The priming effect of 12(S)-hydroxyeicosatetraenoic acid on lymphocyte phospholipase D involves specific binding sites.

We have previously shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE)-enrichment primed human peripheral blood mononuclear cells for phospholipase D activation by mitogens. Given that 12(S)-HETE-enriched cells stimulated with concanavalin A released free 12(S)-HETE in the extracellular medium, and that the priming effect of 12(S)-HETE on phospholipase D was suppressed by the non-permeant drug, suramin, we hypothesized an extracellular mechanism for 12(S)-HETE-induced PLD activation. Using [3H]12(S)-HETE as a ligand and a rapid filtration technique, we have pointed out the presence of specific low-affinity 12(S)-HETE binding sites on intact human mononuclear cells and lymphocytes. [3H]12(S)-HETE binding was efficiently displaced by other monohydroxylated and n-3 fatty acids but not by oleate and arachidonate, and was also significantly inhibited by suramin and pertussis toxin. Furthermore, 12(S)-HETE-induced PLD activation was strongly inhibited by pertussis toxin and genistein, but was not PKC-dependent. In addition, 12(S)-HETE also potentiated the ConA-induced tyrosine phosphorylation of a 46-50 kDa protein, which was inhibited by genistein. Collectively, these results suggest that 12(S)-HETE binding sites on human lymphocytes may be coupled to phospholipase D through pertussis toxin sensitive G-proteins and tyrosine kinases.

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.

Phosphodiesterase inhibitory profile of some related xanthine derivatives pharmacologically active on the peripheral microcirculation.

The cyclic nucleotide phosphodiesterase (PDE) inhibitory profile of four related xanthine derivatives: pentoxifylline (BL 191), propentofylline (HWA 285), torbafylline (HWA 448) and albifylline (HWA 138), pharmacologically active on the peripheral and/or cerebral microcirculation was established using the four main PDE isoforms present in rat heart cytosol. HPLC on a Mono Q ion-exchange column resolved four separate cyclic nucleotide PDE activities: a calmodulin-activated fraction (PDE I), a cGMP-stimulated fraction (PDE II), a cAMP-specific rolipram-sensitive fraction (PDE IV) and a cGMP-inhibited fraction (PDE III). Among the four compounds studies, only torbafylline and pentoxifylline inhibited more efficiently the calcium plus calmodulin-stimulated than the basal activity of PDE I. The four xanthine derivatives inhibited more potently the cGMP-stimulated than the basal activity of the cGMP-stimulatable PDE II, propentofylline being the most inhibitory (IC50: 20 microM). Except for propentofylline, which exhibited a marked selectivity toward the rolipram-sensitive PDE versus the cGMP-inhibited PDE III, the other xanthines modestly (IC50 in the 10(-4) M range) inhibited both cAMP-specific isoforms with similar potency. Propentofylline proved to be the best inhibitor whatever the considered isoform whereas torbafylline exhibited the weakest inhibitory potency with, however, some selectivity for PDE I.

Glutathione peroxidase activity and metabolism of arachidonic acid in peripheral blood mononuclear cells from elderly subjects.

1. Since ageing has been associated with a decrease in both immune responses and antioxidant defences, this study was undertaken to compare the glutathione peroxidase activity in peripheral blood mononuclear cells of healthy elderly and young donors. The mean value of glutathione peroxidase activity was significantly lower in the aged group (-36%) than that observed in the young control group (n = 10). 2. This defect was accompanied by a marked increase (+106%) in the oxygenated metabolism of endogenous arachidonic acid by lipoxygenases as judged by the radiolabel associated with hydroxyeicosatetraenoic acids in [3H]arachidonic acid-prelabelled peripheral blood mononuclear cells, whereas the cyclo-oxygenase activity, estimated by the radiolabel associated with thromboxane B2, was not significantly altered. 3. Upon stimulation by the mitogenic lectin concanavalin A, the radioactivity associated with total eicosanoids (free arachidonic acid plus hydroxyeicosatetraenoic acids plus thromboxane B2) was significantly increased over basal levels in the peripheral blood mononuclear cells of both the elderly and the control groups. However, the mitogen-induced increase was lower in the elderly group (+48%) than in the control group (+131%). Upon concanavalin A stimulation, the radioactivity of hydroxyeicosatetraenoic acids was increased by only 96% in peripheral blood mononuclear cells from the elderly group compared with 350% in control cells. Similarly, the radioactivity associated with thromboxane B2 was increased by only 82% in peripheral blood mononuclear cells from the elderly group compared with 218% in control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Early increase in lymphocyte cyclic nucleotide phosphodiesterase activity upon mitogenic activation of human peripheral blood mononuclear cells.

Cyclic nucleotide phosphodiesterase activity of human peripheral blood mononuclear cells was significantly increased following a short (30 min) incubation with the mitogenic lectin Concanavalin A. Con A stimulated phosphodiesterase activity to the same extent whatever the subcellular compartment (homogenate, cytosol or pellet). Further separation of the Con A-activated mononuclear cells into lymphocyte-enriched and monocyte-enriched populations showed that the Con A-induced increase of phosphodiesterase activity exclusively affected the lymphocyte-enriched population. In lymphocytes, cyclic GMP phosphodiesterase activity was more importantly enhanced by Con A (+275%) than cyclic AMP phosphodiesterase activity (+75%). The increase of both activities occurred as early as from 10 min of Con A incubation and proved to be maximal with Con A doses of 2.5 and 5 micrograms per 10(6) cells, lower and higher doses being less effective. Inhibition experiments with reference inhibitors suggested that, among the high affinity phosphodiesterase isoforms, the cyclic GMP-inhibited enzyme might be more selectively enhanced by Con A than the cyclic AMP-specific, Rolipram-sensitive one. The non-mitogenic lectin Helix pomatia hemagglutinin, was not able to enhance cyclic nucleotide phosphodiesterase activity of human mononuclear cells whereas anti-CD3 monoclonal antibody, although being less effective than Con A, exhibited a significant stimulatory effect. Putting together these results suggest that the early increase in phosphodiesterase activity might be a normal step involved in the mitogenic activation of human lymphocyte.

Dietary polyunsaturated fatty acids modulate fatty acid composition and early activation steps of concanavalin A-stimulated rat thymocytes.

The early biochemical responses to concanavalin A (Con A) of thymocytes from rats fed a saturated (coconut oil), (n-6) (sunflower oil) or (n-3) (fish oil) fatty acid-enriched diet for 3 wk were investigated. Fish oil feeding resulted in greater (n-3) polyunsaturated fatty acid level (PUFA) at the expense of (n-6) PUFA in total and individual thymocyte phospholipids. Such alterations of the fatty acid composition did not affect basal ornithine decarboxylase (ODC), cyclic nucleotide phosphodiesterase (PDE) or gamma-glutamyl transferase activities. However, the fish oil-enriched diet impaired some of the early thymocyte responses to Con A, such as the rapid induction (30 min) of soluble ODC and PDE activities. Synthesis of [3H]20:4(n-6) oxygenated metabolites was not different between the dietary groups; however, the uptake of [3H]20:4(n-6) into phospholipid classes was significantly lower in phosphatidylcholine and greater in phosphatidylethanolamine and phosphatidylinositol after fish oil feeding. Similarly, the Con A-induced remodeling of the [3H]20:4(n-6) esterification in phospholipids differed in sunflower oil- vs. fish oil-fed rats, suggesting a modulation of acyl CoA synthase and/or acyl CoA transferase activities. Thus, the modulation of Con A-induced ODC and PDE stimulation upon in vivo changes of membrane phospholipid fatty acid composition is not related to eicosanoid formation, but rather to the modification of the fatty acid acylation processes, altering phospholipid composition and signal transduction.

Decreased cyclic nucleotide phosphodiesterase activity in human peripheral blood mononuclear cells from elderly women.

1. Both adenosine 3':5'-cyclic monophosphate and guanosine 3':5'-cyclic monophosphate phosphodiesterase activities of peripheral blood mononuclear cells were markedly decreased in elderly women as compared with young control women. 2. In contrast, the ability of these cells to bind guanosine 5'-[beta, gamma-imido]triphosphate, a non-hydrolysable analogue of guanosine 5'-triphosphate, was the same in both groups. 3. These findings are discussed in the context of the decline in immune function which occurs with increasing age.