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The very early management of pulmonary embolism (PE), a part from antithrombotic treatment, has been little studied. Our aim was to compare the effects of diuretic therapy (DT) versus volume expansion (VE) in patients hospitalized for PE with RV dysfunction.We conducted a randomized open-label multicentric study including patients with intermediate high-risk PE. Patients were randomized between diuretics or saline infusion. The primary endpoint was time to troponin (Tp) normalization. Secondary endpoints were time to normalization of B-type natriuretic peptide (BNP), changes in echocardiographic RV function parameters and treatment tolerance. Sixty patients presenting intermediate high-risk PE were randomized. Thirty received DT and 30 VE. We noted no changes in Tp kinetics between the two groups. In contrast, faster normalization of BNP was obtained in the DT group: 56 [28-120] vs 108 [48-144] h: p = 0.05, with a shorter time to 50%-decrease from peak value 36 [24-48] vs 54 [41-67] h, p = 0.003 and a higher rate of patients with a lower BNP concentration within the first 12 h (42% vs 12% p 0.001). RV echocardiographic parameters were unchanged between the groups. One dose 40 mg furosemide was well-tolerated and not associated with any serious adverse events.In the acute management of intermediate high-risk PE, initial therapy including diuretic treatment is well-tolerated and safe. Although changes in Tp kinetics and echocardiographic RV dysfunction parameters did not differ, normalization of BNP is achieved more quickly in the DT group. This finding, which need to be confirmed in trials with clinical end points, may reflects a rapid improvement in RV function using one dose 40 mg furosemide.Clinical Trial Registration NCT02531581.

ObjectivesSeverity of a first pulmonary embolism (PE) is sometimes proposed as a criterion for prolonging anticoagulant treatment. However, little evidence supports this idea. We attempted to determine the connection between severity of first PE and the risk of recurrence.ParticipantsPatients admitted with PE between 2012 and 2018 and for whom anticoagulant treatment had been discontinued were followed. PEs were classified according to the severity into the following two groups: those with associated cardiac involvement (increased cardiac biomarker(s) and/or echocardiographic right ventricular dysfunction) and those with no cardiac involvement which were classified as non-severe. Recurrence-free survivals were estimated using the Kaplan-Meier method and compared using the log-rank test.Results417 patients with PEs (186 with cardiac involvement) were followed for at least 1 year after discontinuation of treatment with a mean follow-up of: 3.5±1.9 years. 72 patients (17.3%) experienced venous thromboembolism recurrence: 24 (5.8%), 44 (12 %) and 72 (28.3 %) respectively, at 1, 2 and 5 years. In 63 patients (88%), recurrence was a PE. Mean time to onset of recurrence was 24.9±19.9 months. At 5 years, the recurrence rate is higher when the first PE was associated with cardiac involvement p=0.043. In contrast, in patients with provoked PE, the recurrence rate is higher when the first PE event was associated with cardiac involvement: p=0.032. Multivariate analysis demonstrates that PE severity is an independent factor of recurrence (HR 1.634 (1.015–2.632), p=0.043).ConclusionWe report for the first time a possible link between a higher recurrence rate and the severity of the first PE. This result which must be confirmed in a dedicated prospective trial could become an important criterion for the duration of anticoagulant therapy after a PE.Trial registration numberNCT04980924.

By presenting antigenic peptides on the cell surface, human leukocyte antigen (HLA) class I molecules are critical for immune defense. Their surface density determines, to a large extent, the level of CD8+ T cell–dependent immune reactions; their loss is a major mechanism of immune escape. Therefore, powerful processes should regulate their surface expression. Here we document the mechanisms used by CD99 to mediate HLA class I modulation. Up-regulation of HLA class I by IFN-γ requires CD99. In the trans Golgi network (TGN), and up to the cell surface, CD99 and HLA class I are physically associated via their transmembrane domain. CD99 also binds p230/golgin-245, a coiled-coil protein that recycles between the cytosol and buds/vesicles of the TGN and which plays a fundamental role in trafficking transport vesicles. p230/golgin-245 is anchored within TGN membranes via its Golgin-97, RanBP1, IMh1p, P230 (GRIP) domain and the overexpression of which leads to surface and intracellular down-modulation of HLA class I molecules.

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.

The VLA-4 integrin supports static cell-cell, cell-matrix adhesion, and dynamic interactions with VCAM-1. Although functions for well-conserved β1 integrin cytoplasmic domains in regulating static cell adhesion has been established, the molecular basis for β1 integrin-dependent arrest on VCAM-1 under flow conditions remains poorly understood. We have transfected the β1 integrin-deficient A1 Jurkat T cell line with β1 cDNA constructs with deletions of the NPXY motifs and specific mutations of tyrosine residues. Deletion of either NPXY motif impaired static adhesion induced by CD2 or CD47 triggering or direct β1 integrin stimulation. In contrast, PMA-induced adhesion to VCAM-1 was unaffected by deletion of the NPIY motif and only slightly impaired by deletion of NPKY. Moreover, deletion of the NPIY motif resulted in enhanced rolling and reduced arrest on VCAM-1 under shear flow conditions. In contrast, deletion of the NPKY motif did not alter arrest under flow. Although tyrosine to phenylalanine substitutions within two NPXY motifs did not alter static adhesion to VCAM-1, these mutations enhanced arrest on VCAM-1 under flow conditions. Furthermore, although deletion of the C′-terminal 5 AA of the β1 cytoplasmic domain dramatically impaired activation-dependent static adhesion, it did not impair arrest on VCAM-1 under flow conditions. Thus, our results demonstrate distinct structural requirements for VLA-4 function under static and shear flow conditions. This may be relevant for VLA-4 activity regulation in different anatomic compartments, such as when circulating cells arrest on inflamed endothelium under shear flow and when resident cells in bone marrow interact with VCAM-1- positive stromal cells.

Objective . Because the absence of immune restoration in HIV-infected patients efficiently treated by highly active antiretroviral therapy (HAART) may be due to excessive immune activation, we prospectively studied the effect of hydrocortisone on T-cell apoptosis in a cohort of patients with satisfactory virologic response. Methods . Apoptosis of T-cell subsets including naive CD45RA + CD4 + T-cells was determined at baseline and at months 1 and 3 after initiation of HAART. A satisfactory immune response was defined as an increase >100/μL CD4 + T-cells at month 3 compared to baseline. Results . Twenty out of 63 patients showed undetectable viral load at month 3, among whom eight exhibited a satisfactory immune response. Down-regulation spontaneous CD4 + T-cell apoptosis was significant in the group of patients with a satisfactory immune response compared to the other patients. However, hydrocortisone up-regulated apoptosis of naive CD4 + CD45RA + T-cells, specifically in group of patients with poor immune response, whatever the time point considered: percentage of apoptotic CD4 T-cells was 16±16% without hydrocortisone and 22±22% with hydrocortisone at month 1, and respectively, 10±9 and 17±15% at month 3 ( P + T-cell apoptosis, whatever the considered group. Conclusion . Our results suggest to not use steroid therapy as adjuvant immunotherapy in patients with less than optimal immunologic response to HAART.

This prospective study investigated the contributions of apoptosis and proliferation of CD4(+) T cells obtained by the introduction of a new antiretroviral treatment for human immunodeficiency virus infection. Virus load; T cell counts; apoptosis of T cell subsets, including naive cells; and proliferation were determined from treatment initiation to the third month in a cohort of patients. An increase in CD4(+) T cell count > or = 100 cells/microL over baseline was considered to be a satisfactory immune reconstitution. Sixty-nine patients completed the protocol, 22 of whom met our definition of a satisfactory immune reconstitution, showing a significantly more pronounced reduction in spontaneous CD4(+) T cell apoptosis at month 1 as well as month 3, compared with the other patients. In contrast, neither Fas-induced apoptosis down-regulation nor Fas-induced increased proliferation capacity was associated with a satisfactory immune reconstitution. Down-regulation of CD4(+) T cell apoptosis by antiretroviral treatment is the main mechanism associated with early CD4(+) T cell increase.

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.

The mechanisms of apoptosis have become better understood, in part with the discovery of Fas/ CD95. We report the case of a patient characterized by a decreased CD4 - T cell count and an overexpression of Fas/CD95 resulting in apoptosis. A 54-year-old man presented with disseminated Mycobacterium xenopi infection. Analysis showed CD4 + T lymphopenia. Tests for human immunodeficiency virus (HIV) types 1 and 2 were negative. We compared the patient with eight healthy controls and five HIV-infected patients in terms of the expression of Fas/CD95 and Fas-mediated apoptosis of peripheral T lymphocytes. The percent of CD95 + cells in lymphocytes was 98% for the patient, and the mean percent of CD95 + cells in lymphocytes ± SD for HIV-infected patients and healthy controls was 75% ± 16% and 36% ± 26%, respectively. The patient had a high level of spontaneous apoptosis, and apoptotic cells were all identified as being CD4 + T cells. Monoclonal antibodies to CD95 dramatically increased apoptosis of CD4 + T cells exclusively. CD4 + T lymphopenia observed in our patient correlated with an overexpression of Fas together with spontaneous and Fas-induced apoptosis.

The effects of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) on intracellular Ca2+ mobilization and cyclic AMP accumulation were investigated using rat brain capillary endothelial cells which express an endogenous P2Y1 receptor, human platelets which are known to express a P2Y1 receptor, and Jurkat cells stably transfected with the human P2Y1 receptor. In endothelial cells, BzATP was a competitive inhibitor of 2-methylthio ADP (2-MeSADP) and ADP induced [Ca2+]i responses (Ki = 4.7 microM) and reversed the inhibition by ADP of adenylyl cyclase (Ki = 13 microM). In human platelets, BzATP inhibited ADP-induced aggregation (Ki = 5 microM), mobilization of intracellular Ca2+ stores (Ki = 6.3 microM), and inhibition of adenylyl cyclase. In P2Y1-Jurkat cells, BzATP inhibited ADP and 2-MeSADP-induced [Ca2+]i responses (Ki = 2.5 microM). It was concluded that BzATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation. In contrast to other P2Y1 receptor antagonists (A2P5P and A3P5P) which inhibit only ADP-induced Ca2+ mobilization, BzATP inhibits both the Ca2+- and the cAMP-dependent intracellular signaling pathways of ADP. These results provide further evidence that P2Y1 receptors contribute to platelet ADP responses.

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.

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.

Insulin-like growth factor-1 receptors (IGF-1R) and insulin receptors (IR) are closely related tyrosine kinases. However, the IR plays a major role in metabolism control, whereas the IGF-1R is mainly involved in growth and differentiation. With these observations in mind, we wished to define the regions of IR and IGF-1R responsible for generation of biological specificity. We constructed a chimeric IGF-1R in which the carboxyl-terminal domain was replaced by that of IR. This receptor (IGF/CTIR) was expressed in NIH3T3 cells, and we compared its biological activity with that of wild-type receptors. The IGF/CTIR was fully functional regarding kinase activity and biological properties. Comparison of insulin and IGF-1 effects on IR and IGF-1R cells, respectively, indicated that the IR is more efficient in stimulating glycogen synthesis and p44mapk activity than is the IGF-1R. Interestingly, in IGF/CTIR16 cells expressing only 250,000 receptors glycogen synthesis was better stimulated than in IGF-1R cells with 600,000 receptors. Similarly, p44mapk activation was slightly higher in IGF/CTIR16 cells than in IGF-1R cells. These results suggest that the carboxyl-terminal domain of IR is more tightly coupled to the stimulation of glycogen synthesis and to the p44mapk pathway than is that of IGF-1R. We propose that this domain plays a crucial role in the transmission of biological effects and could account, at least in part, for receptor specificity.

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.

The CD20 molecule is a phosphoprotein expressed on the surface of B lymphocytes that plays a role in the regulation of B cell proliferation and differentiation. In this study it was found that monoclonal antibodies (mAb) directed to CD20 decrease the expression of IgM at the surface of normal human B lymphocytes and B cell lines. This effect was time-dependent with a half-time of about 5 h. Incubation of B cells with CD20 mAb B1 did not affect the steady-state level of IgM mRNA, suggesting that it acts at a nontranscriptional stage. Phorbol esters also produced inhibitory effect on surface IgM expression. Staurosporine reversed both the phorbol ester- and the CD20-induced down-regulation. Genistein did not reverse the down-regulation induced by the CD20 mAb B1. CD20 most likely triggers a protein kinase C-dependent pathway to down-regulate sIgM. CD20 mAb also counteracted the interleukin-4 (IL-4)-induced up-regulation of sIgM. The ability of anti-IgM to mobilize intracellular calcium was reduced in sIgM down-regulated cells, suggesting that B cells activation through the antigen receptor may be negatively regulated by CD20 and positively by IL-4.

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.

Pertussis toxin (PT) has previously been shown to affect a wide variety of immune responses and to cause lymphocyte proliferation. We have investigated the biochemical basis for the mitogenic activity of PT by using human peripheral blood lymphocytes. PT was found to induce a rapid rise in cytosolic free calcium concentration and an alkalinization of the cytosol through the Na+/H+ antiporter. The toxin was also found to induce expression of IL-2-receptor on CD3+ cells and to stimulate IL-2 production. PT induced proliferation of both CD4+ and CD8+ T cells in the presence (but not in the absence) of accessory cells. PT also stimulated IL-1 production by monocytes but neither IL-1, IL-6 alone nor a combination of the two lymphokines could replace accessory cells suggesting that cell:cell contact is required. Low doses of PT induced ADP-ribosylation of G proteins but this treatment did not affect significantly PHA-induced [Ca2+]i increase and IL-2-induced DNA synthesis suggesting that the substrates of the ADP-ribosyltransferase activity of PT are not involved in the signalling pathways leading to DNA replication.

Triggering the CD3/TCR complex of lymphocytes induces a rapid rise in cytosolic free calcium followed by a slowly declining plateau. The level of this plateau depends on external pH, the more alkalinized media leading to higher values. Neither a pH-dependent binding of mAb, nor a pertubation of internal pH can account for this effect. In a sodium-free medium, or in the presence of dimethylamiloride Ca 2+ , elevation is accompanied by an acidification of the cells, both of them depend, to the same extent, on external calcium concentration. TPA inhibits CD3-, but not ionomycin-induced Ca 2+ and H + raises, indicating that it acts more probably on Ca 2+ influx, rather than on its efflux. These results suggest that intracellular calcium could be regulated by a Ca 2+ /H + ATPase which drives H + in and Ca 2+ out. In the presence of external Na + , H + should return to the medium by the Na + /H + exchanger.

International audience; Here, we report the isolation of a human multipotent adipose-derived stem (hMADS) cell population from adipose tissue of young donors. hMADS cells display normal karyotype; have active telomerase; proliferate >200 population doublings; and differentiate into adipocytes, osteoblasts, and myoblasts. Flow cytometry analysis indicates that hMADS cells are CD44+, CD49b+, CD105+, CD90+, CD13+, Stro-1(-), CD34-, CD15-, CD117-, Flk-1(-), gly-A(-), CD133-, HLA-DR(-), and HLA-I(low). Transplantation of hMADS cells into the mdx mouse, an animal model of Duchenne muscular dystrophy, results in substantial expression of human dystrophin in the injected tibialis anterior and the adjacent gastrocnemius muscle. Long-term engraftment of hMADS cells takes place in nonimmunocompromised animals. Based on the small amounts of an easily available tissue source, their strong capacity for expansion ex vivo, their multipotent differentiation, and their immune-privileged behavior, our results suggest that hMADS cells will be an important tool for muscle cell-mediated therapy.

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.

We have previously shown that laminin-5 is expressed in the human thymic medulla, in which mature thymocytes are located. We now report that laminin-5 promotes migration of mature medullary thymocytes, whereas it has no effect on cortical immature thymocytes. Migration was inhibited by blocking mAbs directed against laminin-5 integrin receptors and by inhibitors of metalloproteinases. Interactions of thymocytes with laminin-5 induced a strong up-regulation of active metalloproteinase-14. However, we found that thymocytes did not cleave the laminin-5 gamma(2) chain, suggesting that they do not use the same pathway as epithelial cells to migrate on laminin-5. Interactions of thymocytes with laminin-5 also induced the release of a soluble fragment of CD44 cell surface molecule. Moreover, CD44-rich supernatants induced thymocyte migration in contrast with supernatants depleted in CD44 by immunoadsorption. CD44 cleavage was recently reported to be due to metalloproteinase-14 activation and led to increased migration in cancer cells. Thus, in this study, we show that laminin-5 promotes human mature thymocyte migration in vitro via a multimolecular mechanism involving laminin-5 integrin receptors, metalloproteinase-14 and CD44. These data suggest that, in vivo, laminin-5 may function in the migration of mature thymocytes within the medulla and be part of the thymic emigration process.We have previously shown that laminin-5 is expressed in the human thymic medulla, in which mature thymocytes are located. We now report that laminin-5 promotes migration of mature medullary thymocytes, whereas it has no effect on cortical immature thymocytes. Migration was inhibited by blocking mAbs directed against laminin-5 integrin receptors and by inhibitors of metalloproteinases. Interactions of thymocytes with laminin-5 induced a strong up-regulation of active metalloproteinase-14. However, we found that thymocytes did not cleave the laminin-5 gamma(2) chain, suggesting that they do not use the same pathway as epithelial cells to migrate on laminin-5. Interactions of thymocytes with laminin-5 also induced the release of a soluble fragment of CD44 cell surface molecule. Moreover, CD44-rich supernatants induced thymocyte migration in contrast with supernatants depleted in CD44 by immunoadsorption. CD44 cleavage was recently reported to be due to metalloproteinase-14 activation and led to increased migration in cancer cells. Thus, in this study, we show that laminin-5 promotes human mature thymocyte migration in vitro via a multimolecular mechanism involving laminin-5 integrin receptors, metalloproteinase-14 and CD44. These data suggest that, in vivo, laminin-5 may function in the migration of mature thymocytes within the medulla and be part of the thymic emigration process.

Multiple failures of antiretroviral treatments, as a result of multidrug-resistant virus, have led to a proposal for structured therapeutic interruptions (STI). However, a significant decrease in CD4 + T cells may occur. The aim of our study was to determine the kinetics of T cell subpopulation changes, T cell apoptosis and peripheral blood mononuclear cell proliferation after STI. The impact of resistance mutation disappearance on T cell apoptosis was also studied. Ten patients were enrolled prospectively, and blood sampling was performed at weeks 0, 2, 4, 6, 8 and 12. The mean increase in viral load was 1.3 log 10 copies/mL, ranging from 0.1 to 3.2. CD4 + T cell count decreased to a mean of 80 cells/mm 3 from baseline to week 12. In the same period, CD8 + T cells decreased to a mean of 139 cells/mm 3 . A significant increase in both T cell apoptosis and proliferation of mononuclear cells was observed. However, proliferation was an early and brief event. The increase in CD4 + T cell apoptosis was obvious in patients exhibiting complete reversion of resistance mutations to antiviral drugs. Our results suggest that during STI, apoptosis is an overwhelming phenomenon compared with proliferation, and may explain the limited immunological impact of this therapeutic option.

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 p56(lck) 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-beta-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca(2+) concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts.

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.

Apoptosis is one of the mechanisms involved in the persistent CD4(+) T cell lymphopenia occurring in human immunodeficiency virus (HIV)-infected patients treated with highly active antiretroviral therapy (HAART). The aim of this study was to look at the relationship between the level of T cell apoptosis in patients on HAART and their HIV viral load measurement. Forty-five patients receiving HAART for a median time of 5 months were included: 13 had an undetectable viral load and 32 had a viral load > or = 200 copies/mL. No relationship was observed between the level of spontaneous T cell apoptosis and the viral load measurement. Patients with a viral load > or = 200 copies/mL exhibited a significantly higher level of CD4-induced apoptosis of CD4(+) T cells, compared with patients with undetectable viraemia (38 versus 15%, respectively, P = 0.03). Activation-induced apoptosis may be involved in the paucity of the immune reconstitution observed in HIV-infected patients treated with HAART.

Aspirin is a commonly used drug with a wide pharmacological spectrum including antiplatelet, anti-inflammatory, and neuroprotective actions. This study shows that aspirin and sodium salicylate, its major blood metabolite, reverse contractile actions of endothelin-1 (ET-1) in isolated rat aorta and human mammary arteries. They also prevent the intracellular Ca(2+) mobilizing action of ET-1 in cultured endothelial cells but not those of neuromedin B or UTP. Inhibition of the actions of ET-1 by salicylates is apparently competitive. Salicylates inhibit (125)I-ET-1 binding to recombinant rat ETA receptors. Salicylic acid promotes dissociation of (125)I-ET-1 ETA receptor complexes both in the absence and the presence of unlabeled ET-1. It has no influence on the rate of association of (125)I-ET-1 to ETA receptors. Salicylates do not promote dissociation of (125)I-ET-1 ETB receptor complexes. Salicylates potentiate relaxing actions of receptor antagonists such as bosentan. It is concluded that salicylates are allosteric inhibitors of ETA receptors. The results also suggest that: 1) irreversible ET-1 binding probably limits actions of receptor antagonists in vivo, and 2) an association of salicylates and ETA receptor antagonists should be used to evaluate the physiopathological role of ET-1 and may be of therapeutic interest in the treatment of ischemic heart disease.

Ligation of Fas with its natural ligand or with anti-Fas antibodies induces an apoptotic program in Fas sensitive cells. We report here the identification of the tyrosine kinase p59Fyn as a substrate for CPP32-like proteinases and more particularly caspase 3 during Fas-mediated apoptosis in Jurkat T cells. Inhibition of CPP32-like proteinases by Ac-Asp-Glu-Val-Asp-aldehyde but not by Ac-Tyr-Val-Ala-Asp-aldehyde prevents CPP32, PARP and p59Fyn cleavage indicating that CPP32 or CPP32-like proteinases are responsible for the cleavage of p59Fyn. Cleavage occurs in the N-terminal domain of p59Fyn between Asp19 and Gly20 and is accompanied by relocation of an active p57Fyn kinase to cytoplasm of Fas-stimulated Jurkat cells as judged by both biochemical and confocal microscopy experiments. Thus, p59Fyn relocation and activity may play an important role during Fas-mediated cell death in human T lymphocytes.

CD20 monoclonal antibody (mAb) B1 is known to inhibit B cell proliferation. We show that B1 reduced both anti-mu + interleukin-4 (IL-4)-induced DNA synthesis and the concomitant expression of CD23 at the surface of human tonsillar B cells. B1 mAb had no effect on CD23 mRNA levels. The disappearance of CD23 molecule from the surface correlates with an increase of soluble CD23 fragments in the culture medium, indicating that CD20 mAb B1 stimulated the cleavage of the molecule. B1 also inhibits IgE production by peripheral blood mononuclear cells cultured in the presence of IL-4. Suppression of IgE synthesis and enhancement of CD23 cleavage are concomitant but appear not to be functionally related.

Mitogenic pairs of CD2 mAb typically transduce activation/proliferation signals within T cells. However, complementary signal(s) provided by accessory cells are required to induce T cell proliferation. We show here that a particular combination of three CD2 mAb, D66 + GT2 + T11.1, leads to the proliferation of highly purified human T lymphocytes, without other complementary signal(s). The CD2 triplets was able to induce CD4+ and, to a lesser extent, CD8+ cells to proliferate. Interestingly, the so-called "naive" T cells (CD45RA+) were strongly stimulated, but more immature cells, such as thymocytes, were not. The proliferative response induced by the CD2 triplet was entirely mediated by the IL-2 autocrine pathway, as shown by the complete inhibition with anti-IL2 Ab. T cells stimulated with the CD2 triplet were also able to secrete TNF alpha. We found no evidence for an unusual secretion of cytokines that might explain the lack of requirement of complementary signal(s). As high as it was, the proliferation induced by the CD2 mAb triplet could be further increased by the addition of IL-1, and this proliferative fraction could be inhibited by antibodies against TNF alpha. The CD2 mAb triplet increased [Ca2+]i, while mitogenic CD2 mAb pairs needed the presence of a cross-linking agent. Thus, our data show that T cells can be activated to fully proliferate by this particular CD2 pathway, in the absence of accessory signal(s).

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 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.

While calcium ions are known to play a prominent role in signal transduction in the activation of T lymphocytes, its mechanism of action, target and function have not been elucidated. One crucial event in the calcium-dependent process is the activation of the CD3 complex, and this, too, is not understood. While studying the release of Ca2+ from intracellular stores by several monoclonal antibodies (mAb) against CD3, we found that one of them, mAb 141, was ineffective unless free Ca2+ was present in the external medium. By flow cytometric analysis of the binding of this mAb to Jurkat T cells and peripheral blood lymphocytes we showed that 141 does not recognize the CD3 complex when external Ca2+ is chelated by EGTA. The binding was restored by addition of Ca2+ but not Mg2+. Finally at least one subunit of the CD3 complex displayed a modified electrophoretic migration rate when immunoprecipitated by Leu-4 in the absence of external free Ca2+. These results suggest that the conformation of the CD3 complex depends on Ca2+, the epitope recognized by 141 being concealed at low Ca2+ concentration.

Different classes of protein kinase inhibitors for protein kinase C, cAMP-dependent protein kinase or protein tyrosine kinases have been studied for their effect on phospholipid metabolism. The results show that among the compounds studied, only 4′-aminohydroxyflavone (AHF), previously described as a specific inhibitor of the protein tyrosine kinase p56lck, markedly increased phosphatidylserine synthesis in Jurkat T cells. The biosyntheses of phosphatidylcholine and phosphatidylethanolamine were not affected. Also, the synthesis of phospholipids from tritium-labeled fatty acid as precursor was left unchanged by the p56lck inhibitor. The decreased phosphatidylserine synthesis induced when triggering the CD3-TCR complex was impaired by AHF, suggesting that p56lck could be implicated in the regulation of the serine-base exchange enzyme system. Direct evidence for the participation of p56lck in the regulation of the serine-base exchange enzyme system was obtained by using p56lck-deficient Jurkat cells (J.CaM 1.6) in which the basal base exchange activity was markedly increased and on the other hand AHF had no effect. In addition, transfection of J.Cam 1.6 cells with p56lck-cDNA allowed recovery of the AHF activity. © 1997 Federation of European Biochemical Societies.

The adapter 3BP2 is involved in leukocyte signaling downstream Src/Syk-kinases coupled immunoreceptors. Here, we show that 3BP2 directly interacts with the endocytic scaffold protein CIN85 and the actin-binding protein HIP-55. 3BP2 co-localized with CIN85 and HIP-55 in T cell rafts and at the T cell/APC synapse, an active zone of receptors and proteins recycling. A binding region of CIN85 SH3 domains on 3BP2 was mapped to a PVPTPR motif in the first proline-rich region of 3BP2, whereas the C-terminal SH3 domain of HIP-55 bound a more distal proline-rich domain of 3BP2. Together, our data suggest an unexpected role of 3BP2 in endocytic and cytoskeletal regulation through its interaction with CIN85 and HIP-55.

Pretreatment of T lymphocytes with anti-CD3 or anti-TCR mAb results in the disappearance of the T cell receptor complex (TCR/CD3) from the cell surface. The mechanisms of this down-regulation have not been fully elucidated. We demonstrate here that the modulation of the CD3/TCR complex can be completely inhibited by hypertonic medium, a condition known to block receptor-mediated endocytosis. Consequently, the sequestration of the CD3/TCR complex associated to relevant mAb in acid vesicles did not occur under hypertonicity condition. Moreover, monensin, which is known to interfere with receptor recycling, was found to amplify the CD3/TCR modulation induced by specific mAb and decrease the uptake of 125I-labeled anti-CD3/TCR mAb by T cells. We therefore propose that mAb-CD3/TCR complexes are internalized via coated pits by a receptor-mediated endocytosis and then transported to acid compartments. MAb are degraded in lysosomes during this process, whereas CD3/TCR molecules recycle back to cell surface.

Although it is well known that the CD3/T-cell receptor (TCR) complex modulates from the surface of T cells upon exposure to monoclonal antibodies (mAb) directed against it, the fate of bound mAb has not been yet elucidated. We therefore perform direct binding experiments of 125I-labeled mAb against CD3 or TCR to investigate their fate in Jurkat T cells. We demonstrated that all mAb were progressively internalized and degraded in Jurkat T cells and that this degradation was inhibited by chloroquine, an inhibitor of lysosomal degradation enzymes. The sequestration of anti-CD3 mAb in acid compartments was furthermore shown using cytofluorometry. All together our results show that antibodies against CD3 or against TCR follow the same endocytic pathway.