Characteristics of leukotriene biosynthesis by human granulocytes in presence of plasma

Abstract

The formation of leukotriene B 4 (LTB 4) by neutrophils stimulated with the ionophore A23187 or physiological stimuli in heparinized plasma was investigated. In comparison with neutrophils stimulated (A23187) in a protein-free buffered salt solution, neutrophils stimulated in plasma produced only trace amounts of LTB 4. The addition of human recombinant LTA 4-hydrolase or erythrocytes to plasma prior to A23187 stimulation strongly and selectively stimulated (>4-fold) the formation of LTB 4 supporting that neutrophils activated in plasma with A23187 release in the extracellular milieu most of LTA 4 formed by the cells, and indicating that plasma proteins drastically slow down the further metabolism of LTA 4 released by neutrophils. The formation of LTB 4 was then investigated in GM-CSF-primed neutrophils stimulated with fMLP in plasma; levels of synthesis were very low and the addition of erythrocytes prior to stimulation strongly enhanced LTB 4 synthesis, demonstrating that agonist-stimulated neutrophils also release most of LTA 4 generated in the extracellular milieu. Investigations on the fate of LTA 4 in plasma revealed that LTA 4 was slowly degraded through an unknown process, i.e. not through the previously described non-enzymic hydrolysis resulting in the formation of dihydroxy derivatives of LTA 4. Using neutrophils labeled with tritiated arachidonate, we also demonstrated that neutrophils stimulated in plasma with fMLP or A23187, almost exclusively use endogenous arachidonate, as opposed to plasma arachidonate, to generate 5-lipoxygenase products. Finally, experiments performed with purified eosinophils indicated that contrary to neutrophils, the eosinophils do not release LTA 4, but directly release LTC 4.