Effect of priming on activation and localization of phospholipase D-1 in human neutrophils.
Phospholipase D (PLD) plays a major role in the activation of the neutrophil respiratory burst. However, the repertoire of PLD isoforms present in these primary cells, the precise mechanism of activation, and the impact of cell priming on PLD activity and localization remain poorly defined. RT-PCR analysis showed that both PLD1 and PLD2 isoforms are expressed in human neutrophils, with PLD1 expressed at a higher level. Endogenous PLD1 was detected by immunoprecipitation and Western blotting, and was predominantly membrane-associated under control and primed/stimulated conditions. Immunofluorescence showed that PLD had a punctate distribution throughout the cell, which was not altered after stimulation by soluble agonists. In contrast, PLD localized to the phagolysosome membrane after ingestion of nonopsonized zymosan particles. We also demonstrate that tumour necrosis factor alpha greatly potentiates agonist-stimulated PLD activation, myeloperoxidase release, and superoxide anion generation, and that PLD activation occurs via a phosphatidylinositol 3-kinase-sensitive and brefeldin-sensitive ADP-ribosylation factor GTPase-regulated mechanism. Moreover, propranolol, which causes an increase in PLD-derived phosphatidic acid accumulation, caused a selective increase in agonist-stimulated myeloperoxidase release. Our results indicate that priming is a critical regulator of PLD activation, that the PLD-generated lipid products exert divergent effects on neutrophil functional responses, that PLD1 is the major PLD isoform present in human neutrophils, and that PLD1 actively translocates to the phagosomal wall after particle ingestion.
Cadwallader
Karen A
KA
Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge, UK. kc220@cam.ac.uk
Uddin
Mohib
M
Condliffe
Alison M
AM
Cowburn
Andrew S
AS
White
Jessica F
JF
Skepper
Jeremy N
JN
Ktistakis
Nicholas T
NT
Chilvers
Edwin R
ER
D020727:ADP-Ribosylation Factors / Q000037:antagonists & inhibitors; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008854:Microscopy, Electron; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010739:Phospholipase D / Q000378:metabolism*; D011233:Precipitin Tests; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015290:Second Messenger Systems; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017931:DNA Primers; D004791:Enzyme Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D010739:Phospholipase D; C106801:phospholipase D1; D020727:ADP-Ribosylation Factors