Triggering of a Phospholipase D Pathway Upon Mitogenic Stimulation of Human Peripheral Blood Mononuclear Cells Enriched with 12(S)‐hydroxyicosatetraenoic Acid

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Abstract
The influence of 12(S)‐hydroxyicosatetraenoic acid (12‐HETE), that we have previously shown to decrease the proliferative response of human lymphocytes to mitogens, on diacylglycerol and phosphatidic acid (PtdOH) formation was investigated in stimulated human peripheral blood mononuclear cells (PBMC). When human PBMC were first enriched with 12‐HETE, then stimulated by the mitogenic lectin concanavalin A (Con A), the production of PtdOH normally associated with Con A stimulation was markedly increased as compared with non‐enriched cells. The Con‐A‐induced rise in the PtdOH mass was markedly decreased by 1 % ethanol in 12‐HETE‐enriched cells, whereas it was unaffected in control cells stimulated by Con A alone. Furthermore, in [3H]arachidonic‐acid‐labelled cells previously enriched with 12‐HETE, the formation of [3H]arachidonic‐acid‐labelled phosphatidylalcohol was significantly increased upon Con A stimulation, no phosphatidylalcohol being synthesized in non‐enriched cells. Collectively, these results suggest that, in the presence of 12‐HETE, Con A stimulates a phospholipase D activity which was not triggered by Con A alone. These data are consistent with the lack of effect of suramin, reported as a phospholipase D inhibitor, which we observed in cells stimulated by Con A alone and with the suramin‐induced decrease of PtdOH mass in 12‐HETE‐plus‐Con‐A‐treated cells. Moreover, 12–[3H]HETE‐enriched PBMC produced a significant amount of 12–[3H]HETE‐containing PtdOH (0.4% of the total PtdOH) in resting conditions. Upon mitogenic Stimulation by Con A, the phorbol ester tetrade‐canoylphorbol acetate or the anti‐CD3 mAb OKT3, this proportion was decreased to 0.1–0.2%, since the total PtdOH mass was more drastically increased than the 12‐HETE‐containing PtdOH species. Although present in relatively low amount in stimulated cells, 12‐HETE‐containing PtdOH species might have been generated in strategic compartments of the membrane bilayer so that the following events involved in the transduction of the mitogenic signal could be impaired. GC analyses have pointed out drastic variations in the fatty acid composition of PtdOH in non‐enriched and in 12‐HETE‐enriched stimulated cells. Especially PtdOH synthesized in 12‐HETE‐enriched cells upon Con A stimulation contained a higher amount of saturated fatty acids and a lower amount of arachidonic acid than that formed in control cells stimulated with Con A alone. Such saturated PtdOH species with a low arachidonic acid content are very likely to have a low mitogenic potential.