Differential Metabolism of Exogenous and Endogenous Arachidonic Acid in Human Neutrophils

Leukotrienes can be produced by cooperative interactions between cells in which, for example, arachidonate derived from one cell is oxidized to leukotriene A4(LTA4) by another and this can then be exported for conversion to LTB4 or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC4 synthase but are known to cooperate with endothelial cells or platelets (which do have this enzyme) to generate cys-LTs. Stimulation of human neutrophils perfusing isolated rabbit hearts resulted in production of cys-LTs, whereas these were not seen with perfused hearts alone or isolated neutrophils. In addition, the stimulated, neutrophil-perfused hearts generated much greater amounts of total LTA4 products, suggesting that the hearts were supplying arachidonate to the neutrophils and, in addition, that this externally derived arachidonate was preferentially used for exported LTA4 that could be metabolized to cys-LTs by the coronary endothelium. Stable isotope-labeled arachidonate and electrospray tandem mass spectrometry were used to differentially follow metabolism of exogenous and endogenous arachidonate. Isolated, adherent neutrophils at low concentrations (to minimize transcellular metabolism between them) were shown to generate higher proportions of nonenzymatic LTA4products from exogenous arachidonate (deuterium-labeled) than from endogenous (unlabeled) sources. The endogenous arachidonate, on the other hand, was preferentially used for conversion to LTB4by the LTA4 hydrolase. This result was not because of saturation of the LTA4 hydrolase, because it occurred at widely differing concentrations of exogenous arachidonate. Finally, in the presence of platelets (which contain LTC4 synthase), the LTA4 synthesized from exogenous deuterium-labeled arachidonate was converted to cys-LTs to a greater degree than that from endogenous sources. These experiments suggest that exogenous arachidonate is preferentially converted to LTA4 for export (not intracellular conversion) and raises the likelihood that there are different intracellular pathways for arachidonate metabolism.