Biomedical subjects

C Aussel

Publications and source records attributed to C Aussel.

At least 55 records · Page 3Linked to original sources

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↗

PubMed1994

Regulation of T cell activation by cytochrome P450 inhibitors.

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.

Biological Transport↗

PubMed1994

Effect of thimerosal on cytosolic calcium and phosphatidylserine synthesis in Jurkat T cells.

1. We investigated the effect of the thiol reagent, thimerosal on calcium movements in the Jurkat T cell line. 2. Thimerosal induced a rise in cytosolic Ca2+ concentration due both to a release of Ca2+ from intracellular stores and a Ca2+ influx. 3. Thimerosal, released Ca2+ from the same intracellular stores than CD3 mAb and ionomycin. 4. Emptying the Ca2+ 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.

Calcium↗

PubMed1993

The inhibition by fatty acids of receptor-mediated calcium movements in Jurkat T-cells is due to increased calcium extrusion.

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.

Antibodies, Monoclonal↗

PubMed1993

Monoclonal antibodies directed against the E2 protein (MIC2 gene product) induce exposure of phosphatidylserine at the thymocyte cell surface.

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.

12E7 Antigen↗

PubMed1993

Suppressive effect of T cell proliferation via the CD29 molecule. The CD29 mAb 1 "K20" decreases diacylglycerol and phosphatidic acid levels in activated T cells.

We had previously reported that the CD29 mAb "K20," presented in a soluble form, blocks peripheral T cell proliferation/activation induced by a CD3 mAb. To better characterize the negative signal delivered by soluble K20, we have investigated its effects on the phospholipid metabolism, both in Jurkat and CD4+ T cells. In CD3-activated T cells, K20 inhibited the increase of diacylglycerol (DAG) and phosphatidic acid levels, but did not modify phosphatidylinositol 4,5-bisphosphate levels, cytosolic Ca2+ raise, and inositolphosphates formation, indicating that K20 did not inhibit phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C-gamma. Moreover, in these conditions, K20 increased phosphatidylethanolamine levels, without variation of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, suggesting that K20 specifically increased the phosphatidylethanolamine biosynthesis from DAG. Thus, the effects of K20 on DAG and phosphatidic acid levels resulted from an accelerated catabolism rather than from a defect of synthesis. That K20 acts solely at an early step of T cell activation, namely before the binding of IL-2 to its receptor, is supported by the observation that adding exogenous rIL-2 increased proliferation in spite of K20. These results suggest that the beta 1 integrin molecules interact with the membrane phospholipid metabolism and they appear to be the hallmark of a peculiar negative pathway of T cell activation, likely to play an important regulatory role mediated via the T cell integrin molecules.

Antigens, CD↗

PubMed1993

Imidazole antimycotics inhibitors of cytochrome P450 increase phosphatidylserine synthesis similarly to K(+)-channel blockers in Jurkat T cells.

The imidazole antimycotics, miconazole, econazole and triclomazole as well as alpha-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 Ca(2+)-influx and membrane potential.

Antifungal Agents↗

PubMed1993

Ca(2+)-ATPase inhibitors induce interleukin-2 synthesis and T cell proliferation.

In Jurkat cells, the three Ca(2+)-ATPase blockers, thapsigargin, cyclopiazonic acid, and di-tert-butylhydroquinone (DtBuHQ) induced both a release of Ca2+ from intracellular stores and a Ca2+ influx. In contrast to CD3 mAb, the Ca(2+)-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 Ca(2+)-ATPase inhibitors induced Jurkat cells to synthesize large amounts of interleukin-2 demonstrating that early signal transduction mechanisms can be bypassed by Ca(2+)-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.

Calcium↗

PubMed1993

CD3 monoclonal antibodies evoke the same cytochrome P450-regulated capacitative entry of calcium as thapsigargin in Jurkat T cells.

In T cells CD3 monoclonal antibodies mediate an elevation of cytosolic Ca2+ concentration due to a release from internal stores and also due to an entry from extracellular medium, the mechanism of which is not clearly elucidated. Previous studies on several cell types have reported that depleting intracellular Ca2+ stores with inhibitors of the reticulum Ca(2+)-ATPase resulted in an increased plasma membrane permeability to calcium ions. It has been suggested that emptying the reticulum triggers a Ca2+ influx from extracellular medium, independent of phosphoinositide hydrolysis. To document the physiological relevance of such a mechanism, we compared CD3- and thapsigargin-induced sustained increase of cytosolic Ca2+ concentration in Jurkat T cells with regard to their sensitivity to internal and external Ca2+ level and to several inhibitors which do not affect the release of internal stores. We show that (1) there was no additivity of the two effects; (2) both CD3- and thapsigargin-evoked Ca2+ influx were inhibited when membrane was depolarized by either gramicidin or a high potassium concentration; and (3) Ca2+ influx was abrogated by cytochrome P450 inhibitors such as lipoxygenase inhibitors or imidazole antimicotic drugs. CD3 mAb and thapsigargin thus triggered the same signaling events, probably involving a cytochrome P450, to transmit information from depleted endoplasmic reticulum to the plasma membrane.

Calcium↗

PubMed1993

Effect of Cinchona bark alkaloids and chloroquine on phospholipid synthesis. K+ channel blockers specifically enhance the activity of the serine base exchange enzyme system in Jurkat T cells.

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.

Cell Line↗

PubMed1993

Regulation of phospholipid metabolism by K+ channel blockers and inhibitors of choline transport in the Jurkat T cell line. Relationships with cell proliferation and interleukin-2 production.

In the human T cell line Jurkat, three drugs generally used as effectors of K+ channels, i.e., quinine, 4-aminopyridine and tetraethylammonium, modify phospholipid metabolism. The drugs inhibited the synthesis of both phosphatidylcholine and phosphatidylethanolamine. The mechanism of such inhibition involves a decreased uptake of choline and ethanolamine by the cells, since the three K+ channel blockers were found to be able to competitively inhibit the high-affinity choline/ethanolamine transport system at the membrane level. In contrast, choline transport-inhibitors such as hemicholinium-3, decamethonium and dodecyltrimethylammonium do not inhibit interleukin-2 synthesis and proliferation of the Jurkat T cell line. This indicates that the inhibition of either phosphatidylcholine and/or phosphatidylethanolamine synthesis is not directly implicated in these processes. The inhibition of interleukin-2 synthesis appeared to be mediated through the inhibition of diacylglycerol production induced by T cell activators. A major role for phosphatidylserine in the regulation of T cell activation emerged, since we demonstrated that a panel of K+ channel blockers enhanced the synthesis of this phospholipid mimicking the previously described effect of exogenously added phosphatidylserine in Jurkat cells, i.e., a blockade of interleukin-2 synthesis probably due to a defect in diacylglycerol production.

4-Aminopyridine↗

PubMed1992

Agonist-induced inhibition of phosphatidylserine synthesis is secondary to the emptying of intracellular Ca2+ stores in Jurkat T-cells.

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.

Antibodies, Monoclonal↗

PubMed1992

Effects of antiarrhythmic drugs on phospholipid metabolism in Jurkat T cells. The potassium channel blocker, clofilium, specifically increases phosphatidylserine synthesis.

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.

Acecainide↗

PubMed1992

IL-1 beta, a strong mediator for glucose uptake by rheumatoid and non-rheumatoid cultured human synoviocytes.

Higher basal 2-deoxy-D-glucose uptake in rheumatoid synovial cells than in non-rheumatoid synovial cells, was found to be associated with an increased interleukin-1 beta (IL-1 beta) secretion (respectively 850 +/- 238 vs. 8.3 +/- 2.4 pg/24 h/10(5) cells, mean +/- S.E.M.). When exogenous human recombinant IL-1 beta was added to cultures, a marked stimulation of 2-deoxy-D-glucose uptake was performed by both human synovial cultured cells, in a time-dependent and dose-dependent manner (IL-1 beta 0-100 ng/ml). In non-rheumatoid synoviocytes, stimulation occurred 1-3 h following the addition of 1 ng/ml interleukin-1 beta and increased up to 24 hours (respectively +150% and +261.4% after 6 and 24 hours association time). Rheumatoid synovial cells were less sensitive to 1 ng/ml IL-1 beta (respectively +80% and +146.4%). IL-1 beta increased significantly the Vmax for 2-deoxy-D-glucose uptake by synovial cells, with no change in the Km. This effect was protein synthesis-dependent, and not secondary to prostaglandin E2 synthesis or cell growth. IL-1 beta possesses an important effect on glucose homeostasis in synovial cells, which could be indirect and/or regulated by the presence of natural inhibitors.

Arthritis, Rheumatoid↗