Immunoglobulin G receptors (FcλRs) on myeloid cells are responsible for the internalisation and degradation of immune complexes. In interferon-λ primed U937 cells, FcλRI is coupled to a novel pathway that involves the sequential activation of PI3 kinases, phospholipase D (PLD) and sphingosine kinase. This study investigated the factors that couple FcλRI to PLD and the role of PLD in the FcλRI activation of NADPH oxidase.

FcλRI was aggregated by immune complexes and PLD activity was measured. Pretreatment of cells with brefeldin A had no effect on PLD activation suggesting ARFs 1Ð5 played no role in receptor-coupled activation of PLD. Cells were then transfected with either a dominant negative form of ARF6 or ARF6 was down-regulated using an antisense oligonucleotide. In both cases, coupling of FcλRI to PLD was significantly inhibited in the treated cells. Aggregation of FcλRI with immune complexes resulted in the translocation of ARF6 from the cytoplasm to membrane fraction as assessed by cell fractionation and Western blotting. Furthermore, following immune complex activation, ARF6 appeared in FcλRI immunoprecipitates and this was abolished in cells pretreated with 50 nM wortmannin. In order to understand the mechanisms coupling PI3 kinase to ARF6, these receptor immunoprecipitates were also probed for ARNO and were found to be positive, but not in cells pretreated with wortmannin. These same immunoprecipiates were then probed for the presence of PLD1 or PLD2. Only PLD1 was found. Pretreatment of cells with 0.3 % butan-1-ol completely abolished the oxidase burst normally observed in these cells following aggregation of FcλRI. The role of PLD1 but not PLD2 was confirmed by the use of antisense oligonucleotides to down-regulate specifically either PLD isozyme. Thus treatment of cells with antisense to PLD1 reduced both the intracellular calcium and oxidase burst responses to FcλRI activation.

Thus FcλRI appears to be coupled to the activation of PLD1 through a PI3 kinase-dependent process that requires ARNO and ARF6. PLD1 is required for the intracellular calcium and oxidase burst responses to FcλRI aggregation.