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

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 (cPLA2α) targets arachidonic acid–containing phospholipids but the role of the other cytosolic enzyme present in macrophages, the Ca2+-independent group VIA phospholipase A2 (iPLA2β) 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 cPLA2α, iPLA2β 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 iPLA2β, with minimal or no involvement of cPLA2α. The other major species produced, stearoyl-glycerophosphocholine, is generated primarily by cPLA2α. Collectively, these findings suggest that cPLA2α and iPLA2β act on different phospholipids during zymosan stimulation of macrophages and that iPLA2β 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.

Group V Secreted Phospholipase A2 Is Upregulated by IL-4 in Human Macrophages and Mediates Phagocytosis via Hydrolysis of Ethanolamine Phospholipids

“…PE and PI molecular species were identified by multiple reaction monitoring experiments on chromatographic effluent by comparison with previously published data (24,(26)(27)(28)(29)(30)(52)(53)(54). Cutoff parameter was set at m/z 150, and fragmentation amplitude was set at one arbitrary unit.…”

“…The gradient was started at 100% solvent A, which was decreased linearly to 75% at 3 min, 55% at 11 min, 40% at 13 min, 25% at 18 min, and 10% at 18.5 min. The last solvent mixture was held for an additional 1.5-min period; finally, the column was re-equilibrated with 100% solvent A for 10 min before the next sample injection (30). The flow rate through the column was fixed at 0.6 ml/min, and this flow entered into the electrospray interface of an API2000 triple quadrupole mass spectrometer (Applied Biosystems).…”

“…The retention time window was set to 120 s. The declustering potential and collision energy for each analyte were optimized by the use of analytical standards. Other parameters were fixed for all analytes: entrance potential, 210 V; focusing potential, 2350 V; and collision cell exit potential, 210 V. Quantification was carried out by integrating the chromatographic peaks of each species and comparing with an external calibration curve made with analytical standards (30).…”

“…In previous work, we investigated the mechanisms of PLA 2 -mediated phospholipid turnover in monocytes and macrophages responding to a variety of stimuli of innate immunity and inflammation (21)(22)(23)(24)(25)(26)(27)(28)(29)(30). In those studies we took advantage of mass spectrometry-based lipidomic approaches to define, at a molecular species level, the phospholipid substrate specificities of the enzymes involved.…”

Cytosolic phospholipase A2: physiological function and role in disease

“…Most cells contain several types of PLA 2 enzymes and studies have suggested that they have distinct functions. For example, a comparison of the function of cPLA 2 ␣ and iPLA 2 ␤ in mast cells and macrophages found that cPLA 2 ␣ , but not iPLA 2 ␤ , selectively hydrolyzes arachidonic acid-containing phospholipids in response to cell stimulation ( 12,13 ).…”