CRYSTAL-INDUCED NEUTROPHIL ACTIVATION .2. EVIDENCE FOR THE ACTIVATION OF A PHOSPHATIDYLCHOLINE-SPECIFIC PHOSPHOLIPASE-D

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-H-3-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 alkyl-phosphatidic 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.