As expected, phagocytic signaling selleck required both Syk and phosphoinositol-3-kinase (PI3K). However, while PI3K is required for serotonin secretion, inhibition of Syk does not reduce secretion. Differences exist in requirements for phagocytic and endocytic signaling [9, 20]. Our observations suggest that, similarly, alternate FcγRIIA signaling pathways exist for phagocytosis and serotonin secretion. Cells and reagents. RBL-2H3 cells were maintained in minimum essential medium containing
15% foetal calf serum (FCS; HyClone, UT, USA) and 1% streptomycin (Gibco BRL, Grand Island, NY, USA). Cells were grown in T-75 tissue culture flasks (Corning; Corning, NY, USA) at 37 °C under 5% CO2. Radiolabeled serotonin (5-HT) was purchased from Perkin Elmer (NET-498) and used within 6 months of Gefitinib in vivo purchase. Anti-FcγRIIA (IV.3) monoclonal
antibody (mAb) was purified from hybridoma supernatants and digested into Fab fragments. Goat anti-mouse F(ab’)2 was purchased from Jackson Immunoresearch (West Grove, PA, USA). The calcium ionophore A23187 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Transfection and selection. RBL-2H3 cells were stably transfected with WT FcγRIIA or mutants of FcγRIIA. Mutations were generated from the WT FcγRIIA ITAM-like sequence Y1QTANGGY2MTLNPRAPTDDDLNIY3LTL. Single and double mutations of tyrosine (Y) to phenylalanine (F) in the FcγRIIA cytoplasmic domain were generated by polymerase chain reaction. The FcγRIIA mutants are designated Y1F, Y2F, Y3F, Y1Y2F, Y1Y3F, Y2Y3F. The cDNA sequences encoding FcγRIIA wild-type or tyrosine mutants were cloned into pcDNA3.1 and transfected into RBL-2H3 cells using Fugene-6 (Roche Applied Science, Indianapolis, IN, USA) per the manufacturer’s instructions and
selected using G-418 (Mediatech, Manassas, VA, USA). Transfected cells were sorted for expression of FcγRIIA using fluorescence-activated cell sorting analysis with anti-FcγRII monoclonal antibody (IV.3) and fluorescein isothiocyanate (FITC)-conjugated Urease goat anti-mouse secondary antibody [FACS Diva (B-D Biosciences); Fig. 1]. These multi-clonal populations of each transfectant were assayed for serotonin secretion. Subsequently, single cell clones were generated by limited dilution. Single cell clones were then re-tested by flow cytometry for FcγRIIA expression, and serotonin secretion experiments were conducted on these single cell clones as described below. Serotonin release assay. One day before assay, 2 × 104 RBL-2H3 cells from single cell clones were plated in triplicate in 96-well plates. Before receptor crosslinking, cells were preloaded with 2 μCi/ml 3H-serotonin at 37 °C for 1 h. Cells were washed, incubated with fresh medium for 1 h and washed again. Washed cells were incubated on ice for 30 min in medium containing F(ab)’2 mAb IV.3 and then incubated an additional 30 min after addition of the secondary goat-anti-mouse antibody GAM F(ab)’2 (IV.3 + GAM).