Objective. To investigate the involvement of phospholipase D in the signaling pathways activated by 2 pathologically relevant inflammatory microcrystals, monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD).Methods. Human peripheral blood neutrophils were used throughout. Phospholipase D activity was monitored by measuring 3 separate indices: 1) the mass of phosphatidic acid, 2) the levels of alkyl-phosphatidic acid, and 3) the levels of formation, in the presence of ethanol, of phosphatidylethanol. The latter 2 parameters were measured in cells labeled with 1-0-3H-alkyl-2-acetyl-sn-glycero-3-phosphocholine. The cells were stimulated with microcrystals of triclinic morphology.Results. Both MSU and CPPD crystals induced a time-and concentration-dependent accumulation of phosphatidic acid mass and elevation in levels of alkylphosphatidic acid and phosphatidylethanol in prelabeled cells. The activation of phospholipase D by the microcrystals was partially sensitive to colchicine and largely resistant to pertussis toxin. Inhibition of phosphatidic acid formation by wortmannin or ethanol reduced the microcrystal-stimulated production of superoxide anions.Conclusion. These results indicate that microcrystals stimulate phospholipase D in human neutrophils and that at least some of the functional consequences of neutrophil-microcrystal interactions may be dependent on this biochemical pathway.A significant body of evidence indicates that an interaction of monosodium urate (MSU) or of calcium pyrophosphate dihydrate (CPPD) crystals with neutrophils is involved in the etiology of gouty arthritis and of articular chondrocalcinosis, respectively ( 1,2). This conclusion is supported, inter alia, by the presence of microcrystals in the synovial environment including the synovial fluid, and by the ability to reproduce the major symptoms of gout and articular chondrocalcinosis by introduction of these crystals into normal joints (3-6).Microcrystals interact with all of the major synovial cell types, including neutrophils, monocyte/

SummaryThe generation of diradylglycerol (DRG) and phosphatidic acid (PdtOH) was investigated in neutrophils primed with granulocyte-macrophage colony-stimulating factor (GM-CSF) . Mass accumulation of DRG and PdtOH was measured using reversed-phase high performance liquid chromatography and thin layer chromatography, respectively. GM-CSF had no direct effect on the levels of PdtOH and DRG, but it increased PdtOH generation and the late phase of DRG accumulation in human neutrophils stimulated with FMLP. The elevation of the mass of PdtOH peaked -100 s and clearly preceded that of DRG, which peaked at 150 s. The diacylglycerol kinase inhibitor R59022 enhanced the sustained increase in DRG but did not produce a parallel inhibition in PdtOH production . GM-CSF was without effect on the level of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and did not affect the liberation of Ins(1,4,5)P3 induced by FMLP. These findings exclude the involvement of the PtdIns(4,5)PZ-specific phospholipase C/diacylglycerol pathway in the sustained phase of DRG accumulation. The early (30-s) appearance ofPdtOH clearly suggests that GM-CSF enhanced FMLP receptor-linked phospholipase D (PLD) generation of PdtOH. PLD was assessed more directly by formation of labeled phosphatidylethanol (PEt) through PLD capacity of catalyzing a trans-phosphatidylation in presence ofethanol. The formation ofPEt associated with a concomitant decrease in PdtOH directly demonstrated that the mechanism by which GM-CSF enhances PdtOH production is activation of a PLD active on phosphatidylcholine. This study provides evidence that the mechanism ofaction ofGM-CSF involves upregulation of PLD activity leading to enhanced generation of PdtOH and DRG in FMLP-stimulated neutrophils. These findings may provide the basis for several of the priming effects of GM-CSF.

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