Ceramide inhibition of phospholipase D and its relationship to RhoA and ARF1 translocation in GTP gamma S-stimulated polymorphonuclear leukocytes.

Phospholipase D (PLD) regulates the polymorphonuclear leukocyte (PMN) functions of phagocytosis, degranulation, and oxidant production. Ceramide inhibition of PLD suppresses PMN function. In streptolysin O-permeabilized PMNs, PLD was directly activated by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulation of adenosine diphosphate (ADP)-ribosylation factor (ARF) and Rho, stimulating release of lactoferrin from specific granules of permeabilized PMNs; PLD activation and degranulation were inhibited by C2-ceramide but not dihydro-C2-ceramide. To investigate the mechanism of ceramide's inhibitory effect on PLD, we used a cell-free system to examine PLD activity and translocation from cytosol to plasma membrane of ARF, protein kinase C (PKC)alpha and beta, and RhoA, all of which can activate PLD. GTP gamma S-activated cytosol stimulated PLD activity and translocation of ARF, PKC alpha and beta, and RhoA when recombined with cell membranes. Prior incubation of PMNs with 10 microM C2-ceramide inhibited PLD activity and RhoA translocation, but not ARF1, ARF6, PKC alpha, or PKC beta translocation. However, in intact PMNs stimulated with N-formyl-1-methionyl-1-leucyl-1-phenylalamine (FMLP) or permeabilized PMNs stimulated with GTP gamma S, C2-ceramide did not inhibit RhoA translocation. Exogenous RhoA did not restore ceramide-inhibited PLD activity but bound to membranes despite ceramide treatment. These observations suggest that, although ceramide may affect RhoA in some systems, ceramide inhibits PLD through another mechanism, perhaps related to the ability of ceramide to inhibit phosphatidylinositol-bisphosphate (PIP2) interaction with PLD.

Regulation of polymorphonuclear leukocyte degranulation and oxidant production by ceramide through inhibition of phospholipase D.

Exogenous C(2)-ceramide has been shown to inhibit polymorphonuclear leukocyte (PMN) phagocytosis through inhibition of phospholipase D (PLD) and downstream events, including activation of extracellular signal-regulated kinases 1 and 2, leading to the hyphothesis that the sphingomyelinase pathway is involved in termination of phagocytosis. Here it is postulated that increased PLD activity generating phosphatidic acid and diacylglycerol (DAG) is essential for superoxide release and degranulation and that ceramide, previously shown to be generated during PMN activation, inhibits PLD activation, thereby leading to inhibition of PMN function. When PMNs were primed with granulocyte colony-stimulating factor (G-CSF) and then activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP), C(2)-ceramide (10 microM) completely inhibited release of superoxide, lactoferrin, and gelatinase; the DAG analog sn-1,2-didecanoylglycerol (DiC10) (10 microM) restored oxidase activation and degranulation in the ceramide-treated cells. Similarly, C(2)-ceramide inhibited oxidase activity and degranulation of PMNs treated with cytochalasin B followed by FMLP, and DiC10 restored function. In contrast, C(2)-ceramide did not inhibit phosphorylation of p47phox or p38 mitogen-activated protein kinase, or translocation of p47phox, PLD-containing organelles, adenosine diphosphate-ribosylation factor 1, RhoA, protein kinase C (PKC)-beta or PKC-alpha to the plasma membrane in G-CSF or cytochalasin B-treated, FMLP-activated PMNs. PLD activity increased by 3-fold in G-CSF-primed PMNs stimulated by FMLP and by 30-fold in cytochalasin B-treated PMNs stimulated by FMLP. Both PLD activities were completely inhibited by 10 microM C(2)-ceramide. In conclusion, superoxide, gelatinase, and lactoferrin release require activation of the PLD pathway in primed PMNs and cytochalasin B-treated PMNs. Ceramide may affect protein interactions with PLD in the plasma membrane, thereby attenuating PMN activation.