Agonist-stimulated alveolar macrophages: apoptosis and phospholipid signaling.
Bovine alveolar macrophages (BAM) were labeled with [3H]-choline or [3H]-ethanolamine and exposed to quartz dust, metal oxide-coated silica particles, Escherichia coli-derived lipopolysaccharide (LPS) or tumor promotor 12-O-tetradecanoyl phorbol 13-acetate (PMA). The activation of phospholipases A2, C and D (PLA2, PLC and PLD) acting on phosphatidylcholine and phosphatidylethanolamine was determined by high performance liquid chromatography (HPLC) separation and liquid scintillation counting of water- and lipid-soluble phospholipid metabolites. Exposure of BAM to quartz dust, metal oxide-coated silica particles, and LPS led to a transient PLD activation while treatment with PMA caused a prolonged rise in PLD activity. LPS and quartz dust induced a short-term increase of PLC cleavage products. All agonists caused a transient activation of PLA2. To induce apoptosis, BAM were stimulated with C8-ceramide, calcium-ionophore 23187, or gliotoxin. Apoptosis was investigated by qualitative and quantitative methods like flow cytometry, propidium iodide/Hoechst 33258 double staining, Cell Death Detection ELISA, and electrophoretical detection of DNA fragmentation. All three agonists led to apoptosis of BAM in a time- and concentration-dependent manner. After stimulation with gliotoxin an increase in ceramide and a drastic decrease in sphingosine-1-phosphate levels were observed, suggesting an involvement of these sphingolipids in gliotoxin-mediated apoptosis.
Lütjohann
J
J
Department of Biochemistry and Molecular Biology, University of Hamburg, Germany.
Spiess
A N
AN
Gercken
G
G
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002417:Cattle; D002794:Choline / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004391:Dust; D019856:Ethanolamine / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D008670:Metals / Q000633:toxicity; D010087:Oxides / Q000633:toxicity; D010743:Phospholipids / Q000378:metabolism / Q000502:physiology*; D011791:Quartz / Q000633:toxicity; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D012822:Silicon Dioxide / Q000633:toxicity; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014316:Tritium
D004391:Dust; D008070:Lipopolysaccharides; D008670:Metals; D010087:Oxides; D010743:Phospholipids; D014316:Tritium; D011791:Quartz; D019856:Ethanolamine; D012822:Silicon Dioxide; D002794:Choline; D013755:Tetradecanoylphorbol Acetate