Triggering of a phospholipase D pathway upon mitogenic stimulation of human peripheral blood mononuclear cells enriched with 12(S)-hydroxyicosatetraenoic acid.

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 tetradecanoylphorbol 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.

Esterification of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid into the phospholipids of human peripheral blood mononuclear cells: inhibition of the proliferative response.

12-hydroxy-eicosatetraenoic acid (12-HETE), the lipoxygenase metabolite of arachidonic acid produced by activated platelets, has been shown to accumulate in peripheral blood mononuclear cells (PBMC) of elderly people. 12-HETE being antimitogenic for lymphocytes, its accumulation in blood cells might be involved in the well-known decline in immune function which accompanies aging. Because HETEs have been shown to be rapidly metabolized and/or incorporated into cellular lipids in a variety of cell types, we have investigated the uptake, metabolism, and intracellular distribution of exogenous 12-HETE by human PBMC. [3H]-12-HETE was dose and time dependently incorporated by PBMC and also metabolized to more polar products. These polar metabolites were mainly released extracellularly and only marginally esterfied in phospholipids. Although [3H]-12-HETE radiolabel was preferentially associated with phosphatidylcholine, especially after prolonged labeling incubations or following successive short labeling pulses, a substantial amount of radiolabel was also found associated with phosphatidylinositol (20-50% of the labeled phospholipids). The stability of 12-HETE in the phospholipid pool was comparable to that reported for most other cell types, with 50% of the initial radiolabel being still present after 18 hr. Upon exposure to mitogenic activation, 12-HETE-labeled PBMC released unmodified 12-HETE from phosphatidylinositol. In addition, 12-HETE dose dependently inhibited the proliferative response of PBMC to Con A stimulation. These results suggest that 12-HETE esterification in phospholipids might lead to the generation of unusual lipid second messengers with impaired capacity to transduce activation signals, thus decreasing lymphocyte function.

Influence of polyunsaturated fatty acids on lipid metabolism in human blood mononuclear cells and early biochemical events associated with lymphocyte activation.

n-3 and n-6 polyunsaturated fatty acids are involved in the regulation of the immune response. Although different hypotheses related to modifications of arachidonic acid metabolism or alterations at the level of the cell membrane have been put forward to explain their suppressive effect on the lymphocyte growth, their mechanism of action remains largely unknown. Cyclic nucleotide phosphodiesterase (PDE) has been shown to be an important target involved in the control of lymphocyte proliferation. The present study aimed to determine whether in vitro addition of a physiological concentration (5 microM) of n-6 (20:3n-6) or n-3 (18:4n-3, 20:5n-3, 22:6n-3) fatty acids to human peripheral blood mononuclear cells (PBMC) was able to alter the PDE activity of these cells, and especially the PDE increase in response to Con A stimulation. Pretreatment of human PBMC for a short period of time (90 min) with 5 microM of either 20:3n-6, 20:5n-3 or 22:6n-3 was sufficient to induce a significant enrichment of cellular phospholipids in the corresponding fatty acid, whereas 18:4n-3 was poorly incorporated. Either fatty acid significantly increased both cAMP- and cGMP-PDE activities in the cytosolic compartment, the particulate PDE activities being less sensitive to their stimulatory effect. In contrast, they significantly lowered the PDE increase to Con A stimulation. Except 20:5 n-3, the three other fatty acids did not alter significantly the basal or Con A-induced oxygenated metabolism of arachidonic acid (AA), appreciated by the measurement of radioactive eicosanoids formed in [3H]AA-labelled cells. Furthermore, only 20:5n-3 significantly inhibited the lymphoproliferative response to Con A, whereas 16:0, 18:0, 18:1n-9, 20:3n-6 and 20:4n-6 were inactive. The inhibitory effect was not prevented by antioxidant vitamins C and E. The present results suggest that the lymphocyte growth suppressive effect of 20:5n-3 20:5n-3 is very likely to be independent on both the cAMP system and eicosanoid synthesis, and does not seem to involve their conversion to peroxidised products.

Increased glutathione peroxidase activity in human blood mononuclear cells upon in vitro incubation with n-3 fatty acids.

Fish oil-enriched diets have been shown to increase the n-3 polyunsaturated fatty acid (PUFA) content of cell membranes, in vivo, and to simultaneously enhance the glutathione peroxidase (glutathione: H2O2 oxidoreductase, EC 1.11.1.9) (GSH-Px) activity of platelets and erythrocytes both in animals and humans. The present study aimed to determine whether in vitro enrichment of human peripheral blood mononuclear cells (PBMC) with either eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) might have similar influence on the GSH-Px activity of these cells. Pretreatment of human PBMC for a short period of time (90 min) with 5 microM of either EPA or DHA was sufficient to induce a significant enrichment of cellular phospholipids in the corresponding fatty acid. This was accompanied by an increased GSH-Px activity of these cells. This stimulatory effect proved to be specific of n-3 fatty acids since it was not observed with saturated, monounsaturated or n-6 polyunsaturated fatty acids. Neither EPA nor DHA had a direct influence on the GSH-Px activity of PBMC in cell-free preparations. Both fatty acids had no influence on the distribution of total proteins between the cytosolic and the particulate compartments. EPA did not significantly alter the GSH-Px kinetic behavior thus suggesting an increased expression of the enzyme. In addition, DHA slightly but significantly decreased the proliferative response of PBMC to the mitogenic stimulation by Con A whereas EPA only tended to do so.

Diethyldithiocarbamate (ditiocarb sodium) effect on arachidonic acid metabolism in human mononuclear cells. Glutathione peroxidase-like activity.

Diethyldithiocarbamate (DTC), a thiol delivery agent, has been shown to significantly reduce the frequency of primary opportunistic infections in HIV-infected patients. This therapeutic effect has been related to the capacity of DTC to reverse the deleterious effects of the oxidative stress occurring in HIV infection. The influence of DTC on the oxygenated metabolism of arachidonic acid (AA) was investigated in mitogen-stimulated human peripheral blood mononuclear cells (PBMC). Upon incubation with PBMC previously labelled with [3H]AA, Concanavalin A (Con A) markedly increased cyclooxygenase and lipoxygenase activities, within 30 min, as judged by thromboxane B2 (TxB2) and hydroxyeicosatetraenoic acid (HETE) production. Con A activation of [3H]AA platelets also increased 12-HETE production but did not induce any TxB2 synthesis. Micromolar concentrations of DTC, added simultaneously with the mitogen, significantly enhanced the synthesis of HETEs above the Con A-induced level while TxB2-induced synthesis was inhibited but only at DTC concentrations higher than 50 microM. In the presence of nordihydroguaiaretic acid, a lipoxygenase inhibitor, which inhibited the Con A-induced synthesis of HETEs by 78%, DTC no longer stimulated HETE production above the Con A-induced level. Reverse phase HPLC analysis showed that Con A increased the PBMC production of 5-, 12- and 15-HETEs. In the presence of 5 microM DTC, 5-HETE production was entirely suppressed whereas the 15-HETE level was markedly enhanced, 12-HETE production by the contaminating platelets remained unchanged. In vitro experiments indicated that DTC alone did not significantly influence 15-hydroperoxyeicosatetraenoic (15-HPETE) production by the soybean 15-lipoxygenase but, in the presence of added reduced glutathione, DTC markedly reduced 15-HPETE into 15-HETE. In addition, DTC was able to substitute for cellular extract in the glutathione peroxidase (GPx) assay system. Taken together, these results indicate that DTC, through its "GPx-like" activity is able to modify the lipoxygenase cascade. Its ability to selectively reduce 15-HPETE known to stimulate immunosuppressive T-cells might help to explain its positive regulatory effect upon the immune system.