The subcellular distribution of platelet-activating factor in stimulated human neutrophils

Exposure of human peripheral blood neutrophils to a variety of phagocytic and soluble stimuli is known to induce the synthesis and secretion of platelet-activating factor (PAF), a unique ether-linked phospholipid. It has recently been observed in this laboratory, that whereas some PAF is secreted to the exterior of the cell, the majority of the newly synthesized PAF is retained intracellularly. This observation led us to investigate the subcellular distribution of intracellular PAF in stimulated human neutrophils, and to question the possible intracellular role of this molecule. Approximately 2 x 10(8) neutrophils were exposed to either the phagocytic stimulus, opsonized zymosan particles (25 particles/cell), the soluble stimulus, Ca2(+)-ionophore A23187 (5 micrograms/ml), or were left unstimulated for up to 30 min. After disruption, the cells were fractionated into nuclei, phagolysosomes, specific granules, azurophil granules, membranes, and cytosol. Fractions were analyzed for representative organellar markers, as well as for total protein, total phospholipid phosphorous, and PAF. In cells that had been exposed to opsonized zymosan particles, the majority of the PAF was localized to the phagolysosomal fraction, with lesser amounts being detected in the membranous and granular fractions of the cells. In neutrophils that had been exposed to A23187, the major portion of the PAF was detected in the membranous fractions with smaller amounts being seen in fractions corresponding to the specific granules. On the basis of these data, combined with the known physicochemical properties of PAF, it is speculated that the PAF detected at discrete intracellular locations in stimulated human neutrophils may play an important role in the endocytic and/or secretory functions of neutrophils.