Antibodies and Reagents
Biotinylated goat anti-human and anti-mouse AR antibodies, and recombinant human (rh) IL-3 were obtained from R&D Systems (Minneapolis, MN). Antibodies specific for human CD3ε (OKT3), IL-3 (BVD8-3G11), CD69 (FN50, Pacific Blue), CD123 (6H6, PE-Cy7), and CD203c (NP4D6, PE) were purchased from BioLegend (San Diego, CA). Antibodies specific for human CD28 (CD28.2), CD4 (RPA-T4, APC-AF750 or PE-Cy7), CD19 (HIB19, PE-Cy5), and mouse Ly-6G (Gr-1, RB6-8C5, AlexaFluor700), IL-4 (BVD6-24G2, PE-Cy7), and FcεRIα (MAR-1, PE) were obtained from eBioscience (San Diego, CA). Antibodies specific for human CD8α (3B5, PE-Texas Red), CD14 (TüK4, PE-Cy5), mouse CD4 (RM4-5, AF405), and mouse CD19 (6D5, PE-Texas Red) were obtained from CALTAG (Carlsbad, CA). APC-conjugated anti-human CD303 was a generous gift from Dr. Ernest Wang. Polyclonal goat anti-human IgE (ε-chain specific) was obtained from Sigma (Saint Louis, MO). 7AAD was obtained from Calbiochem (Gibbstown, NJ). The basophil isolation kit II was obtained from Miltenyi Biotec (Auburn, CA).
Human PBMC activation and cell surface staining
Heparinized blood was obtained from healthy donors under a protocol approved by the University of Rochester Medical Center Research Subjects Review Board. PBMC were isolated by Ficoll-Hypaque (Cellgro, Herndon, VA) density gradient centrifugation. Cells were suspended in RPMI-1640 medium containing 100U penicillin/streptomycin (Invitrogen, Carlsbad, CA) supplemented with 8% heat-inactivated fetal calf serum (FCS, HyClone, Logan, UT). 106 PBMC per well were stimulated with medium alone or 5 μg/ml anti-CD3ε + 1μg/ml anti-CD28 in round-bottom 96-well plate (Costar, Corning Inc., Corning, NY) for 6 hours at 37°C. After stimulation, the cells were stained for cell surface markers AR, CD4, CD8, CD14, CD19, CD69, CD123, CD203c, CD303 and live/dead 7AAD staining, then with APC-conjugated streptavidin (BD Bioscience) at 4°C for 15min, and fixed with 2% paraformaldehyde. Samples were analyzed using an LSR II flow cytometer (BD Bioscience), and FlowJo software (Tree Star Inc., Ashland, OR).
Human basophils preparation and activation
Peripheral blood basophils were purified by cell sorting or enriched by negative selection. For studying the direct induction of AR in human basophils by IL-3, basophils were sorted from fresh PBMC by surface markers CD4−CD8−CD14−CD19−7AAD−CD123+ on a FACSAria (BD Bioscience, San Jose, CA). Purified basophils were treated with medium alone; 5μg/ml anti-CD3ε + 1μg/ml anti-CD28 (as a negative control); and 5ng/ml rhIL-3 for 16 hours. Cells were stained with 7AAD and antibodies against AR, CD123, CD203c, and CD69.
For the remaining experiments, basophils were enriched from peripheral blood by depletion of magnetically labeled cells according to the manufacturer’s instructions (Basophil isolation kit II, Miltenyi Biotec, Auburn, CA). The purity of live basophils was about 20~80%. Enriched basophils were suspended in IMDM medium supplemented with 5% FCS, 1x MEM non-essential amino acids (Invitrogen), ~105 cells/well in 96-well plates, and treated for 0.5, 1, 4 or 16 hours with medium alone, 10ng/ml rhIL-3 or 10ng/mL anti-IgE. Supernatants (4 hrs) were collected to measure histamine release. Half of the cells were collected for extraction of total RNA, and the remaining cells were stained with 7AAD and antibodies against AR, CD123, CD203c, FcεRIα, and CD69 to measure surface AR expression.
Quantitative real time PCR for gene expression
Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. cDNA was prepared by reverse transcription from total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and Oligo(dT)12-18 (Invitrogen) as primer. Real-time PCR was performed using Applied Biosystems 7900HT Sequence Detection System. Primers and probes specific for AR, IL-3, IL-4, IL-13, HB-EGF and GAPDH were obtained from the TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). mRNA amounts were normalized to endogenous GAPDH gene expression. Samples were run in triplicate and data were analyzed using SDS software (Applied Biosystems).
Histamine release assay
Histamine in enriched basophil supernatants was measured in duplicate by the Histamine enzyme immunoassay kit (Cayman, Ann Arbor, MI) according to the manufacturer’s protocol.
Measurement of AR release
Enriched basophils were treated with medium alone, 10ng/mL rhIL-3 or 10ng/mL anti-IgE, with or without 5μg/mL anti-IL3Rα (R&D) or 50μM TAPI-1, an ADAM17 protease inhibitor 18
(Calbiochem). After 24 hours, supernatant AR was measured using the human Amphiregulin DuoSet ELISA Development kit (R&D, detection limit 7.8 pg/mL). This kit may underestimate the absolute levels of AR, because the ELISA standard and the immunogen for the polyclonal antibody reagent were unglycosylated bacterial recombinant human AR, whereas natural AR is heavily glycosylated. However, studies on AR produced by eosinophils and mast cells used the same ELISA kit 12, 13
, so our results can be directly compared with these other studies.
Intracellular staining of mouse cells
AR deficient (AR−/−) mice 8
, originally obtained from David Lee were backcrossed to AR C57BL/6.PL (B6/PL) mice for ten generations and then maintained by homozygous breeding 9
. C57BL/6.PL mice were bred in our facility.
Peripheral blood from mice was obtained by cardiac puncture. Red cells were lysed by ACK lysis buffer (0.8% NH4Cl, 10mM KHCO2 and 0.1mM EDTA), and the remaining cells were stimulated with medium alone, 20ng/ml recombinant mouse IL-3 (R&D Systems Minneapolis, MN) or 20ng/mL rat anti-mouse IgE (R35-92, BD Bioscience) in a 96-well plate at 106 cells/well. After 16 hours stimulation (with 2μM monensin present for the last 6 hours), the cells were stained with antibodies specific for CD4, CD19, Gr-1, FcεRIα and 7AAD, then fixed and permeabilized using Fix-Perm (BD Bioscience), and stained with anti-AR and anti-IL-4 intracellularly. Cells were analyzed using an LSR II flow cytometer, and FlowJo software.