Flow cytometric kinetic measurements of neutrophil phospholipase A activation.
The interrelationships between activation of phospholipases and neutrophil stimulus-induced Ca2+ responses remain unclear. We report here that immune complexes activate a phosphatidylcholine-specific phospholipase A in a neutrophil only after the cytoplasmic Ca2+ transient has been initiated in the same cell, while chemotactic peptide activation does not proceed via such a phospholipase A-mediated mechanism. Measurements of [Ca2+] changes and of phosphatidylcholine-specific phospholipase A activity were made by flow cytometry, using Indo-1 for Ca2+ indication, and a new fluorescent probe, bis-BODIPY-phosphatidylcholine, localized in the inner leaflet of the plasma membrane, to measure phospholipase A activation. Both 100 nM formyl-methionyl-leucyl-phenylalanine (with or without cytochalasin B) and 60 micrograms/ml insoluble immune complexes elicited cytoplasmic Ca2+ transients, but only insoluble immune complexes stimulated phospholipase A activation in a subpopulation of cells exhibiting an elevation of [Ca2+]in. Phospholipase A activation followed the Ca2+ transient, starting, in each cell, after [Ca2+]in had begun to decrease as Ca2+ redistributed in the activated cell. The products of this phospholipase activation were confirmed by thin layer chromatography. We conclude that neutrophils respond to immune complexes with an elevated cytoplasmic Ca(2+)-requiring phosphatidylcholine-specific phospholipase A activation and to chemotactic peptides by a different mechanism.
Meshulam
T
T
Boston University School of Medicine, Massachusetts 02118.
Herscovitz
H
H
Casavant
D
D
Bernardo
J
J
Roman
R
R
Haugland
R P
RP
Strohmeier
G S
GS
Diamond
R D
RD
Simons
E R
ER
D001896:Boron Compounds; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*
D001896:Boron Compounds; D005456:Fluorescent Dyes; D008081:Liposomes; D010713:Phosphatidylcholines; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010741:Phospholipases A; D002118:Calcium