PRIMED NEUTROPHILS REQUIRE PHOSPHATIDIC-ACID FOR MAXIMAL RECEPTOR-ACTIVATED ELASTASE RELEASE

Background. Priming of neutrophils (PMNs) for protease release is beli eved to be central to the pathogenesis of PMN-mediated tissue injury o bserved in ARDS/ MOF. Defining the intracellular signaling pathways in volved with this excessive protease release may aid in establishing fu ture therapies for ARDS. Phospholipase D (PLD) production of phosphati dic acid (PA) is thought to be pivotal in reactive oxygen species gene ration but its role in degranulation (i.e., protease release) remains unclear. Our hypothesis was that primed neutrophils require PLD produc tion of PA for maximal activated release of elastase. Methods. Isolate d human PMNs were incubated with a well described antagonist of PA pro duction, ethanol (ETOH, 100-1000 mg/dL), and then primed (PAF, 200 nM) followed by activation (fMLP, 1 mu M). To mimic fMLP receptor-depende nt activation, PMNs were primed and then directly activated with exoge nous dioctanoyl L-alpha-phosphatidic acid (PA(8), 0.5-200 mu M) To con firm the importance of PA in elastase release, PA(8) was given to prim ed-activated PMNs after ethanol pretreatment in an attempt to recover the maximal response. Elastase release was measured by the cleavage of AAPV-pNA. Results. PA blockade with ETOH attenuated PAF-primed/fMLP-a ctivated PMN elastase release in a dose-dependent manner. Exogenous PA (8) reproduced maximally primed-activated PMN elastase release, and fu rthermore, PA(8) was able to restore maximal elastase release followin g ethanol attenuation. Conclusions. Elastase release from PAF-primed/f MLP-activated neutrophils is dependent on PA production. Thus, PA prod uction appears to be involved in both oxidant-dependent and independen t mechanisms of neutrophil cytotoxicity and may be a potential therape utic target in the treatment of hyperinflammatory diseases such as ARD S/MOF. (C) 1998 Academic Press.