Abstract: Phospholipase A2s generate lipid mediators that constitute an important component of the integrated response of macrophages to stimuli of the innate immune response. Because these cells contain multiple phospholipase A2 forms, the challenge is to elucidate the roles that each of these forms plays in regulating normal cellular processes and in disease pathogenesis. A major issue is to precisely determine the phospholipid substrates that these enzymes use for generating lipid mediators. There is compelling evidence that group IVA cytosolic phospholipase A2 (cPLA2?) targets arachidonic acid-containing phospholipids but the role of the other cytosolic enzyme present in macrophages, the Ca2+-independent group VIA phospholipase A2 (iPLA2?) has not been clearly defined. We applied mass spectrometry-based lipid profiling to study the substrate specificities of these two enzymes during inflammatory activation of macrophages with zymosan. Using selective inhibitors, we find that, contrary to cPLA2?, iPLA2? spares arachidonate-containing phospholipids and hydrolyzes only those that do not contain arachidonate. Analyses of the lysophospholipids generated during activation reveal that one of the major species produced, palmitoyl-glycerophosphocholine, is generated by iPLA2?, with minimal or no involvement of cPLA 2?. The other major species produced, stearoyl- glycerophosphocholine, is generated primarily by cPLA2?. Collectively, these findings suggest that cPLA2? and iPLA 2? act on different phospholipids during zymosan stimulation of macrophages and that iPLA2? shows a hitherto unrecognized preference for choline phospholipids containing palmitic acid at the sn-1 position that could be exploited for the design of selective inhibitors of this enzyme with therapeutic potential.

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