Abstract
Leukotrienes can be produced by cooperative interactions between cells in which, for example, arachidonate derived from one cell is oxidized to leukotriene A(4) (LTA(4)) by another and this can then be exported for conversion to LTB(4) or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC(4) 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 LTA(4) products, suggesting that the hearts were supplying arachidonate to the neutrophils and, in addition, that this externally derived arachidonate was preferentially used for exported LTA(4) 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 LTA(4) products from exogenous arachidonate (deuterium-labeled) than from endogenous (unlabeled) sources. The endogenous arachidonate, on the other hand, was preferentially used for conversion to LTB(4) by the LTA(4) hydrolase. This result was not because of saturation of the LTA(4) hydrolase, because it occurred at widely differing concentrations of exogenous arachidonate. Finally, in the presence of platelets (which contain LTC(4) synthase), the LTA(4) 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 LTA(4) for export (not intracellular conversion) and raises the likelihood that there are different intracellular pathways for arachidonate metabolism.
Highlights
Plicated as a critical step in the production of mediators of inflammatory events [1,2,3]
In agreement with previous results [16], an fMLP challenge of the same number of neutrophils in the reperfused, spontaneously beating, isolated rabbit heart resulted in the production of large amounts of cysteinyl leukotrienes (cys-LTs) (Fig. 2C), presumably as a result of neutrophil-endothelial cell cooperative synthesis of cys-LT
The data suggested that neutrophil interaction with the coronary vasculature of the heart led to the new synthesis of cys-LTs and to a 4-fold increase in total leukotriene A4 (LTA4) metabolites, likely because of additional sources of externally derived arachidonate
Summary
Plicated as a critical step in the production of mediators of inflammatory events [1,2,3]. Neutrophils by themselves do not synthesize cys-LT but, in cooperation with endothelial cells (or platelets), lead to the production of these mediators by the exchange of newly synthesized LTA4 to the endothelial cell or platelet that contains constitutively active leukotriene synthase [13, 14]. This cooperative formation of cys-LT has been shown to cause coronary vasoconstriction and severe inflammatory changes in the rabbit heart [15, 16]. The study demonstrates that the metabolic fate of arachidonate released from the endogenous pool of neutrophil phospholipids is different from that of arachidonate derived from exogenous sources
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