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, USA), glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), pyruvate (1 mM), nonessential amino acids, hepes (10 mM), and 2-ME (50 μM) . BMMCs were cultured in the presence of IL-3 (20 ng/ml) (Peprotech, Inc. Rocky Hill, USA) for the first three weeks, and with both IL-3 (20 ng/ml) and SCF (10 ng/ml) (Peprotech, Inc. Rocky Hill, USA) during week 4. After four weeks of culture, BMMCs were examined for FcεRI, and c-Kit expressions by flow cytometry analyses as we have previously demonstrated [18, 21] and were found to be double positive (95-97%) for FcεRI and c-Kit at this time point. 4-8 weeks old BMMCs were used for all experiments.
Peritoneal mast cell analyses
Mice were anesthetized and a hypodermic needle were inserted into the abdominal cavity (18 gauge). Sterile saline (5 ml) was injected through a needle placed near the sternum and the abdominal cavity was gently massaged for 1 minute and the fluid removed. The peritoneal lavage was centrifuged at 1200 rpm for 5 minutes at room temperature. The supernatant was poured off and the cell pellet resuspended in FACS buffer (PBS/1% FCS). The single-cell suspensions were washed with FACS buffer (PBS/1% FCS) and incubated with combinations of the following Abs: PE anti-mouse-FcεRI (clone R35-72; 0.5 μg/106 cells; BD Pharmingen; San Jose, CA), FITC anti-mouse FcγRII/III (clone 2.4G2; 0.2 μg/106 cells; BD Pharmingen; San Jose, CA), APC-Alexafluor-750 anti-mouse CD117 (c-kit) (0.2 μg/106 Ebioscience; San Diego, CA). The following Abs were used as appropriate isotype controls: FITC rat IgG2a and APC-AlexaFluor 750 rat IgG2a (BD Pharmingen; San Jose, CA). Cells were analyzed on FACSCalibur (BD Immunocytometry Systems; San Jose, CA), and analysis was performed using Flow Jo software (Tree Star; Ashland, OR).
BMMC degranulation was measured by hexosaminidase release as described previously . BMMCs (5 × 105 cells each) were sensitized with IgE anti-TNP mAb (10 μg/ml) (BD Biosciences, San Jose, CA) for 2 hours. Sensitized BMMCs in 100 μl suspension were challenged with BSA-TNP (10, 100 or 1000 ng/ml) (Biosource Technologies, USA) in different concentrations for 15 minutes. Supernatant was examined for hexosaminidase activity by colormetric determination using 4-Nitrophenyl N-acetyl-β-D-glucosaminide, PNAD (Sigma, St.Louis, USA) at 405 nm. Degranulation is expressed as the percentage of total hexosaminidase activity. The BMMC lysate was used to estimate total hexosaminidase activity. In some experiments BMMCs (5 × 106/ml) were sensitized with IgE anti-TNP mAb (10 μg/ml) for an hour, and challenged with BSA-TNP (100 ng/ml) for 3 hours. Supernatant was analyzed for IL-6, IL-13 and TNF-α by ELISA as described by manufacturer (R&D systems, Minneapolis, USA).
BMMCs (2 × 106) were stained with CFSE (5 μM) (Invitrogen, Carlsbad, USA) for 10 min in PBS with 0.1% BSA and cultured in the presence of IL-3 (20 ng/ml) and SCF (10 ng/ml). The cells were examined by flow cytometry for CFSE mean fluorescence intensity on the 4th and 8th day. CFSE positive cells were analyzed with a proliferation software tool (Flowjo, Ashland, USA) .
Apoptosis was determined using annexin V-allophycocyanin and 7-aminoactinomycin D (7AAD), as we have previously described . Early apoptosis was defined as annexin V-positive, 7AAD negative. Briefly, BMMCs (2 × 106) were washed and resuspended in annexin V-binding buffer and stained with annexin V-allophycocyanin and 7AAD. Samples were then incubated at room temperature for 15 minutes and analyzed by flow cytoemetry. Active Caspase 3 expression, a biomarker for cells in apoptosis, was examined by flow cytometry using PE-Rabbit Anti-Active Caspase 3 antibody (BD Pharmingen, San Diego, USA .
ELISA measurements for OVA-specific IgG1 and IgE were done as previously reported . Total IgE was estimated as described by the manufacturer using a mouse IgE Elisa Kit BD-Opt EIA™ (BD Biosciences, New Jersey, USA). Total IgG1 and IgG2a concentrations (1/10000) were compared between BALB/c and 129S5 mice. Serum IgG was captured with goat anti-mouse IgG (1:1000 Dilution) (Southern Biotech, Birmingham, USA). After capture, serum IgG1 and IgG2a were detected by using different secondary antibodies; namely goat anti-mouse IgG1-HRP (Southern Biotech, Birmingham, USA) and goat anti-mouse IgG2a-HRP (Southern Biotech, Birmingham, USA), respectively.
Plasma Histamine determinations
Plasma was collected 5 min after anti-IgE-induced passive anaphylaxis (10 μg/200 μl saline i.v). Plasma was collected in EDTA-coated tubes and plasma histamine levels were determined with an histamine immunoassay kit (Immunotech, Marseille Cedex, France) as described by the manufacturer.
Data are expressed as mean ± standard error of the mean (SEM), unless otherwise stated. Statistical significance comparing different sets of mice was determined by Student's t-test. In experiments comparing multiple experimental groups, statistical differences between groups were analyzed using the one-way ANOVA parametric and a Tukey's multiple comparison post-test. In experiments comparing two experimental groups, statistical differences between groups were determined using a Students T-test. P < 0.05 was considered significant. All analyses were performed using Prism 5.0 software.