Coupling of FcγRI to downstream intracellular signaling pathways requires PLD1 and not PLD2.

(A) Intracellular cytosolic calcium changes following aggregation of FcγRI. Responses were compared in control cells and cells pretreated with antisense oligonucleotides (10 μM) to either PLD1 or PLD2. Traces shown are as follows: left, XL FcγRI control cells = FcγRI aggregation in IFN-γ–primed control cells; upper right panel, FcγRI aggregation in IFN-γ–primed cells pretreated with antisense to PLD1 (XL FcγRI a.s.PLD1); lower right panel, FcγRI aggregation in IFN-γ–primed cells pretreated with antisense to PLD2 (XL FcγRI a.s.PLD2). The arrow marks the addition of the goat antihuman IgG antibody to create cell surface immune complexes. Traces are typical from fura-2–loaded cells from 3 separate experiments. (B) FcγRI coupling to sphingosine kinase. Following aggregation of FcγRI in IFN-γ–primed U937 cells, cells were harvested at given time points to measure sphingosine kinase activity. Sphingosine kinase activity was assayed from basal control cells (basal control); following FcγRI aggregation in control cells (XL control) and in cells pretreated with antisense oligonucleotides (10 μM) for either PLD1 (XL a.s.PLD1) or PLD2 (XL a.s.PLD2). Lysates from these cells were treated with phosphatidic acid (L-α-phosphatidic acid (1,2-diacyl-sn-glycero-3-phosphate) in vitro to ensure sphingosine kinase activity (P.A.a.s.PLD1 and P.A.a.s.PLD2).33 Results are the mean ± SD for triplicate measurements and are representative of the results from at least 3 separate experiments.