AGONIST-STIMULATED ALVEOLAR MACROPHAGES - APOPTOSIS AND PHOSPHOLIPID SIGNALING

Bovine alveolar macrophages (BAM) were labeled with [H-3]-choline or [ H-3]-ethanolamine and exposed to quartz dust, metal oxide-coated silic a particles, Escherichia coli-derived lipopolysaccharide (LPS) or tumo r promotor 12-O-tetradecanoyl phorbol 13-acetate (PMA). The;activation of phospholipases A(2), C and D (PLA(2), PLC and PLD) acting on phosp hatidylcholine and phosphatidylethanolamine was determined by high per formance liquid chromatography (HPLC) separation and liquid scintillat ion counting of water- and lipid-soluble phospholipid metabolites. Exp osure 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 transien t activation of PLA(2). To induce apoptosis, BAM were stimulated with C-8-ceramide, calcium-ionophore 23187, or gliotoxin. Apoptosis was inv estigated 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-depende nt manner. After stimulation with gliotoxin an increase in ceramide an d a drastic decrease in sphingosine-1-phosphate levels were observed, suggesting an involvement of these sphingolipids in gliotoxin-mediated apoptosis. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved .