Release of platelet-activating factor and the metabolism of leukotriene B₄ by the human neutrophil when studied in a cell superfusion model

Stimulated human neutrophils are known to synthesize large quantities of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 5,12-dihydroxy-6,14-cis-8,10-trans-transeicosatetraenoic acid (LTB₄). However, in an isolated cell suspension the majority of synthesized PAF appears to remain cell associated. In addition, LTB₄ is rapidly metabolized to an ω-oxidation product (20-OH-LTB₄). Experiments were designed to test the hypothesis that the degree of association of PAF with the neutrophils and the metabolism of LTB₄ by the neutrophils is a result of the in vitro condition used during cell activation. Here we have compared in paired experiments ionophore A23187-induced production of PAF and LTB₄ by human neutrophils in a concentrated cell suspension, a diluted cell suspension and in a system in which the cells are placed on a matrix and superfused with buffer at a constant flow rate (dynamic system). There was little difference in the amount of PAF synthesized in the concentrated cell suspension and the dynamic system. However, less PAF was produced by neutrophils in the dilution system. The percent of PAF released was consistently greater in the dynamic and dilution systems than in the concentrated cell suspension. For example, more than 40% of PAF measured by incorporation of [³H]acetate or gas chromatography/mass spectrometry was released in the dynamic system and dilution systems. In contrast, less than 15% of the PAF synthesized was released from the cells in the concentrated cell suspension. 1-0-Hexadecyl-2-acetyl-3-GPC was primarily released from the neutrophils. By contrast both 1-0-hexadecyl and 1-0-octadecyl linked species of PAF were found within the cells. Exogenous PAF added to neutrophils in the dynamic or dilution systems was taken up and metabolized at a significantly lower rate than that added to cells in the concentrated cell suspension. Most of the leukotrienes synthesized by the neutrophil during A23187 stimulation were released from the cells. However, studies of LTB₄ metabolism revealed differences between the dynamic and concentrated cell suspension designs. By 20 min, most of the LTB₄ was recovered as 20-OH-LTB₄ in the concentrated cell suspension, whereas in the dynamic system little 20-OH-LTB₄ was found in the superfusate over 20 min. These experiments suggest that a large proportion of PAF synthesized by neutrophils may be released. They also suggest that the ω-hydroxylation of LTB₄ by neutrophils occurs after synthesized LTB₄ is released and taken back up by the cell.