Cytosolic Group IVA and Calcium-Independent Group VIA Phospholipase A2s Act on Distinct Phospholipid Pools in Zymosan-Stimulated Mouse Peritoneal Macrophages

Phospholipase A(2)s 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 A(2) 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 A(2) (cPLA(2)alpha) targets arachidonic acid-containing phospholipids but the role of the other cytosolic enzyme present in macrophages, the Ca2+-independent group VIA phospholipase A(2) (iPLA(2)beta) 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 cPLA(2)alpha, iPLA(2)beta 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 iPLA(2)beta, with minimal or no involvement of cPLA(2)alpha. The other major species produced, stearoyl-glycerophosphocholine, is generated primarily by cPLA(2)alpha. Collectively, these findings suggest that cPLA(2)alpha and iPLA(2)beta act on different phospholipids during zymosan stimulation of macrophages and that iPLA(2)beta 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.