©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calmodulin, a Junction between Two Independent Immunosuppressive Pathways in Jurkat T Cells (*)

(Received for publication, November 17, 1994; and in revised form, January 24, 1995)

Claude Aussel (§) Jean-Philippe Breittmayer Claudette Pelassy Alain Bernard

From the Interaction Cellulaires et Moléculaires en Immunologie, INSERM U 343, Hôpital de l'Archet, BP.79, 06202 Nice cédex 03, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 Ca 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 Ca 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 Ca 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.


INTRODUCTION

T cell activation via the T cell receptor complex involves a transduction pathway that begins with the phosphorylation on tyrosine of phospholipase C(1)(1, 2, 3, 4, 5) . This phospholipase hydrolyzes phosphatidylinositol bisphosphate into diacylglycerol and inositol trisphosphate. This second messenger is responsible for the release of Ca from intracellular stores(6, 7) . Emptying of the Ca stores is followed by a decreased synthesis of phosphatidylserine (PtdSer)(^1)(8, 9, 10) , since the Ca-dependent base exchange enzyme system responsible for the synthesis of this phospholipid is mainly located in the endoplasmic reticulum (the major Ca store) in T cells as in other cell types(11, 12) . The release of Ca from the endoplasmic reticulum is also followed by an influx of Ca through the plasma membrane(13) . The changes in the Ca concentration both in the endoplasmic reticulum and in the cytoplasm occurring during T cell activation modify the activity of calmodulin. Calmodulin, a Ca-binding protein, located both in the cytosol and the particulate fraction of mammalian cells, interacts with several proteins including calcineurin (14, 15, 16) and the base exchange enzyme system(17, 18) . Cyclosporin A (CsA) inhibits T cell activation through an interaction with cyclophilin (19, 20) that in turn interacts with calcineurin, a phosphatase that is regulated by calmodulin. In previous reports, we have shown that a number of inhibitors of interleukin-2 synthesis such as K channel blockers (21, 22, 23) , cytochrome P-450 inhibitors(24) , and diacylglycerol kinase inhibitors (25) reversed the CD3-induced inhibition of PtdSer synthesis. Here we have examined the effects of calmodulin inhibitors on IL-2 production and PtdSer synthesis, and we have found that chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, HCl (W7) inhibited IL-2 production and increased PtdSer synthesis. By contrast, the CsA-inhibiting pathway has been found to be independent of changes in PtdSer synthesis. The CaMbulletCa complex, through its interaction with the base exchange enzyme system, thus appears as a target for some immunosuppressive drugs.


MATERIALS AND METHODS

Cells

The human T cell line Jurkat was kindly supplied by Dr. A. M. Schmitt-Verhulst (Centre d'Immunologie, Marseille-Luminy, France). Cells were cloned by limiting dilution. Clone D was selected on the basis of its IL-2 production when activated with phytohemagglutinin and the phorbol ester PMA. Cells were cultured in RPMI 1640 medium (Seromed, Lille, France) supplemented with 5% fetal calf serum, 50 units/ml penicillin, 50 µg/ml streptomycin, 2 mML-glutamine, 1 mM pyruvate.

Chemicals

The Ca ionophore (ionomycin), the blocker of Ca-ATPase (thapsigargin), oleylamine, and stearylamine were purchased from Sigma. The calmodulin antagonists trifluoperazine, chlorpromazine, and W7 were from Calbiochem. [^3H]Serine (0.37-1.1 TBq/mmol), [methyl-^3H]choline (2.8-3.1 TBq/mmol), [1-^3H]ethan-1-ol-2-amine hydrochloride (0.18-1.1 TBq/mmol), and myo-[2-^3H]inositol (370-740 GBq/mmol) were purchased from Amersham Corp. Cyclosporin A was a gift from Sandoz.

Phospholipid Synthesis in Jurkat Cells

Jurkat cells (2 times 10^6) were maintained in 0.5 ml of a buffer (pH 7.4) containing 137 mM NaCl, 2.7 mM KCl, 2.5 mM glucose, 20 mM Hepes, 1 mM MgCl(2), and CaCl(2) (as indicated in the figure legends) at 37 °C in the presence of 4 µCi/ml of [^3H]serine, [^3H]choline, [^3H]ethanolamine, or [^3H]inositol and effectors (see concentrations in the figure legends). After an incubation period varying from 0 to 2 h the cells were rapidly sedimented in an Eppendorf centrifuge, the supernatants were discarded, and the cell phospholipids were extracted using chloroform/methanol according to Bligh and Dyer(26) . This two-step extraction procedure allowed the determination of total ^3H-labeled products incorporated into the cells by measuring a 25-µl sample of the chloroform/methanol extract. Then the addition of chloroform and water allowed the separation of the organic and aqueous phases. The lipid extracts (organic phases) were analyzed by thin layer chromatography in a solvent system composed of chloroform/methanol/acetic acid/water (75:45:12:3). Authentic phospholipid standards (Sigma) were run in parallel and detected with iodide vapors. Radioactivity in lipid spots was determined by using an automatic linear radiochromatography analyzer (Berthold).

Phosphatidylserine Synthesis in an Acellular System

Jurkat cells (2.5 times 10^7) were centrifuged at 1000 rpm for 5 min and then washed with phosphate-buffered saline and frozen at -20 °C. After thawing in 2 ml of the buffer described above, the cells were sonicated two times for 2 min. One hundred µl of homogenate were incubated with 0.5 µCi of [^3H]serine in the presence or absence of effectors for 1 h at 37 °C. CaM inhibitors were added at time 0. Phospholipids were extracted and analyzed as described above.

Cell Activation

Cell activation was performed as described previously(27) . Briefly, Jurkat cells were incubated for a 24-h period in 24-well plates (Nunc, Roskilde, Denmark) in the presence of 10 ng/ml PMA and 2 µg/ml CD3 mAb, IOT3 (Immunotech, Marseille, France). After the incubation period, cells and their medium were transferred into centrifuge tubes. After centrifugation (300 times g for 5 min) the medium was collected and assayed for IL-2. The cells were submitted to trypan blue exclusion assay in order to verify their viability in the presence of the different drugs.

IL-2 Assay

The method of enzyme immunoassay was that previously described(28) . Briefly, the lymphokine to be assayed was first extracted from supernatants of Jurkat cells by anti-recombinant IL-2 rabbit IgG insolubilized onto polystyrene microtiter plates and revealed by an anti-IL-2 Fab` fragment conjugated to peroxidase. Peroxidase activity was assayed by using 0.1 M phosphate citrate buffer (pH 5.5), hydrogen peroxide (0.02%), and orthophenylene diamine (3 mg/ml) (final pH, 5.0). The standard used was recombinant IL-2.

Measurements of Changes in Cytosolic Ca, [Ca](i)

The assay of cytosolic Ca was performed by using Indo-1 (Calbiochem)(29) . Cells (5 times 10^6 cells/ml) were loaded by using 5 µM Indo-1 at 37 °C in the dark for 1 h and then washed and resuspended in albumin-free medium. The analyses were performed on a fluorescence-activated cell sorter (FACStar Plus, Becton Dickinson). The fluorescence intensity at 480 nm corresponding to the free Indo-1 concentration as well as the fluorescence at 400 nm corresponding to the complex Ca-Indo-1 was measured. Calculation of the ratio of fluorescence at 400 nm to fluorescence at 480 nm allows the evaluation of changes in cytosolic free Ca concentration independently of the cell size and the intracellular Indo-1 concentration.


RESULTS

CaM Antagonists Inhibit IL-2 Synthesis by Activated Jurkat Cells

Jurkat T cell activation by a combination of CD3 mAb and the phorbol ester PMA results in IL-2 synthesis. CaM inhibitors TFP, CPZ, and W7 each induced a marked, concentration-dependent decrease of T cell activation as demonstrated by measuring IL-2 synthesis in the presence of these drugs ( Fig. 1and Table 1). Since it is acknowledged that CaM is activated by Ca and since T cell activation with CD3 mAb results in an increase of the cytosolic free Ca concentration, we have tested whether CaM antagonists modify either Ca movements or a well known Ca-dependent event such as PtdSer synthesis.


Figure 1: Effect of W7 concentration on phosphatidylserine synthesis (left panel) and interleukin-2 production (right panel) in Jurkat T cells activated with CD3 monoclonal antibody (2 µg/ml) and the phorbol ester PMA (10 ng/ml). For IL-2 synthesis measurements, cells were incubated for 24 h as described under ``Materials and Methods.'' For phosphatidylserine synthesis, cells were incubated with the activators, various concentrations of W7, and [^3H]serine at time 0. Then after 2 h at 37 °C, phospholipids were extracted, separated by thin layer chromatography (tlc), and quantified with a tlc analyzer.





CaM Antagonists Increase PtdSer Synthesis in Jurkat Cells

The incorporation of [^3H]serine into PtdSer was measured in nonactivated Jurkat cells in the presence of different concentrations of W7, TFP, or CPZ. A typical concentration-dependent response curve obtained with W7 is depicted in Fig. 1. A similar increase in PtdSer synthesis (not shown) was observed with the three other CaM antagonists. The EC calculated from the dose-response curves are depicted in Table 1. The kinetics of the increase in PtdSer synthesis induced by W7 (Fig. 2) indicated that this drug acts very rapidly (within a few minutes). Similar results (not shown) were obtained with TFP and CPZ. The increase in PtdSer synthesis observed was not due to an increased incorporation of [^3H]serine by the cells, since the drugs slightly decreased the uptake of [^3H]serine (Table 2). In the presence of W7, the synthesis of the other phospholipids was either unchanged (phosphatidylinositol) or markedly decreased (phosphatidylcholine and phosphatidylethanolamine). The decrease of phosphatidylcholine and phosphatidylethanolamine synthesis was due to a large decrease of the total uptake of either choline or ethanolamine by the cells (Table 2). Similar results (not shown) were obtained with CPZ and TFP. This pattern of phospholipid synthesis has been previously described by us as an effect of K channel blockers on Jurkat and THP1 cells(21, 23) . Furthermore, we have also demonstrated (35) that both the rise in PtdSer synthesis and the decreased IL-2 synthesis caused by K channel blockers in Jurkat cells are not the result of the decreased synthesis of phosphatidylcholine and phosphatidylethanolamine. This was assessed by using three inhibitors of the choline/ethanolamine transport process, i.e. hemicholinium, decamethonium, and dodecyltrimethylammonium(35) .


Figure 2: Kinetics of phosphatidylserine synthesis in control and 50 µM W7-treated Jurkat T cells. Nonactivated cells were treated or not treated with W7 in the presence of [^3H]serine for 2 h. Phospholipids were extracted, separated by tlc, and then quantified as in Fig. 1. Results are expressed as counts/min ± S.D. (n = 6 from two experiments with triplicate points) of [^3H]serine incorporated into phosphatidylserine.





Lack of Effect of CaM Antagonists on CaMovements in Control and Activated Jurkat Cells

As shown in Fig. 3, none of the CaM antagonists modified cytosolic Ca concentration in nonactivated cells. When cells were activated with CD3 mAb, a marked increase in [Ca](i) was observed in the presence of EGTA, indicating that CD3 mAb released Ca from intracellular stores. Upon addition of 1 mM CaCl(2) at 5 min, i.e. when the cytosolic Ca concentration had returned to the base line, a Ca influx was observed. Similar results were obtained in the presence of TFP, CPZ, or W7 (Fig. 3), indicating that CaM antagonists were unable to modify significantly both the release of Ca from intracellular stores and the Ca influx generated by the activation signal. The increase of [Ca](i) evoked either by ionomycin or thapsigargin (not shown) was also found not to be affected by CPZ, TFP, or W7. Given the fact that CPZ, TFP, and W7 had no effect on the biosynthesis of phosphatidylinositol and that Ca movements generally attributed to the formation of inositol trisphosphate were unchanged by the three drugs, it is very likely that CaM inhibitors play their role in a later event.


Figure 3: Changes in cytosolic Ca concentration induced by CD3 monoclonal antibody. At time 0, the cells were incubated with 2 mM EGTA and 2 µg/ml CD3 mAb. Changes in the cytosolic Ca concentration due to the release of Ca from intracellular stores were monitored for 5 min. At 5 min, the Ca concentration in the medium was adjusted to 1 mM in order to study the CD3-induced Ca influx. Calmodulin inhibitor CPZ, TFP, or W7 (50 µM) was added at time 0. The figure is representative of at least five independent experiments.



Effect of CaM Antagonists on PtdSer Synthesis in CD3- or Thapsigargin-activated Cells

We have previously shown (8) that the inhibition of PtdSer synthesis observed in phytohemagglutinin + PMA-activated cells was only due to the lectin, phytohemagglutinin, and that the PKC activator, PMA, does not participate in this inhibition. The CaM inhibitors were able to reverse the phytohemagglutinin-induced inhibition of PtdSer synthesis(9) . We show here that TFP, CPZ, and W7 are able to reverse the inhibition of PtdSer synthesis induced after CD3 activation (Fig. 4). Thapsigargin is a blocker of the endoplasmic reticulum Ca-ATPase(13, 36) . This compound has been shown to be able to replace CD3 mAb with the object of activating T cells(37) . We show here that in thapsigargin-treated cells, the three drugs also remained able to increase PtdSer synthesis 2-3-fold (Fig. 5). Since CaM inhibitors did not modify changes in [Ca](i) induced by T cell activators, the effect of CPZ, TFP, or W7 is most likely attributable either to an effect on K channels or to a true interaction with CaM.


Figure 4: Inhibition of phosphatidylserine synthesis by 2 µg/ml CD3 mAb (panel A) and reversal of this inhibition by three anti-calmodulin drugs (panel B). In panelA, cells were either nonactivated (Control) or activated with CD3 mAb for 2 h. In panelB, cells activated with CD3 for 2 h were treated with 50 µM CPZ, TFP, or W7, and phosphatidylserine synthesis was monitored for an additional 1 h.




Figure 5: Effect of CPZ, TFP, and W7 on phosphatidylserine synthesis in thapsigargin-treated Jurkat cells. Cells were incubated for 2 h in the presence of [^3H]serine, 0.1 µM thapsigargin, and 50 µM of either CPZ, TFP, or W7 when indicated.



Effect of CaM Antagonists on PtdSer Synthesis in an Acellular System

Since TFP and CPZ have been described as blocking K channels (30, 31) and since we have previously demonstrated that a number of K channel blockers induce a rise in PtdSer synthesis in Jurkat cells(21, 22, 23) , we have tested whether CPZ, TFP, and W7 increase PtdSer synthesis in an acellular system. As shown in Fig. 6, both in intact and homogenized Jurkat cells, the three compounds remained able to induce an increased PtdSer synthesis. In addition, experiments done either in the presence of 1 mM CaCl(2) or without calcium (2 mM EGTA) indicated that TFP, CPZ, and W7 work only when Ca is present, suggesting that their effect is Ca-dependent as expected if their effect is mediated through CaM.


Figure 6: A, cells were maintained either in 1 mM EGTA (no calcium, whitebars) or in 1 mM Ca-containing medium (blackbars) in the absence or presence of 50 µM TFP, CPZ, or W7. B, 100 µl of Jurkat cell extract (prepared as described under ``Materials and Methods'') were used. As in A, blackbars represent experiments done in 1 mM Ca- containing medium, and whitebars represent experiments done in the presence of 1 mM EGTA. Results are expressed as percentage versus control in order to facilitate the comparison of the two different sets of experiments. The values, ± S.D. (n = 6), correspond to three experiments with duplicate points.



CaM Antagonists Work Differently from Cationic Amphiphilic Compounds

It has been proposed that cationic amphiphilic drugs (32, 33, 34) increase PtdSer synthesis by exposing the ethanolamine moiety of phosphatidylethanolamine (the substrate for PtdSer synthesis) to a hydrophilic environment accessible to the catalytic site of the base exchange enzyme. In order to test this hypothesis, we have compared the ability to increase PtdSer synthesis of the three calmodulin antagonists versus two cationic amphiphilic compounds, oleylamine and stearylamine. In cells incubated in 1 mM Ca-containing medium, the three CaM antagonists as well as the two amphiphilic compounds strongly increased PtdSer synthesis (not shown). Nevertheless, a major difference within the two classes of compounds was the strict Ca dependence observed for the calmodulin inhibitors. Indeed, TFP, CPZ, and W7 were unable to increase PtdSer synthesis in the absence of calcium ions, while oleylamine and stearylamine enhanced PtdSer synthesis by 3-4-fold in cells totally depleted of Ca with EGTA. (Fig. 7).


Figure 7: Left panel, comparison of the effect of 50 µM TFP, CPZ, or W7, 5 µM oleylamine, or 5 µM stearylamine on phosphatidylserine synthesis in Jurkat cells maintained for 2 h in 2 mM EGTA-containing medium. [^3H]Serine was added at time 0, and phosphatidylserine synthesis was measured at 2 h. Under these experimental conditions, the Ca concentration in the intracellular Ca stores was undetectable, as shown in the inset. The inset represents changes in the Ca cytosolic concentration elicited by 10M ionomycin in control cells maintained for 2 h in 1 mM Ca-containing medium (box) and in cells maintained in 2 mM EGTA for 2 h () (2 mM EGTA was added at time 0, just before ionomycin, in order to evaluate the Ca content of the intracellular stores for cells maintained in Ca-containing medium (box)). The rightpanel represents PtdSer synthesis in the absence or presence of either oleylamine or stearylamine (5 µM) in cells incubated in 1 mM Ca-containing medium.



Cyclosporin A Does Not Change PtdSer Synthesis

Cyclosporin A (CsA), a well known immunosuppressive drug, acts through an interaction with cyclophilin (19, 20) that in turn interacts with calcineurin, a phosphatase that is regulated by calmodulin. It was thus of interest to study whether CsA interferes with PtdSer synthesis. It was found (Fig. 8) that CsA within the 1-1000 nM range of concentration that totally inhibits IL-2 synthesis had no effect on PtdSer synthesis.


Figure 8: Lack of effect of CsA on phosphatidylserine synthesis. Cells were treated with CD3 mAb (2 µg/ml) in the absence or presence of different concentrations of CsA and [^3H]serine for 2 h. At the end of this incubation period, phospholipids were extracted, separated by tlc, and then quantified. Results are expressed as counts/min ± S.D. of [^3H]serine incorporated into phosphatidylserine (n = 6 from two experiments done in triplicate).




DISCUSSION

A key phenomenon in T cell activation is the rise of cytosolic Ca concentration. Early events leading to this phenomenon represented by the ``tyrosine kinase pathway'' have been extensively investigated. Yet the events that occur in the ``postcalcium period'' are less known. One of these events is the inhibition of PtdSer synthesis that has been attributed to the release of Ca from intracellular stores(11) . The exact role of PtdSer inhibition is not known, although changes in PtdSer synthesis seem to be implicated in T cell signaling pathways since a number of inhibitors of IL-2 synthesis reverse this process. Among the drugs tested in past years, potassium channel blockers(21, 22, 23) , diacylglycerol kinase inhibitors (25) , and cytochrome P-450 inhibitors (24) enhanced PtdSer synthesis and inhibited IL-2 production and T cell proliferation with similar dose-response curves, indicating that these events may be linked. We show herein that three calmodulin antagonists increase PtdSer synthesis and inhibit IL-2 synthesis in Jurkat T cells with similar concentration-dependent effects. The stimulation of PtdSer synthesis by calmodulin antagonists we have observed in Jurkat cells confirms the findings of previous works performed in acellular systems either with human leukocyte membranes by Niwa and Tanigushi (18) or with rat brain microsomes by Buchanan and Kanfer(17) . CaM inhibitors have been shown to be able to interact with K channels(31) . Since K channel blockers increase PtdSer synthesis and inhibit IL-2 synthesis, we have tested the effect of CaM inhibitors on an acellular system in which changes in K concentration or changes in K channel activity are unlikely. In this acellular system, CaM inhibitors remained able to increase PtdSer synthesis, suggesting that CaM could be the target of the drugs used. In order to confirm this point we have tested whether the increase of PtdSer caused by the drugs is calcium-dependent. As expected, CPZ, TFP, and W7 increased PtdSer synthesis in control, CD3-activated, and thapsigargin-treated cells but were inactive in EGTA-treated cells. We have verified that after 2 h in the presence of 2 mM EGTA the cells were depleted of Ca as shown in Fig. 7(inset). Furthermore, as shown in Fig. 6, the Ca dependence of CaM antagonists was also observed in the acellular system. On the other hand, it has been suggested that amphiphilic cations increase PtdSer synthesis by exposing the ethanolamine moiety of phosphtidylethanolamine (the substrate for PtdSer synthesis) to a hydrophilic environment accessible to the catalytic site of the base exchange enzyme(32, 33) . This interpretation appears unlikely, since as shown in Fig. 7the two cationic amphiphilic drugs tested (oleylamine and stearylamine) strongly increased PtdSer synthesis but in a calcium-independent process. Furthermore, our previous work on K channel blockers (22) in which we tested five antiarrhythmic drugs belonging to the cationic amphiphilic drug family indicated that only the potassium channel blocker, clofilium, increased PtdSer synthesis. From this study and the results presented herein it can be concluded that increasing PtdSer synthesis is not a general property of cationic amphiphilic drugs.

Altogether, our results strongly support the hypothesis that CPZ, TFP, and W7 play their role through an interaction with the CaMbulletCa complex.

A second event in the postcalcium period of T cell activation is the formation of Ca-calmodulin complexes that in turn bind to calcineurin A subunit(14) . This pathway is inhibited by cyclosporin A through an interaction of cyclophilin-CsA complexes to calcineurin (14, 15, 16) . We have tested whether CsA modifies PtdSer synthesis, and it was found (Fig. 8) that this immunosuppressive drug had no effect on PtdSer even at concentrations far above the doses necessary to totally abrogate IL-2 synthesis.

CaMbulletCa complexes thus appear as a junction leading to at least two independent pathways of regulation of T cell activation, one involving the calcineurin phosphatase and the other involving the base exchange enzyme system responsible for PtdSer synthesis.


FOOTNOTES

*
This work was supported by INSERM and the Association pour la Recherche contre le Cancer (ARC Grant 6879). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 33-93-9215-77-09.

(^1)
The abbreviations used are: PtdSer, phosphatidylserine; CPZ, chlorpromazine; TFP, trifluoperazine; W7, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide HCl; CaM, calmodulin; CsA, cyclosporin A; IL-2, interleukin-2; PMA, phorbol myristate; mAb, monoclonal antibody.


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