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The inhibition of human CD4+ T lymphocyte activation and proliferation by cholera toxin B-subunit (CTB) is a well-established phenomenon; nevertheless, the exact mechanism remained unclear. In the present study, we propose an explanation for the rCTB-induced inhibition of CD4+ T lymphocytes. rCTB specifically binds to GM1, a raft marker, and strongly modifies the lipid composition of rafts. First, rCTB inhibits sphingomyelin synthesis; second, it enhances phosphatidylcholine synthesis; and third, it activates a raft-resident neutral sphingomyelinase resembling to neutral sphingomyelinase type 1, thus generating a transient ceramide production. We demonstrated that these ceramides inhibit protein kinase Cα phosphorylation and its translocation into the modified lipid rafts. Furthermore, we show that rCTB-induced ceramide production activate NF-κB. Combined all together: raft modification in terms of lipids, ceramide production, protein kinase Cα inhibition, and NF-κB activation lead to CD4+ T cell inhibition.

Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56lck and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-β-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca2+ concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts.

CD5 is a transmembrane coreceptor that modulates activation and differentiation signals mediated by the Ag-specific receptor present on both T and B1a lymphocytes. CD5 lacks intrinsic catalytic activity, and its immunomodulatory properties result from intracellular interactions mediated by the CD5 cytoplasmic tail. The nature of these interactions is currently a matter of investigation. Here, we present a selective mutagenesis analysis of two conserved threonine residues (T410 and T412) located at the membrane-proximal cytoplasmic region of CD5. These residues are contained within consensus phosphorylation motifs for protein kinase C and are shown here to be critical for in vivo protein kinase C-mediated phosphorylation of CD5. Functional studies revealed that the integrity of T410 and T412 is also critical for CD5-mediated phosphatidylcholine-specific phospholipase C (PC-PLC) activation and phorbol ester-mediated inhibition of Ab-induced internalization of CD5. These results strongly argue in favor of a role for T410 and T412 in the signaling mediated by CD5.

Early in the apoptotic process, CD95 induces a translocation of phosphatidylserine (PtdSer) from the inner to the outer leaflet of the cellular plasma membrane. In mammalian cells, PtdSer is only synthesized through a calcium-dependent exchange of the polar head group of pre-existing phospholipids, either phosphatidylcholine or phosphatidylethanolamine, by a serine. Using a pharmacological approach, we examined the influence of PtdSer synthesis on CD95-induced PtdSer exposure at the surface of Jurkat cells. We found that CD3/TCR triggering or thapsigargin treatment of Jurkat cells was accompanied both by a decreased PtdSer synthesis and by a strong reduction of CD95-induced PtdSer at the cell surface, as monitored by fluorescence-activated cell sorting (FACS) analysis of annexin V-fluorescein isothiocyanate (FITC)-labeled cells. PtdSer synthesis through the serine-base exchange enzyme system thus appeared as one of the mechanisms implicated in the recently discovered CD3/TCR-induced down-regulation of CD95-induced apoptosis. Conversely, increasing the activity of the serine-base exchange enzyme system with different drugs, either the K+ channel blocker quinine, the cationic amphiphil stearylamine, or three different calmodulin antagonists, chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), resulted in an increased appearance of PtdSer at the surface of CD95-treated cells. Both PtdSer synthesis and CD95-induced annexin V-FITC reactivity were abrogated in ATP-depleted cells. Also, modifying the membrane potential with valinomycin (hyperpolarization) or either gramicidin or KCl (depolarization) demonstrated a strong relationship between PtdSer synthesis and annexin V-FITC reactivity in CD95-treated cells. Together, our results indicate that CD95-induced exposure of PtdSer at the cell surface could be regulated by the activity of the serine-base exchange enzyme system.

Sodium ortho-vanadate (Na3VO4), an inhibitor of protein tyrosine phosphatase, induces a rapid (15 min) and strong inhibition of phosphatidylserine synthesis with an IC50 = 100 microM. The mode of action of Na3VO4 was compared to that of CD3 mAbs. It was found that Na3VO4 bypasses the major CD3-induced T cell activation signals including protein tyrosine phosphorylation, p56lck activation and the generation of second messengers including inositol phosphates and its subsequent Ca2+ mobilization as well as diacylglycerol production. These facts were confirmed by using a panel of Jurkat clones that differs by the expression of either tyrosine kinases involved in the CD3-induced T cell activation pathway such as p56lck, p72syk and ZAP-70 or some cell surface receptors such as the CD3/TCR complex or the CD45 phosphatase.

Phosphatidylserine synthesis as measured by the incorporation of [(3)H]serine into phosphatidylserine (PtdSer) through the serine-base exchange enzyme system (serine-BEES) is markedly inhibited in Jurkat cells treated with caffeine. The caffeine-induced inhibition was compared to that observed in cells treated with either CD3 mAb or thapsigargin. While CD3- and thapsigargin-induced inhibition was related to the release of Ca(2+) from the endoplasmic reticulum (ER), a process that deprives the serine-BEES of its major cofactor, caffeine modified PtdSer synthesis in the absence of decreased Ca(2+) content of ER. Using Jurkat clones differing by the expression of cell surface markers or protein tyrosine kinases implicated in the CD3/TCR signal transmission pathway, we have shown that CD3 mAb-induced inhibition of PtdSer synthesis necessitates the expression of both the CD3/TCR and the protein tyrosine phosphatase CD45 at the cell surface as well as the presence of p56(lck) and ZAP-70 protein tyrosine kinases. By contrast, thapsigargin, a blocker of the Ca(2+)-ATPase of the ER, known for its Ca(2+) releasing properties, inhibited PtdSer synthesis in all the Jurkat clones tested, indicating that this compound bypasses the CD3/TCR-induced signals. Despite its lack of effect on Ca(2+) release from ER and on protein tyrosine phosphorylations, caffeine inhibited PtdSer synthesis in all the Jurkat clones. The use of several cAMP-inducing drugs and of others xanthine derivatives indicated that caffeine modify PtdSer synthesis either by a direct action on the serine-BEES or by a modification of the structure of the phospholipids used as substrate by the enzyme.

CD95 (Fas, APO-1)-induced programmed cell death (apoptosis) in T cell lines is accompanied by a rapid flip-flop of phosphatidylserine (PtdSer). Externalization of this phospholipid has been previously recognized as one of the early detectable events of cells undergoing apoptosis. We show here that CD95 induces a rapid (detectable at time

Phosphatidylserine (PtdSer) is synthesized by an exchange of the polar head group of phospholipids for a serine residue. The enzyme responsible for this reaction, the serine-base exchange enzyme system (serine-BEES) is inhibited during lymphocyte activation. We show here that triggering the CD4 cell surface molecule in several CD4+ T-cell lines regulates the serine-BEES activity, thus resulting in marked changes in PtdSer synthesis. CD4 ligands able to generate an activating signal in T-cells such as the lectin jacalin, down-regulate the synthesis of PtdSer. In contrast, monoclonal antibodies (mAbs) directed against the CD4 molecule, such as IOT4 and IOT4a, which have previously been described as generating an inhibitory signal to T-cells, induced an up-regulation of the serine-BEES and impaired CD3-induced inhibition of PtdSer synthesis. Similarly, the HIV-gp120 envelope glycoprotein, in both soluble and cross-linked forms, induces an increase in PtdSer synthesis. The protein tyrosine kinase p56lck participates in the regulation of serine-BEES activity because the effect of CD4 mAbs was additive to that of amino-hydroxyflavone, an inhibitor of p56lck. Also, CD4 mAbs were inactive in J Cam 1.6 cells or when the CD3 signals were bypassed by using thapsigargin. These results demonstrate that the CD4 surface molecule can transmit both activating and inhibiting intracellular signals depending on the CD4 ligand used. We suggest that PtdSer synthesis would be one of the intracellular signals that could explain the opposite effects of different CD4 ligands on T-cells.

Inhibitors of Ca 2+ -calmodulin (CaM)-dependent protein kinases strongly modify phospholipid metabolism. Two compounds, KN62 and KT5926 recognized as blockers of Ca 2+ -CaM-dependent protein kinase II, induced a specific increase in phosphatidylserine (PtdSer) synthesis without noticeable changes in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) biosynthesis. The increase of PtdSer synthesis was dependent on the presence of Ca 2+ in the incubation medium and was impaired in cells whose Ca 2+ stores were depleted by pretreatment with CD3 mAb, thapsigargin or EGTA. The mechanism of the stimulation of PtdSer synthesis by these two compounds seems to involve an accumulation of Ca 2+ into the endoplasmic reticulum, possibly due to an increased activity of the endoplasmic reticulum Ca 2+ -ATPase. By contrast, ML-7 and ML-9, two inhibitors of the myosin light chain kinase (MLCK), another Ca 2+ -CaM-dependent kinase, were both capable of increasing PtdSer synthesis and decreasing PtdCho and PtdEtn synthesis, reproducing the effect previously described with CaM-antagonists. The increase of PtdSer caused by ML-7 and ML-9 was Ca 2+ -dependent while the inhibition of PtdCho and PtdEtn synthesis was not. The use of these four protein kinase inhibitors thus suggests the possible existence of two CaM-dependent pathways that differentially regulates phospholipid metabolism in T cells.

The calcium release-activated channel (CRAC) opened in Jurkat cells activated either with CD3 monoclonal antibody or the endoplasmic reticulum Ca2+-ATPase blocker, thapsigargin, is blocked by La3+ with an IC50 of 20 nM. Similarly, the entry of Mn2+, used as a surrogate for Ca2+, is also blocked by submicromolar La3+ concentrations. La3+ seems to play its role simply by plugging the CRAC because this ion does not penetrate the cells, as demonstrated by chelation experiments with EGTA. Blocking the Ca2+ influx in activated Jurkat cells results in a lack of expression of CD25, a chain of the interleukin-2 receptor and of CD69, a marker of T-cell activation. By contrast, the very early steps of the T-cell signalling pathway such as the release of Ca2+ from intracellular stores and the subsequent inhibition of phosphatidylserine synthesis are not affected by La3+.

An CD11a/CD18-dependent homotypic aggregation pathway is induced by triggering CD45 molecules on human thymocytes. By contrast, a CD11a/CD18-independent homotypic aggregation process is induced by triggering the CD99 molecule (E2, MIC2 gene product) expressed at the surface of either Jurkat T cells or human thymocytes. A new quantitative method based on FACS analysis of aggregated cells was used and allowed to show that both types of aggregation (CD11a/CD18-dependent and CD11a/CD18-independent) were inhibited with sphingosine, oleylamine or stearylamine. These three compounds had no effect on the expression of CD99, CD45, CD11a, CD18 or other [correction of others] known integrins expressed at the surface of the cells studied.

Oleylamine and stearylamine, two cationic amphiphilic drugs, strongly increase phosphatidylserine synthesis in human T cells. The two compounds had little effect on either phosphatidylcholine or phosphatidylethanolamine synthesis. A decrease in the formation of phosphatidylethanolamine through decarboxylation of phosphatidylserine was observed, but this effect is only marginally involved in increased phosphatidylserine synthesis. The high incorporation of [ 3 H]-serine into phosphatidylserine is a protein kinase C independent process and is due to a synergy of either oleylamine or stearylamine with calcium ions.

Calmodulin (CaM) antagonists chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide HCl inhibit Jurkat T cell activation, as monitored by measuring interleukin-2 synthesis in cells treated by a combination of CD3 monoclonal antibody and phorbol myristate acetate. T cell activation with CD3 monoclonal antibody is accompanied by a decreased synthesis of phosphatidylserine due to the release of Ca2+ from the endoplasmic reticulum. CaM antagonists reverse the phosphatidylserine (PtdSer) inhibition induced by CD3. This increase of PtdSer synthesis was observed in the absence of any modification of CD3-induced Ca2+ movements. Both in intact cells and in an acellular system, the increase of PtdSer synthesis induced by CaM antagonists was abolished in the presence of EGTA, indicating that the base exchange enzyme system responsible for PtdSer synthesis is regulated by CaM provided that Ca2+ is present. By contrast, cyclosporin A that inhibits T cell activation through the interaction of cyclophilin-cyclosporin A complexes with the calmodulin-activated phosphatase, calcineurin, had no effect on PtdSer synthesis. Calmodulin thus appears as a junction leading to at least two independent pathways of regulation of T cell activation, one involving the calcineurin phosphatase and the other the base exchange enzyme system responsible for PtdSer synthesis.

Cytochrome P450 inhibitors such as alpha-naphthoflavone, the imidazole antimycotics, econazole, clotrimazole, and miconazole and the lipoxygenase inhibitors, nordihydroguaiaretic acid and eicosatetraynoic acid, strongly diminished CD3-induced human T cell proliferation. This effect is due to a marked inhibition of IL-2 synthesis. The mechanism leading to the in vitro immunosuppressive effect of cytochrome P450 inhibitors appears to be a consequence of a blockade of Ca2+ influx induced by CD3 mAb. The drugs tested did not interfere with the release of Ca2+ from the endoplasmic reticulum as demonstrated by Ca+ measurements in the presence of the Ca2+ chelator, EGTA. Measurements of CD3-induced changes in phosphatidylserine synthesis, which reflect the status (full/empty) of intracellular Ca2+ stores confirmed that CD3 mAb remained able to empty the Ca2+ stores either alone or in the presence of cytochrome P450 inhibitors. Altogether, our results support the hypothesis that a cytochrome P450 regulates Ca2+ influx in T cells and are consistent with the proposal that impairing Ca2+ influx leads to the inhibition of IL-2 synthesis and subsequent T cell proliferation.

1. 1. We investigated the effect of the thiol reagent, thimerosal on calcium movements in the Jurkat T cell line. 2. 2. Thimerosal induced a rise in cytosolic Ca 2+ concentration due both to a release of Ca 2+ from intracellular stores and a Ca 2+ influx. 3. 3. Thimerosal, released Ca 2+ from the same intracellular stores than CD3 mAb and ionomycin. 4. 4. Emptying the Ca 2+ intracellular stores was accompanied by a marked decrease of phosphatidylserine synthesis indicating that phosphatidylserine synthesis occurs within or close to the endoplasmic reticulum Ca 2+ -stores as previously described in CD3-, ionomycin- or Ca 2+ -ATPase inhibitor-treated lymphocytes.

CD99, a unique integral membrane protein present on the surface of all human T cells, has previously been shown to regulate cell function and fate. In peripheral T cells, it triggers immediate activation of alpha4b1 integrin and cell arrest on inflamed vascular endothelium, whereas it mediates an apoptotic signal in double-positive thymocytes undergoing the selection process. Two isoforms of CD99 exist, a long form corresponding to the full-length protein and a short form harboring a deletion in the intracytoplasmic segment. Here, we show that while peripheral T cells display exclusive expression of the long form, double-positive thymocytes express both isoforms. Moreover, differential expression of these two CD99 molecules can lead to distinct functional outcomes. Expression of the long form in a CD99-deficient Jurkat T cell line is sufficient to promote CD99-induced cell adhesion, whereas coexpression of the two isoforms is required to trigger T-cell death. When coexpressed, the two proteins form covalent heterodimers, which locate within glycosphingolipidic rafts and induce sphingomyelin degradation. Cholesterol depletion experiments show that this localization is required for the induction of apoptosis. Thus, the surface expression pattern of CD99 isoforms determines T-cell functional outcomes.

The effects of quinine, quinidine, cinchonine, cinchonidine and chloroquine on phospolipid synthesis in the Jurkat T cell line have been compared. It was found that the two potassium channel blockers, quinine and quinidine, markedly enhanced phosphatidylserine synthesis and strongly decreased both phosphatidylcholine and phosphatidylethanolamine synthesis. The inhibition of phosphatidylcholine and phosphatidylethanolamine synthesis was due to the inhibition of the uptake of [3H]choline or [3H]ethanolamine, respectively, by the cells. This effect was also observed when using either cinchonine, cinchonidine and chloroquine. In contrast, these three drugs were unable to modify phosphatidylserine synthesis, indicating that the K+ channel blockers, quinine and quinidine, specifically interfere with the synthesis of this phospholipid.

Five antiarrhythmic drugs (bretylium, clofilium, propranolol, N-acetylprocainamide and amiodarone) were tested for their ability to modify phospholipid metabolism in Jurkat T lymphocytes. The five drugs, decreased in a dose-dependent mode the biosynthesis of both phosphatidylcholine and phosphatidylethanolamine, this effect was essentially due to impairment of either choline or ethanolamine uptake by the cells. The efficiency of the drugs to inhibit phosphatidylcholine and phosphatidylethanolamine synthesis was in the order: clofilium greater than amiodarone much greater than propranolol = bretylium much greater than N-acetylprocainamide. The IC50 varied from 3-5 microM for clofilium to greater than 200 microM for N-acetylprocainamide. In contrast, only clofilium, a voltage-gated K(+)-channel blocker, was able to increase phosphatidylserine synthesis with an EC50 = 50 microM. The effect of clofilium on phosphatidylserine synthesis thus mimics the effect of three other K(+)-channel blockers, quinine, 4-aminopyridine and tetraethylammonium, suggesting close relationships between phosphatidylserine synthesis and K+ channel activity.

Diacylglycerol (DAG) plays a prominent role in several activation processes because of its ability, in the presence of calcium ions and phosphatidylserine, to activate C kinase. In T lymphocytes, however, DAG, produced in response to activating mAb or lectins, is rapidly metabolized into phosphatidic acid (PA) which may participate in further steps of activation. We thus investigated the involvement of a DAG kinase in several events subsequent to the activation of Jurkat T cells by CD3 mAb or phytohemagglutunin (PHA). We showed that three inhibitors of DAG kinase abbrogated PA production and impaired calcium release from intracellular compartment, restored phosphatidylserine synthesis, and finally blocked IL-2 production in CD3- and PHA-stimulated cells. Postactivation DAG levels were not modified and DAG kinase inhibitors lowered IL-2 secretion even in the presence of phorbol ester that directly activates the C kinase. These results clearly demonstrate that, beside the effect of DAG on C kinase, DAG kinase dependent production of PA is crucial for further steps of T cell activation.

Diacylglycerol (DAG) prodution induced after stimulation with either CD3 mAb, a pair of CD2 mAbs or phytohaemagglutinin has been monitored in Jurkat T-cells prelabelled to isotopic equilibrium with seven [3H]- or [14C]fatty acids. It was found that CD3 induced a high production of arachidonic acid-labelled DAG and a modest production of oleic acid-DAG. The reverse was observed when using CD2 as activator. Phytohaemagglutinin induced a high production of these two DAG subspecies and in addition induced the production of linolenic acid-labelled DAG. Whatever the activator used no changes were observed in DAG production when cellular phospholipids were prelabelled with either myristic, palmitic, stearic or linoleic acids. All together our results strongly suggest that the three activation pathways previously described in T-lymphocytes might differ either at the level of the transduction mechanism or the phospholipid pools solicited during the activation process.

T lymphocytes contain two kinetic pools of cholesterol extractable with methyl-β-cyclodextrin (m-β-CD): a fast pool (31.5%, t 1 / 2 = 17 s) and a slow pool (68.5%, t 1 / 2 = 15 min). Purification of detergent-resistant membranes (DRMs) shows that the fast pool corresponds to buoyant cholesterol. Cholesterol extraction of the fast pool (i.e. cholesterol from rafts) still allows the buoyancy of signaling proteins and their phosphorylation under CD3 stimulation. Cholesterol depletion of the slow pool (i.e. cholesterol from membranes other than rafts) is accompanied by the extraction of the whole raft followed by the inhibition of CD3-induced tyrosine-phosphorylations. Cholesterol oxidase (COase) allows a specific oxidation of raft cholesterol into cholestenone. Cholestenone leaves the DRMs and accumulates as Triton X-100-soluble material. Specific cholesterol-rich raft disruption by COase does not inhibit the activation of either Jurkat cells or T CD4 + lymphocytes. Our study challenges the real role of cholesterol-rich rafts in CD3/TCR signaling and suggests that a cholesterol-poor subtype of rafts is involved in signal transmission via the TCR.

Incubation of Jurkat cells in the presence of H2O2 either directly added to the culture medium or generated with glucose oxidase, menadione or the couple xanthine/xanthine oxidase induced a marked decrease of phosphatidylserine synthesis in the absence of changes in the synthesis of phosphatidylcholine and phosphatidylethanolamine. Concentration dependent response curves indicated that H2O2 induced inhibition of phosphatidylserine synthesis with an IC(50)=5 microM while both induction of tyrosine phosphorylation of proteins and Ca(2+) signals were obtained with an EC(50)=300 microM. The tyrosine kinase and Ca(2+) independent mechanism was confirmed by comparing the H2O2-induced and the CD3-induced inhibition of phosphatidylserine synthesis using several Jurkat clones differing in the expression of cell surface receptors such as CD3/TCR and CD45 and protein tyrosine kinase such as p72syk, ZAP-70 and p56lck. While CD3-induced inhibition of phosphatidylserine synthesis necessitates protein tyrosine phosphorylation and Ca(2+) signals, H2O2 provoked its effect in all the clones studied independently of the presence or absence of the proteins previously shown to be key elements in T cell signal transduction. Conversely, the antioxidant molecule, butylated hydroxanisole, generates an increased PtdSer synthesis, suggesting that the synthesis of this phospholipid is regulated by the redox status of the cells.

Sphingomyelinase induces a marked rapid decrease of cytosolic Ca2+ concentration in Jurkat T cells treated with either CD3 monoclonal antibody; the Ca(2+)-ATPase inhibitor, thapsigargin; or the Ca2+ ionophore, ionomycin. Sphingomyelinase treatment of Jurkat cells results in a net decrease of cellular sphingomyelin content. Among the products generated by the catabolism of sphingomyelin, sphingosine displayed exactly the same effect as sphingomyelinase. Sphingosine decreases the cytosolic Ca2+ concentration in cells treated with CD3, thapsigargin, or ionomycin. Studying the effect of sphingosine in CD3-activated cells showed that this compound does not modify Ca2+ mobilization from intracellular stores but strongly inhibited the Ca2+ influx induced by the monoclonal antibody. The fact that sphingosine inhibits Ca2+ influx generated by thapsigargin and ionomycin supports the hypothesis that the drug activates the Ca2+ extrusion process. Derivatives of sphingosine such as erythrosphinganine and threosphinganine share the same inhibitory properties.

Phenylarsine oxide (PAO), an inhibitor of tyrosine phosphatases, has been found to inhibit the early elevation in cytosolic Ca2+ concentration ([Ca2+]i), related to the CD3 activation pathway in Jurkat T cells. This inhibition was dose-dependent, consistent with previously reported effects of PAO on tyrosine phosphatases, and reversed by dimercaptopropanol. By contrast, okadaic acid, an inhibitor of serine/threonine phosphatases, had no effect on CD3-induced Ca2+ flux. PAO was compared with phorbol 12-myristate 13-acetate (PMA), which caused a similar, although less potent, inhibition as previously described. The two reagents produced additive inhibition of the CD3-induced [Ca2+]i rise, but did not affect thapsigargin- or ionomycin-driven Ca2+ flux in Jurkat cells. PAO and PMA prevented cells from complete depletion of intracellular Ca2+ stores by an anti-CD3 monoclonal antibody (mAb) and restored, at least partially, the ionomycin-sensitive pool, when added after anti-CD3 mAb. Moreover, the CD3-induced inhibition of phosphatidylserine synthesis, due to depletion of internal Ca2+ stores, is reversed by PAO and PMA. Anti-phosphotyrosine immunoblot analysis show that these effects cannot be accounted for by an inhibition of CD3-induced tyrosine phosphorylations. We propose that PAO and, to a lesser extent, PMA allow the refilling of internal compartments by Ca2+, which consequently abrogates a capacitative entry of external Ca2+.

Numerous studies on the molecular basis of the mechanism of action of fatty acids have demonstrated their action in cell signaling and particularly on the regulation of cytosolic Ca2+ concentration. Stimulation of Jurkat T cells with CD3 monoclonal antibody results in an increase of intracellular calcium concentration, [Ca2+]i due both to a release of Ca2+ from intracellular stores and a Ca2+ influx. [Ca2+]i increase represents a dynamic balance between Ca2+ influx and efflux. Fatty acids, either saturated (C14:0), monounsaturated (C16:1), or polyunsaturated, belonging to the C18 and the C20 series induce a marked decrease of CD3-induced [Ca2+]i rise. This property of fatty acid is independent of the position of the carbon-carbon double bond but specific of the cis stereoisomeric form. Fatty acids does not block CD3-induced signals but greatly stimulates the Ca2+ extrusion process probably by activating the plasma membrane Ca(2+)-ATPase. This was documented by the observation that fatty acids, reduced to the same extent as [Ca2+]i, elicited either with CD3 monoclonal antibody the calcium ionophore ionomycin or the Ca(2+)-ATPase inhibitor thapsigargin.

Monoclonal antibodies (mAbs) directed against E2, a 32-kDa transmembrane protein encoded by the MIC2 gene located in the pseudoautosomal region, induce a transbilayer movement of phosphatidylserine and, to a lesser extent, phosphatidylethanolamine in human thymocytes and a Jurkat T lymphocytes. The translocation of phosphatidylserine has been evidenced by using either derivatization of anionic phospholipids with trinitrobenzenesulfonate (TNBS) or cytofluorimetry after labeling of cells with antiphosphatidylserine antibodies. The perturbation of membrane phospholipids induced by anti-E2 mAbs was further evidenced by labeling the cells with merocyanine 540. The specificity of anti-E2-induced perturbations of membrane asymmetry was tested by using a number of mAbs able to activate T cells, including CD3 and CD2. The results strongly suggest that anti-E2-induced changes in PtdSer are related to cell aggregation since the same mAbs specifically induce the aggregation of both thymocytes and Jurkat cells and since the E2 molecule has been previously implicated in the adhesive properties of human T cells with erythrocytes.

In Jurkat cells, the three Ca2+-ATPase blockers, thapsigargin, cyclopiazonic acid, and di-teributylhydroquinone (DtBuHQ) induced both a release of Ca2+ from intracellular stores and a Ca2+ influx. In contrast to CD3 mAb, the Ca2+-ATPase inhibitors did not induce the formation of inositol trisphosphate from the hydrolysis of phosphatidylinositides. Emptying intracellular Ca2+ stores was accompanied by a decrease of phosphatidylserine (PtdSer) synthesis as previously observed in PHA- or CD3 mAb-treated Jurkat cells. In the presence of a phorbol ester able to activate protein kinase C, TPA, the three Ca2+-ATPase inhibitors induced Jurkat cells to synthesize large amounts of interleukin-2 demonstrating that early signal transduction mechanisms can be bypassed by Ca2+-ATPase inhibitors. In purified human peripheral blood T lymphocytes, the same inhibitors induced moderate if any cytosolic Ca2+ rise, in the absence of external calcium. Nevertheless analysis of PtdSer synthesis suggested that intracellular stores were efficiently depleted by DtBuHQ and cyclopiazonic acid but not by thapsigargin. In contrast, the three compounds induced similar Ca2+ influx. However, in the presence of TPA, cyclopiazonic acid and DtBuHQ induce highly purified T cells to proliferate while thapsigargin did not, suggesting that the status of internal Ca2+ store may have a decisive role in T cell activation.

The imidazole antimycotics, miconazole, econazole and triclomazole as well as α-naphtoflavone, known as powerful inhibitors of cytochrome P450 and previously recognized as K+ channel blockers are shown to be potent activators of the base exchange enzyme system responsible for the biosynthesis of phosphatidylserine in Jurkat T cells. The inhibition of CD3-induced Ca2+ influx by antimycotics but not by K+ channel blockers, demonstrated that the rise in phosphatidylserine synthesis caused by the two classes of drugs, was independent of Ca2+ influx in the cells. In addition, we show that the action of these drugs on phosphatidylserine synthesis was not mimicked by modifications of membrane potential. The regulation of both K+ channels and the base exchange enzyme system thus occurs through a similar (or common) pathway that is independent of Ca2+-influx and membrane potential.

The importance of phospholipids in biological systems has become increasingly recognized in recent years. In T cells as in other cells, a majority of investigators have focused their research on the role of the phosphoinositide (Ptdlns) cycle as a system that generates the lipidic second messengers. Our studies on T lymphocytes have shown that other phospholipids and particularly phosphatidylserine (PtdSer) could play an essential role in T cell activation monitored by the measurement of interleukin-2 synthesis. The present paper takes stock of our current knowledge on the regulation of PtdSer synthesis during T cell activation and on its possible role in the regulation of interleukin-2 synthesis in Jurkat cells, a leukemic T cell line widely used as a model for the studies on lymphocyte activation.

The biosynthesis of phosphatidylserine (PtdSer) by the serine base-exchange enzyme system, in Jurkat T-lymphocytes, was inhibited in intact cells maintained in low-Ca(2+)-containing buffer (< 10 microM-Ca2+) by using Ca2+ ionophores (A23187 or ionomycin). The rise in cytosolic Ca2+ concentration under these experimental conditions was only due to the release of Ca2+ from intracellular compartments, suggesting that the inhibition of PtdSer synthesis was correlated with the emptying of intracellular Ca2+ pools. This was further studied in saponin-permeabilized cells, in which PtdSer synthesis was found to be inhibited by EGTA, Ca2+ ionophores (A23187 or ionomycin) and Ca(2+)-ATPase inhibitors [thapsigargin or 2,5-di-(t-butyl)-benzohydroquinone]. Since Ca(2+)-ATPase inhibitors impaired refilling of the Ca2+ stores with Ca2+, and since in CD3-activated Jurkat T-cells the Ca2+ stores remained empty after 1 h of treatment with anti-CD3 monoclonal antibodies, we suggest that PtdSer synthesis is mainly regulated by the level of Ca2+ in the intracellular compartments and that the Ca(2+)-dependent serine base-exchange system responsible for PtdSer synthesis is probably located within or close to a Ca(2+)-storage organelle.

Quinine, 4-aminopyridine and tetraethylammonium, three compounds generally used as effectors of K+ channels, strongly modify phospholipid metabolism. In the human monocytic cell line THP1, the three drugs enhanced the incorporation of [3H]serine into phosphatidylserine and that of [3H]inositol into phosphatidylinositol in the absence of significant changes in the uptake of the 3H labels. On the contrary, the biosynthesis of both phosphatidylcholine and phosphatidylethanolamine was strongly inhibited. This inhibition appeared to be mainly due to the inhibition of both [3H]choline and [3H]ethanolamine uptake by the cells, by impairment of choline transport in a competitive mode.

Modification of phospholipid metabolism during T cell activation has been repeatedly reported. Recently, we have shown that phytohaemagglutinin, CD3 and CD2 mAbs, which are potent in vitro activators of helper T lymphocytes, markedly inhibit phosphatidylserine synthesis concomitantly as they induce the secretion of IL-2. In this paper, we show evidence that in T lymphocytes K+ channels, which have been shown to participate in the cell activation process, are also reciprocally related to phosphatidylserine synthesis. In fact, in resting T cells the drugs affecting the activity of K+ channels, such as quinine and 4-aminopyridine, induce a rise of phosphatidylserine synthesis. In activated cells, quinine and 4-aminodopyridine also caused a rise in phosphatidylserine synthesis which paralleled a decreased production of IL-2, strongly suggesting that these two events are correlated in a reciprocal manner. More precisely, phosphatidylserine synthesis was stimulated by drugs which have been reported to inhibit potassium channels in lymphocytes, e.g. quinine, 4-aminopyridine, tetraethylammonium. These data suggest that the decreased PS synthesis observed during T cell activation intervenes in the cascade of events leading to IL-2 secretion. The decrease in the biosynthesis of this phospholipid seems to be dependent on the activity of K+ channels.

During chemical carcinogenesis by N-2-fluorenylacetamide, the hormonal status of female Sprague Dawley rats is largely modified. Thyroids present a modified activity which is evidenced by the histological study as well as T4 and T3 assays. In hypophysis, thyrotropic cells were found in an hyperactive state, a fact which is in agreement with the changes observed in thyroid follicles size and epithelial cells demonstrating an hyperactivity of thyroid glands. Despite this histological result, T4 serum level was lowered. T3 level was also decreased but to a lesser extent. We suggest that the low thyroid hormone level play a role in the relative protection of females versus males toward liver cancer induction by chemicals.

The presence of two high affinity estrogen binding proteins in the uterine cytosol of the immature rat has been observed. Besides the 8 S cytosol estrogen receptor , there is a 4–5 S fraction binding estradiol and estrone with a large capacity. In fact, the two binding systems have a different affinity for estradiol and estrone, the receptor binding more the former and the 4–5 S fraction more the latter. Exposure of the cytosol to specific anti-α1-Fetoprotein antibodies suppresses a large part of the 4–5 S binder, if not the totality. Moreover the estrogen binding 4–5 S fraction decreases with increasing age until puberty, while the receptor increases. These results suggest therefore that the estradiol-estrone binding 4–5 S peak of the uterine cytosol is mainly made up of Estradiol Binding Plasma Protein-α1-Fetoprotein (EBP-AFP). Also they confirm that “cytosol” should be taken as an operational fraction which may include extracellular components. During the course of these experiments, it has been observed that the increase of the estradiol receptor is more rapid than the other uterine cytosol proteins until the 8th day, and that there is a second period of growth when it follows the development of the uterus and of the animal, as if it had reached a constant number of binding sites per cell.

Alpha-fetoprotein (AFP),is able to modify arachidonic acid metabolism In P388DI macrophage - like cells. AFP has been shown to induce mobilization of arachidonic acid and consequently an enhanced production of leukotrienes.The action of AFP on the cyclooxygenase pathway appears, however, more complex, as this protein simultaneously decreases the total amount of cyclooxygenase products and raises the production of PGE2, 6-keto-PGF1α and to a lesser extent of thromboxane B2. On the other hand, indomethacin abolishes the synthesis of PGE2 but has no effect on 6-keto-PGFlα and thromboxane B2. Indomethacin used in combination with AFP partly loses its inhibiting effect on PGE2 synthesis and paradoxically leads to a “superactivation” of P388D1 cells demonstrated by a very high arachidonic acid mobilization, an enhancement of both leukotriene synthesis and cyclooxygenase activity. Evidence for a binding of indomethacin to AFP was obtained that partly explains these results. In addition, it is shown that the effects of AFP and AFP+Indomethacin on cyclooxygenase activity might be explained by the endogenous synthesis of leukotrienes.

Thin layer chromatography with four different solvent systems enabeled us to show that ovarian extracts contained nonesterified unsaturated fatty acids able to compete with estradiol on the α-fetoprotein binding site. Gas chromatography demonstrated a high level of oleic and linoleic acids and the presence of arachidonic acid, a strong competitor of the rat α-fetoprotein-estrogen interaction. Arachidonic acid is a precursor of prostaglandins thus we suggest that its binding to AFP might play a role in the AFP-mediated ovarian regulation previously demonstrated.

Rat alpha-foetoprotein (AFP) was shown to inhibit the formation of water soluble metabolities of oestrone and oestradiol by incubation with microsomes from rat liver in the presence of NADPH. The results support the proposal that in young animals the low activity of enzymes responsible of oestradiol metabolism may be due in part to the presence of AFP and not only to the low level of these enzymes.

Evidence is present that alpha-fetoprotein (AFP), a serum globulin, accounts mainly, if not entirely, for the high estrogen-binding properties of uterine cytosols from immature rats. By the use of specific immunoadsorbents to AFP and by competitive assays with unlabeled steroids and pure AFP, it has been demonstrated that in hypotonic cytosols AFP is present partly as free protein with a sedimentation coefficient of about 4-5 S and partly in association with some intracellular constituent(s) to form an 8S estrogen-binding entity. The AFP leads to 8S transformation results in a loss of antigenic reactivity to antibodies against AFP and a significant change in binding specificity. This change in binding specificity is manifested by an increase in binding affinity for estradiol, estriol, diethylstillbestrol, and nafoxidine ( a non-steroidal anti-estrogen), and by a concomitant decrease in estrone binding. Both the antigenic and binding properties of native AFP are recovered after dissociation of the 8S complex in 0.4 M KC1. An AFP-mediated mechanism of early intracellular events associated with estrogen entry in target cells is suggested and discussed with regard to current views on steroid action.

Uriel et al. (1976) suggested that alpha-fetoprotein (AFP) may be the major uterine binder for estrogen in the immature rat. AFP ultrastructural localization was studied using the immunoperoxidase technique in order to determine if this localization fits with the hypothesis according to which AFP is a hormonal receptor. The localization of AFP was compared with that of another plasma protein, albumin. Both proteins were localised in the extracellular position. AFP or albumin were never found intracytoplasmic or intra-nuclear. Thus this method did not provide further evidence of the role of AFP as a physiological receptor in rats.

Adult female rats, each injected with 760 microgram alpha-fetoprotein (AFP) weekly, presented after the third week a decrease of ovarian activity evidenced by a decreased number of maturing follicles, corpora lutea, and a drop in ovarian weight. The level of progesterone was low and agreed with the decreased number of corpora lutea. Conversely, total and free estradiol-17 beta levels remained normal despite the known ability of AFP to bind estrogenic hormones. A regulatory role of AFP during the postnatal stage of sexual maturation is proposed.

Fatty acids interfere with rat alpha-fetoprotein-estrogen interaction. We present here a quantitative study of the association constants for the binding to alpha-fetoprotein of different fatty acids. It can be concluded that fatty acids bind more strongly if the number of carbon atoms increases with the number of double bonds. The maximum binding ability occurred with the C20 fatty acids having three or four double bonds. As these fatty acids are the precursors in the synthesis of prostaglandins, we tested some compounds in this series and showed that prostaglandins presented no particular affinity for rat alpha-fetoprotein.

Human alpha-fetoprotein (HAFP) has three binding sites for polyunsaturated fatty acids with association constant Ka − 1.8×10 7 M −1 . One of these binding sites overlaps with a retinoid binding site with Ka = 2.6×10 6 M −1 . Competition experiments with bilirubin showed that this compound does not compete neither with fatty acids nor with retinoids. Thus, the two bilirubin binding sites previously demonstrated appear as two additional binding sites on HAFP. Nevertheless, the close proximity of two fatty acid binding sites and two bilirubin binding sites resulted in a modification of the binding constants for fatty acids. It is hypothesised that the binding properties of HAFP reflect the three domain structures of the protein recently deduced from the study of the nucleotide sequence of HAFP mRNA and AFPcDNA segments.

Activation of Jurkat T lymphocytes to produce IL2 is accompanied by a strong inhibition of phosphatidylserine (PS) synthesis. This inhibition was obtained either with the mitogenic lectin PHA, anti-CD3 monoclonal antibodies (mAb), anti-CD2 mAb or anti-Ti mAb. Bypassing membrane receptor signalling, by using a Ca2+ ionophore or a protein phosphatase inhibitor, sodium ortho-vanadate, also results in a marked inhibition of PS synthesis. Activators of phospholipid -Ca2+ dependent protein kinase C (PKC) did not significantly modify PS synthesis, suggesting that the observed changes only involve the transduction of the first activation signal. PS being a necessary cofactor for PKC, our results strongly suggest that the inhibition of PS synthesis induced by receptor triggering exerts a feed back control on PKC therefore leading to a transient activation of the enzyme upon full lymphocyte activation.

α-Fetoprotein (AFP) is able to bind specifically polyunsaturated fatty acids, especially arachidonic acid, the major precursor for prostaglandin and leukotriene synthesis. In P388D1 macrophages, AFP was found to reduce prostaglandin synthesis. This reduced synthesis was counterbalanced by a higher release of unmetabolized arachidonic acid and an enhanced production of leukotrienes. The same results were obtained with unactivated and activated cells irrespective of the activator used: lipopolysaccharide, Ca2+ ionophore A23187, phorbol myristate acetate, interferon-γ, silica, or zymozan particles. The stimulation of leukotriene synthesis by AFP in macrophages thus appears to be a possible mechanism for the in vitro immunosuppressive effects Of this Oncofetal protein.

During a period of 200 days, the chronological changes of polyamine levels (putrescine, spermidine and spermine) were observed in the liver of adult female Sprague Dawley rats submitted to hepatocarcinogenesis by N-2-fluorenylacetamide (FAA). Three groups of 70 rats each were used: (1) Control 1: normal diet; (2) Control 2: low protein and low riboflavin diet; and (3) EXPERIMENTAL: 0.06% FAA added to the diet. No significant differences were noted for tissue levels of the three polyamines when the two control groups were compared. In contrast, considerable variations of these molecules were observed as a function of time in the FAA treated group: (a) an early and constant rise was seen in putrescine, with 3 maxima at days 10, 60 and 150. This last peak was the highest: 25 +/- 6 nmol/g (8 times the value for the controls at this time), and coincided with the appearance of cancerous lesions. (b) While spermidine levels varied during the experiment, no significant differences were noted in comparison with the control groups. Mean levels (nmol/g) were: 535 +/- 108 Control 1; 552 +/- 95 Control 2; 633 +/- 160 FAA-treated group. (c) Spermine levels were significantly lowered, with 3 minima corresponding to the putrescine maxima. The lowest minima was observed on day 60: 114 +/- 67 nmol/g, i.e., 4 times lower than the controls. This work shows that polyamine metabolism is profoundly modified during chemical carcinogenesis, but the possible effect of polyamines on tumorigenesis itself cannot be assessed at this point since modifications of polyamine levels are probably also associated with phenomena of liver necrosis and compensatory tissue proliferation observed during the experiment.

Horseradish peroxidase and horse heart microperoxidase can bind estradiol to human or bovine serum albumin in the presence of hydrogen peroxide. However, we have shown here that, in the absence of serum albumin, the hormone was fixed by the enzyme molecule itself. Evidence is presented that (a) the hormone is transformed into a water-soluble and dialysable derivative of estradiol; (b) this new product is easily separated from the enzyme by gel filtration chromatography. It appears to have a high affinity for the chromatographic gel. The implications of the binding of an estradiol derivative to peroxidases are discussed.

Adult female Sprague-Dawley rats fed with 0.06% N-2-fluorenylacetamide presented an augmentation of plasma alpha-fetoprotein 2 days after the beginning of the experiment. This very early synthesis of alpha-fetoprotein was concomitant with the penetration of N-2-fluorenylacetamide detected by fluorescence microscopy, in the cytoplasm of the hepatocytes.