Abstract
The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.
Highlights
The role of a cytosolic phospholipase A2-␣ in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA2-␣ activity was blocked with the specific cPLA2-␣ inhibitor, Pyrrolidine-1, or by cPLA2-␣ gene disruption. cPLA2-␣ inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, Fc␥II/ III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro
We demonstrated that cPLA2-␣ catalyzes arachidonic acid release, initiates PAF biosynthesis, and partially regulates Escherichia coli killing by neutrophils in vitro and that exogenous arachidonic acid reverses the defect in neutrophil E. coli killing induced by the inhibition or genetic deletion of cPLA2-␣
CPLA2-␣ Mediates Neutrophil Arachidonate Release—To ascertain the importance of cPLA2-␣ in arachidonic acid release, human neutrophils incubated with the specific cPLA2-␣ inhibitor Pyrrolidine-1, which does not inhibit cPLA2-␥, calciumindependent group VI PLA2 or group IIA, group V- or group X-secreted PLA2 activity [19], or cPLA2- in vitro,2 were stimulated with various agonists and the liberation of arachidonic acid was quantified by gas chromatography/mass spectrometry
Summary
Materials—d8-Arachidonic acid is from BioMol. d3-PAF was synthesized using 1-O-hexadecyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids Inc., Alabaster, AL) and d6-acetic anhydride (Aldrich) using standard methods. Murine neutrophils from PLA2g4a(ϩ/ϩ) or PLA2g4a(Ϫ/Ϫ) mice were resuspended in HBSSϩϩ at 107 cells/ml and incubated with vehicle (Me2SO, 0.1%), MK886 (1 M), or U75302 (10 M) for 5 min at 37 °C and incubated with Me2SO (0.1%), arachidonic acid (5 M), PAF (500 nM), or LTB4 (10 nM) for 5 min at 37 °C as indicated in the figure legends. Cells were incubated with cytochalasin D (10 M) and aggregated human IgG or with ionomycin (2 M) for a further 10 min at 37 °C, fixed with paraformaldehyde, rinsed, and incubated with either 1:20 ␣N-RAS (irrelevant IgG), 1:50 ␣CD63, or 1:100 ␣CD66b primary antibodies for 1 h on ice. After washing, neutrophils were incubated with 1:1000 goat anti-mouse Alexa 488 secondary antibody for 1 h on ice, washed again, and analyzed by flow cytometry (FACScan, 10,000 events for each sample). A p value Ͻ 0.05 was considered to be significant
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