Biomedical subjects

C Aussel

Publications and source records attributed to C Aussel.

At least 19 recordsLinked to original sources

PubMed1997

The protein tyrosine kinase p56(lck) regulates the serine-base exchange activity in Jurkat T cells.

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 p56(lck), 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 p56(lck) inhibitor. The decreased phosphatidylserine synthesis induced when triggering the CD3-TCR complex was impaired by AHF, suggesting that p56(lck) could be implicated in the regulation of the serine-base exchange enzyme system. Direct evidence for the participation of p56(lck) in the regulation of the serine-base exchange enzyme system was obtained by using p56(lck)-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 p56(lck)-cDNA allowed recovery of the AHF activity.

CD3 Complex↗

PubMed1997

Regulation of phospholipid biosynthesis by Ca(2+)-calmodulin-dependent protein kinase inhibitors.

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 Ca2+ in the incubation medium and was impaired in cells whose Ca2+ 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 Ca2+ 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.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

Action of anti-inflammatory drugs on interleukin-1 beta-mediated glucose uptake by synoviocytes.

Synovial cell cultures prepared from samples taken from osteoarthritic and rheumatoid patients were treated with different anti-inflammatory agents (cortisol, indomethacin, ibuprofen and piroxicam) to determine their 'anti-interleukin-1 beta' action, using inhibition of interleukin-1 beta-mediated glucose uptake stimulation as a biological test. Confluent cells were treated for 24 h with different concentrations of these drugs (10(-5), 10(-6) and 10(-7) mol/l) to study their effect on the inflammation process. 6 h before glucose uptake studies, interleukin-1 beta (1 ng/ml) was added. Whereas non-steroid anti-inflammatory agents were inefficient, cortisol inhibited the action of interleukin-1 beta on glucose uptake. In osteoarthritic cells, cortisol, 10(-5) mol/l, reduced interleukin-1 beta-mediated glucose uptake by 27% after a 24-h incubation. In rheumatoid cells, stimulated 2-deoxy-D-glucose uptake was reduced by 40.6%. Results were similar when interleukin-1 beta and cortisol were added simultaneously, 6 h before glucose uptake was measured. This rapid effect of cortisol was protein synthesis-dependent (inhibited by cycloheximide). Cortisol decreased glucose uptake by synoviocytes by acting on basal and interleukin-1 beta-mediated glucose uptake. This effect was more pronounced in rheumatoid synovial cells. The inhibition of interleukin-1 beta-mediated glucose uptake could be proposed as a new model for studying the anti-interleukin-1 beta effects of anti-rheumatic drugs.

Anti-Inflammatory Agents↗

PubMed1996

Tyrphostin A9 inhibits calcium release-dependent phosphorylations and calcium entry via calcium release-activated channel in Jurkat T cells.

The mechanism by which calcium-depleted intracellular stores may trigger an external calcium influx through a calcium release-activated channel was investigated by analyzing the effects of several protein tyrosine kinase inhibitors on calcium movements in Jurkat T cells. Tyrphostin A9, an inhibitor of the kinase activity of the platelet-derived growth factor (PDGF) receptor, dramatically impaired the sustained elevation of cytosolic calcium concentration, induced by either CD3 mAbs, thapsigargin, ionomycin at low (10(-7) M) concentration, or passive depletion of intracellular stores; other tested tyrphostins, lavendustin, genistein, and compound 5 lacked significant effect. Tyrphostin A9, added during the plateau phase, was able to return cytosolic calcium to resting concentration. Likewise, it abrogated manganese entry in cells stimulated by CD3 or thapsigargin, measured by the quenching of the fluorescence of Indo-1. However, it did not measurably modify kinetics of intracellular calcium releases monitored in the absence of extracellular calcium, nor did it reverse the inhibition of phosphatidylserine that occurs as a consequence of emptying intracellular stores. Analyses of tyrosine phosphorylations demonstrated that A9 inhibited the phosphorylation of proteins, which occurred every time that internal calcium stores were depleted. These phosphorylations were not impaired by chelation of external Ca2+, nor by La3+ that inhibits calcium release-induced calcium entry. We concluded that their inhibition was not a consequence, but may be a cause, of the blockade of calcium release-activated channel by tyrphostin A9.

Calcium↗

PubMed1996

Submicromolar La3+ concentrations block the calcium release-activated channel, and impair CD69 and CD25 expression in CD3- or thapsigargin-activated Jurkat 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+.

Antibodies, Monoclonal↗

PubMed1996

Dependence of adaptative regulation for IL-1 beta action on system A activity in human synovial cells.

Human synovial cells are a suitable model for estimating the physiopathological effects of IL-1 beta (IL-1) in joint. Given the importance of this cytokine in the modulation of cell metabolic activities, we set out to study the action of IL-1 on the neutral amino acid transport A system, using the methyl (aminoisobutyric) acid (MeAIB), the most highly specific and nonmetabolizable substrate for the A system. Stimulation of system A activity by adaptative regulation is a prerequisite to obtain an increase of MeAIB uptake in IL-1-treated cells, since cells which had been grown in a normal medium did not express stimulation of system A activity when IL-1 was added. The IL-1-mediated MeAIB uptake is independent of protein synthesis, since cycloheximide (CHX) did not inhibit MeAIB uptake, and characterized by a decrease in the Michaelis constant K(m) (0.147 vs. 0.270 mmol/l, IL-1 vs. control) and a slight increase in maximal velocity (Vmax) (4.59 vs. 3.89 nmol/mg prot/10 min, IL-1 vs. control). These observations indicate that IL-1 induces modifications in both system A transporter affinity and number. Moreover, we indicate that system A should be responsive in vivo to IL-1 in the same way since derepression and IL-1 action occurred in the presence of human synovial fluid.

Amino Acids↗

PubMed1996

alpha-Ketoglutarate uptake in human fibroblasts.

Glutamine requirements are increased during injury, in particular to sustain the needs of rapidly growing cells. This includes fibroblasts involved in wound healing. alpha-Ketoglutarate (alpha-KG) has been proved to be a potent precursor of glutamine. However, little is known about the process of its cell uptake. Since this first step could be crucial in alpha-KG metabolism, we have characterized alpha-ketoglutarate uptake in fibroblasts. Total uptake of alpha-ketoglutarate was linear up to 1 mmol and temperature independent. Rate of uptake was independent of the presence of Na+ in the medium. Competition studies with another ketoacid demonstrated the nonspecificity of alpha-ketoglutarate uptake. In addition, 4-hydroxy-alpha-cyanocinnamate, a known inhibitor of anion transport, was ineffective on alpha-ketoglutarate uptake. Taken as a whole, these data provide evidence that alpha-ketoglutarate uptake in fibroblast occurs by an unmediated diffusion process. This suggests that alpha-ketoglutarate uptake is not the controlling step in fibroblasts, i.e. only the availability of extracellular alpha-ketoglutarate. This could be an advantage since during injury, cell membrane depolarization and dissipation of Na+ gradient may limit cellular glutamine uptake.

Amniotic Fluid↗

PubMed1996

Effect of membrane potential on phosphatidylserine synthesis and calcium movements in control and CD3-activated Jurkat T cells.

CD3 mAb induced calcium movements are unaffected by hyperpolarization of the membrane in Jurkat T cells treated with valinomycin. By contrast, the CD3 induced Ca2+ influx was impaired by depolarization of the membrane with either gramicidin or by equimolar substitution of KCl for NaCl in the medium. In depolarized cells, the synthesis of phosphatidylserine was strongly diminished as a result of impaired transport of the [3H]serine substrate. In depolarized cells, the CD3-induced release of Ca2+ from intracellular stores (endoplasmic reticulum) was unaffected. Emptying of the Ca2+ stores by CD3 was shown by the lack of effect of additional treatment of the cells with the Ca2+ ionophore, ionomycin. The empty status of the calcium stores was also confirmed by measurements of phosphatyidylserine synthesis through the Ca2+ -dependent base exchange enzyme system that was found to be significantly decreased despite the low amount synthesized in the presence of a defective [3H]serine transport in depolarized cells.

Antibodies, Monoclonal↗

PubMed1995

Calmodulin, a junction between two independent immunosuppressive pathways in Jurkat T cells.

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.

CD3 Complex↗

PubMed1995

The E2 molecule (CD99) specifically triggers homotypic aggregation of CD4+ CD8+ thymocytes.

We have previously described E2 as a 32-kDa transmembrane glycoprotein displaying an isomorphism, as two epitopes (defined by mAbs O662 and L129) are widely distributed on T cells whereas two epitopes are restricted to T cell subsets (defined by mAbs D44 and 12E7). E2, the MIC-2 gene product, is involved in T cell adhesion because anti-E2 mAbs against pan T epitopes block spontaneous T cell rosettes. Pan T E2 mAbs are also able to induce exposure of the phosphatidylserine at the thymocyte surface but not at the surface of mature T lymphocytes, an event most likely linked to adhesion phenomena. We now show here that the anti-E2 mAbs (0662 and L129) that block rosettes and induce phosphatidylserine exposure at the thymocyte surface, and not those reacting with epitopes not involved in adhesion, also trigger aggregation of certain immature T cell lines and no other cell lines tested. Among the normal cells tested, anti-E2 mAbs exclusively induce homotypic aggregation of CD4+ CD8+ human thymocytes. This phenomenon is temperature, energy, and Mg++ dependent, and requires an intact cytoskeleton. These adhesion properties are rather characteristic of integrins. Nevertheless, mAb against beta 1, beta 2, and beta 3 integrin chains, as well as those against alpha-chains known to be present on thymocytes, are unable to block corticothymocyte aggregation. We conclude that E2 triggers on corticothymocytes and no other T cells a homotypic adhesion pathway most likely mediated by an uncharacterized integrin.

12E7 Antigen↗

PubMed1995

No evidence for a tumor necrosis factor alpha stimulated 2-methylaminoisobutyric acid uptake in hepatocyte monolayer.

This study investigates the short-term effects of glucagon and human recombinant tumor necrosis factor alpha (TNF alpha) singly and in association on 2-methylaminoisobutyric acid (MeAIB) transport in hepatocyte monolayers. As expected, glucagon induced a time-dependent stimulation of MeAIB transport. In our experimental conditions, TNF alpha did not induce cytolysis. A 2 hour exposure to TNF alpha (0.05-500 ng/l) with or without glucagon (10(-9) to 10(-6) M) did not modify the basal or glucagon-stimulated MeAIB transport. Varying the duration of exposure to TNF alpha 5 ng/l up to 6 h was equally ineffective. The presence of hydrocortisone potentiated the glucagon-stimulated transport, but TNF alpha remained ineffective. Finally, the association of interferon (IFN gamma) with TNF alpha and/or glucagon was unable to modify the transport activity. These data demonstrate that TNF alpha does not exert a direct effect on MeAIB transport in hepatocytes, at least on a short-term basis.

Animals↗

PubMed1995

Sphingosine, oleylamine and stearylamine inhibit both CD11a/CD18-dependent and -independent homotypic aggregation: demonstration by cytofluorimetry.

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.

12E7 Antigen↗

PubMed1995

Oleylamine and stearylamine increase phosphatidylserine synthesis in T cells by synergy with calcium ions.

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 [3H]-serine into phosphatidylserine is a protein kinase C independent process and is due to a synergy of either oleyamine or stearylamine with calcium ions.

Amines↗

PubMed1994

Engagement of the CD45 molecule induces homotypic adhesion of human thymocytes through a LFA-1/ICAM-3-dependent pathway.

Cell-cell interactions play a central role during differentiation and development of the immune system. T or B lymphocyte homotypic adhesions can be induced via several surface molecules which, in some cases, are known to trigger the LFA-1/ICAM-1 adhesion pathway. We show here that mAbs reacting with the CD45 common epitopes or restricted RO epitope lead to a strong and rapid aggregation of all human thymocytes, and of T cell lines with an immature phenotype, but not of peripheral T lymphocytes. Aggregation requires energy, a physiologic temperature, Mg2+ divalent cations, and an intact cytoskeleton. It is LFA-1 dependent because CD11a and CD18 mAbs inhibit homotypic aggregation, whereas CD11b and CD11c mAbs do not. Homotypic thymocyte adhesion, however, is not decreased by CD54 mAb (anti-ICAM-1) but is inhibited by CDw50 mAb (anti-ICAM-3). Soluble CD22 fails to induce thymocyte adhesion, suggesting that CD45-induced aggregation is triggered by another ligand. Finally, because inhibitions observed with mAbs against LFA-1 and ICAM-3 are only partial, it can be assumed that another adhesion pathway is involved in thymocyte adhesion. The adhesion event specific for thymocytes we describe here is likely to play an important role in T cell differentiation.

Antibodies, Monoclonal↗

PubMed1994

Gas chromatography applied to the lactulose-mannitol intestinal permeability test.

Intestinal permeability can be modified by various illnesses, trauma and sepsis. Alterations of the intestinal wall can facilitate the diffusion of potentially harmful substances such as endotoxins, as well as bacterial translocation. We describe the validation of a capillary gas chromatographic method for the determination of mannitol and lactulose, used as intestinal permeability probes. The method is linear up to 3 g/l for mannitol and 300 mg/l for lactulose; recovery from overload samples is between 92 to 110%. Intra-assay coefficients of variation (C.V.s) were 2.7 and 6.8% for mannitol and lactulose, respectively, and inter-assay C.V.s were 8.9 and 9.3%. Normal values for 25 healthy subjects (mean +/- S.D.) were 14.5 +/- 3.1% and 0.27 +/- 0.15% for mannitol and lactulose, respectively. The GC method presented is rapid and precise.

Adult↗

PubMed1994

Regulation by sphingomyelinase and sphingosine of Ca2+ signals elicited by CD3 monoclonal antibody, thapsigargin, or ionomycin in the Jurkat T cell line.

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.

Antibodies, Monoclonal↗

PubMed1994

Phenylarsine oxide and phorbol myristate acetate inhibit the CD3-induced rise of cytosolic Ca2+ in Jurkat cells by refilling internal Ca2+ stores.

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+.

Arsenicals↗

PubMed1994

Tumor necrosis factor alpha, a cytokine coregulating sugar uptake by cultured human synovial cells.

Confluent cultures of osteoarthritic and rheumatoid human synovial cells were treated with human recombinant tumor necrosis factor alpha (TNF-alpha). The cytokine increased uptake of 2-deoxy-D-[1-3H]-glucose (2-DOG) in a time- and concentration-dependent manner. In synovial cells obtained from osteoarthritic patients (OA cells), the stimulation of 2-DOG uptake occurred 3 hours following addition of TNF-alpha (1 ng/ml) and was maximal by 24 hours. Rheumatoid synovial cells (RA cells) appeared less sensitive to the cytokine: 2-DOG uptake stimulation was only significant after 6 hours of incubation. In both OA and RA cells, the effect was protein synthesis-dependent, and was not secondary to prostaglandin E2 synthesis or cell growth. Interleukin-1 beta was more efficient than TNF-alpha for 2-DOG uptake stimulation. The two cytokines seemed to act in an additive manner.

Arthritis, Rheumatoid↗