CHARACTERISTICS OF LEUKOTRIENE BIOSYNTHESIS BY HUMAN GRANULOCYTES IN PRESENCE OF PLASMA

The formation of leukotriene B-4 (LTB4) by neutrophils stimulated with the ionophore A23187 or physiological stimuli in heparinized plasma w as investigated. In comparison with neutrophils stimulated (A23187) in a protein-free buffered salt solution, neutrophils stimulated in plas ma produced only trace amounts of LTB4. The addition of human recombin ant LTA(4)-hydrolase or erythrocytes to plasma prior to A23187 stimula tion strongly and selectively stimulated (> 4-fold) the formation of L TB4 supporting that neutrophils activated in plasma with A23187 releas e in the extracellular milieu most of LTA(4) formed by the cells, and indicating that plasma proteins drastically slow down the further meta bolism of LTA(4) released by neutrophils. The formation of LTB4 was th en investigated in GM-CSF-primed neutrophils stimulated with fMLP in p lasma; levels of synthesis were very low and the addition of erythrocy tes prior to stimulation strongly enhanced LTB4 synthesis, demonstrati ng that agonist-stimulated neutrophils also release most of LTA(4) gen erated in the extracellular milieu. Investigations on the fate of LTA( 4) in plasma revealed that LTA(4) was slowly degraded through an unkno wn process, i.e. not through the previously described non-enzymic hydr olysis resulting in the formation of dihydroxy derivatives of LTA(4). Using neutrophils labeled with tritiated arachidonate, we also demonst rated that neutrophils stimulated in plasma with fMLP or A23187, almos t exclusively use endogenous arachidonate, as opposed to plasma arachi donate, to generate 5-lipoxygenase products. Finally, experiments perf ormed with purified eosinophils indicated that contra to neutrophils, the eosinophils do not release LTA(4), but directly release LTC4. (C) 1998 Elsevier Science B.V.