1989;49:877-882. Published online February 1, 1989.Cancer Res P. M. Kimball and Stewart Sell ImbalanceCyclosporin Immunosuppression Mediated by Calcium/Sodium  

  

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(CANCER RESEARCH 49, 877-882, February 15, 1989]

Cyclosporin Immunosuppression Mediated by Calcium/Sodium Imbalance1

P. M. Kimball2 and Stewart Sell

Departments of Surgery, Immunology and Organ Transplantation [P. M. K.], and Pathology/Laboratory Medicine [S. S.J, University of Texas Health Science Center,Houston, Texas 77030

ABSTRACT

Cyclosporin immunosuppression is mediated by a calcium/sodiumexcess during Go which inhibits further cell cycle progression. Theconsequences of cyclosporin on electrolyte content were measured in I -lymphocytes stimulated with concanavalin A. Cyclosporin caused anexcessive accumulation of extracellular calcium for the first 4 h of lectinstimulation. The nonpermissive calcium content resulted from a reductionin the rate of calcium efflux from the cell. Because cyclosporin did notaffect calcium translocation via ATPase but did permit excessive amountsof sodium to enter the resting cell we hypothesized that the calciumexcess is caused by a shut-down of the ( V'VNa+ antiport during the first

hours of lectin stimulation. The subsequent normalization of calciumcontent is coincident with the onset of mRNA synthesis, which suggestsdevelopment of compensatory mechanisms to alleviate the calcium burden. The Go calcium excess did not affect other transductive events suchas ligand recognition, phosphatidyl inositol metabolism, or adenylatecyclase activation. This study points to the causative mechanism ofcyclosporin immunosuppression and emphasizes the dynamic role of ionsas modulators of normal cell proliferation.

INTRODUCTION

Introduction of cyclosporin A to the medical community inthe 1970s revolutionized the field of organ transplantation (1,2). The drug also has proven instrumental in illuminatingregulatory constraints during normal cell activation (1-6). Despite widespread use of CsA3 in both clinical and investigative

arenas, the mechanism of action and cell cycle dependency ofthe drug have remained controversial (3, 6-9). Recent reportsprovided clear evidence that resting (G0) lymphocytes are theexclusive target of CsA action (3-5). The authors concludedthat CsA prevented activation by blocking one or more of theearliest transductive events that couple ligand stimulation tocell cycle progression (4-5).

The emerging role of electrolytes in proliferative regulationand activation signaling (10-14) led us to speculate that CsAaltered the availability of an essential ion at the onset of ligandstimulation. Consistent with this hypothesis were reports ofCsA-induced changes in membrane polarization and membranepotential of resting cells (1, 15, 16). Preliminary investigationspointed to a specific defect in calcium utilization (1,6, 7). Thiswas particularly intriguing because of the role of calcium as asecond messenger during lymphoid activation. Calcium enjoysa dual role in T-cell activation where it regulates both initiationand perpetuation of activation signals. Intracellular mobilization of calcium (Ca¡2+)is an ubiquitous response to ligand

binding which initiates cellular activation (10, 12, 13, 17, 18).In T-lymphocytes, this is followed by a sustained entry ofcalcium from the extracellular space (Cao2+)which perpetuatesongoing signals and drives the cell to mitosis (19-22). We

Received 7/12/88; revised 9/30/88; accepted 11/7/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by NIH Grant A121721.2To whom requests for reprints should be addressed, at Department of

Surgery, Immunology and Organ Transplantation, University of Texas HealthScience Center, Houston, TX 77030.

3The abbreviations used are: CsA, cyclosporin A; Con A, concanavalin A;cAMP, cyclic AMP; CsC, cyclosporin C.

hypothesized that CsA interfered with either intracellular orextracellular mobilization of calcium in T-lymphocytes during

Con A stimulation.This report shows that immunosuppression by CsA is me

diated by an excessive accumulation of extracellular calcium.The calcium excess, which does not affect ligand recognition orgeneration of Ca¡2+,restricts the cell to G0 and prohibits cell

cycle progression. The nonpermissive calcium concentrationresults from a sodium excess produced by CsA during Go whichreduces the rate of calcium efflux during lectin stimulation.Although the mechanism for the calcium/sodium excess isrestricted to Go, the biological consequences of an aberrationin electrolyte balance can be manifested throughout the cellcycle and can delineate the dynamic role of ions in normalproliferative modulation.

MATERIALS AND METHODS

Animals. New Zealand White male rabbits were obtained from RayNichols Rabbitry (Lumberton, TX) at an initial weight of 3 kg. Rabbitshoused in the animal facility at the University of Texas Medical Schoolwere fed daily, watered ad libitum, and maintained in accordance withfederal guidelines for humane care and treatment of laboratory animalsas approved by the Animal Welfare Committee of the University ofTexas Health Science Center at Houston.

Lymphocyte Proliferation Assay. Peripheral blood withdrawn fromthe central ear artery was defibrinated with wooden sticks and sedi-mented in an equal volume of 3.5% pigskin gelatin in phosphate-buffered saline for 30 min at 37°C.The lymphocyte-rich supernatant

was washed extensively prior to RBC lysis by the addition of 4.5 mlwater followed by 0.5 ml 10 x RPMI (Gibco, Grand Island, NY) (4, 5,20). The final cell suspension was comprised of approximately 80% T-lymphocytes, less than 1% monocytes, and 20% B-cells (4, 5, 20, 23).That the effects of Con A and CsA on the mixed population selectivelyreflect T-cell response was shown by T-cell depletion experiments.Isolated B-cells proliferated in response to anti-immunoglobulin stimulation but were not activated by Con A. Also, the restive calcium levelof enriched B-cells was unchanged by Con A and/or cyclosporin.Lymphocytes were resuspended at 106/ml in RPMI supplemented with1% heat-inactivated rabbit serum, 2 HIMglutamine, 100 units/ml penicillin, and 100 ng/m\ streptomycin, and cultured in 96-well microculture plates (Costar, Cambridge, MA) in 0.2 ml medium with or without10 MgCon A. Cells were cultured for 24 h under humidified conditionsat 37°Cin 5% CO2 in air, pulsed with 0.1 uCi [I25l]iododeoxyuridine

(ICN, Irvine, CA) for an additional 24 h, and harvested onto glass fiberfilter paper (Whatman, Clifton, NJ) (4, 5, 20, 23).

Lectin Recognition. Concanavalin A was radiolabeled with '"I by thelodo-Gen procedure (24). Functional integrity was maintained following iodination as shown by the ability of 125I-ConA to induce blasttransformation in cultured cells. Surface binding of '"l-labeled Con Awas measured by incubating cells (2 x 104)in 0.1 ml phosphate-bufferedsaline at 4°Cwith various concentrations of lectin for 0-60 min.Unbound material was removed by centrifugation of washed cells. Cell-associated radioactivity was measured. Lectin endocytosis was measured by incubating cells with 125I-ConA at 37'C for 0-60 min. Excess

label was removed by washing and the amount of label remaining withthe cell pellet was quantitated. Inclusion of the competitive inhibitormethyl mannoside or excess unlabeled Con A prevented binding anduptake of radiolabeled ligand (23, 25).

Phosphatidyl Inositol Catabolism. Cells (IO8)were suspended in 1 mlbalanced salt solution containing 0.1% glucose, 25 ITIM4-(2-hydroxy-

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ELECTROLYTE BALANCE IN RESTING LYMPHOCYTES ALTERED BY CsA

ethyl)- 1-piperazineethanesulfonic acid, 1% fetal bovine serum, 60 pCi/ml [3H]myoinositol and incubated at 37°Cfor 60 min. Cells werewashed twice and resuspended at 2.5 x 107/0.5 ml of BBS with 0.1%glucose, 25 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid,and 5% fetal bovine serum. Cells were stimulated with Con A for 0-20min. Addition of 120 n\ of 0.22 N HC1 terminated the reaction. Cellswere extracted with chloroform: methanol and fractions were collectedonAGl-X8(17, 18).

Quantitation of cAMP. cAMP was quantitated by commercial radio-immunoassay (Rianen, DuPont). Cells were homogenized at 4°Cwith

6% trichloroacetic acid and pelleted at 2500 x g for 15 min. Thesupernatant was extracted 4 times with 5 volumes of water saturatedether. The aqueous sample was evaporated to dryness, dissolved inassay buffer, and acetylated. A trace amount of [3H]cAMP added to all

samples showed that cAMP extraction recovery exceeded 90%.Measurement of Cellular Calcium Equilibrium. The calcium equilib

rium maintained by resting cells with the extracellular environment isa relatively stable ratio of incoming versus outgoing calcium. Lectinstimulation increases the cell calcium content by increasing the rate ofinflux (20, 22). Inclusion of a trace concentration of isotopie calciuminto complete culture media containing physiological amounts of unlabeled calcium permits quantitation of changes in the cell calciumequilibrium at any time during mitogenesis (20). Lymphocytes (105/0.2ml) were equilibrated in complete medium containing 1 ¿iCi45Ca2+(ICN) for 30 min at 37°Cprior to stimulation with Con A for 0-48 h.Unbound 45Ca2+was removed by rapid washing, the cell pellet was

lysed with 0.5 ml water and freeze-thawed. Scintillation cocktail wasadded and the radioactivity was quantitated. Confirmation of viabilityshowed that increased cell-associated calcium does not reflect calciumsequestration by cell-free organelles. Cell calcium concentration wasextrapolated from the proportion of bound versus free isotope and thecalcium concentration of complete medium which was measured rolorimetrically. The effect of CsA on the sodium equilibrium was similarlydetermined by substituting 0.6 n('i isotopie sodium for isotopie calcium.

Calcium Influx. Resting lymphocytes (2 x 105/0.2 ml) were equilibrated with complete medium for 30 min at 37°C.Con A was addedfor 0-60 min. Two min prior to collection, 1 pCi 45Ca2+was added.

Cells were rapidly pelleted in a microfuge, the supernatant was removedby vacuum withdrawal, and the cell pellet was suspended in scintillationcocktail.

Chemicals. Con A was purified from Jack beans by ammoniumsulfate precipitation and affinity chromatography on Sephadex G-25(5, 20). Cyclosporins A and C (Sandoz, NJ) were dissolved at 1 mg/mlin absolute ethanol containing 20% Tween 80, and diluted 10-fold bythe dropwise addition of RPMI and stored at -TV in the dark. Verapamil(Sigma, St. Louis, MO) was dissolved in medium, protected from light,and used the day it was dissolved (26). "Ça2*(specific activity 8) and"Na* (specific activity 200) (ICN) were diluted in RPMI and stored at4°C.

RESULTS

Lectin Recognition. CsA did not affect binding or uptake of125I-ConA. Maximal surface binding of 125I-ConA to cells heldat 4°Cwas achieved within 10 min. Binding was linear with

respect to Con A concentration and did not increase with time(Fig. 1/1). Inclusion of 1 mg/ml methyl mannoside, specificinhibitor of Con A reactivity to glycoconjugates, eliminatedcell-associated radioactivity (4, 18, 27). Incubation at 37°Cfor

0-60 min showed a time-dependent increase in cell-associatedradioactivity and indicated endocytosis of bound material followed by binding of new material at the cell surface (Fig. \B).Inclusion of 20 ng CsA reduced DNA synthesis by 98 ±2%(SD) without affecting viability. Surface binding of 125I-ConA

was unchanged by the presence of 20 ng CsA. The presence ofCsA did not alter the amount or rate of endocytosis of 125I-Con

A measured between 0 and 60 min (Fig. 1C).Phosphatidylinositol Metabolism. Ligand binding triggers

EQ.

ÊO.

£Q.O

4 -

2 -

.04 .12 .20 .28125I-ConA

.36 .44

16 i

14 -

12 -

10 -

8 -

6 •¿�

4 -

2 -

.12 .20 .28125I-ConA

.36 .44

2 -

.04 .44

Fig. 1. I.fleet of CsA on Con A binding and endocytosis. Lymphocytes (2 xIO4)were incubated with various concentrations of '"I-labeled Con A for 10 (•),30 (O), or 60 (. !) min. Excess label was washed away and the amount of cell-associated radioactivity was measured. Binding was measured by incubating cellsat 4'C (A) and endocytosis was measured by incubating cells at 37*C (B). C, IMI-

Con A binding (lower line. •¿�)and endocytosis (upper line, •¿�)were measured after60 min in the presence of 20 ng CsA (O).

phosphatidylinositol catabolism with the subsequent release ofsoluble inositol triphosphate into the cytosol (17, 18). BecauseCsA did not prevent Con A binding, we did not expect CsA toinhibit the formation of inositol phosphate metabolites. Formation of phosphatidylinositol metabolites was measured after0, 10, and 20 min of stimulation with Con A. Inositol mono-phosphate, inositol diphosphate, and inositol triphosphate in-

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ELECTROLYTE BALANCE IN RESTING LYMPHOCYTES ALTERED BY CsA

creased 110 ±5, 130 ±10, and 150 ±10% above resting cellsafter 10 min of Con A stimulation, and 200 ±15, 200 ±15,and 250 ±20% after 20 min of stimulation. In the presence of20 ng CsA, inositol monophosphate, inositol diphosphate, andinositol triphosphate increased 150 ±20, 150 ±10, and 160 ±5% above resting cells after 10 min of Con A stimulation and240 ±20,250 ±15, and 270 ±10% after 20 min of stimulation(Fig. 2).

Effect of CsA on cAMP Formation. Lymphocytes do nottypically use cyclic nucleotides as second messengers duringactivation (18, 27). An elevation in cAMP content can downregulate the inositol triphosphate-Ca¡2+ cascade, probably bynegative modulation of the G-proteins (10, 28). The effect ofCsA on cAMP content was measured after 0, 10, and 20 minof stimulation with Con A. Con A stimulation did not increasethe cAMP content above the 1.6-pmol/million cells value ofresting cells (Table 1). cAMP content of resting or lectin-activated cells was unaffected by 20 ng CsA which abrogatedDNA synthesis.

Effect of CsA on Calcium Promotional Signaling. Maximalactivation of DNA synthesis in lymphocytes requires continuedlectin-receptor interaction for 18 to 24 h (4, 18, 20, 23). Wehave shown that T-lymphocyte activation by Con A is accompanied by two episodes of sustained calcium entry from theextracellular space (Ca„2+).The first episode is regulated by

Con A interaction, drives the cell from Go to G i and must occurwithin particular concentration limits. The second episode appears to be regulated by growth factor interactions and pushesthe cell from Gìthrough M. Both episodes are verapamilinhibitable and essential for mitogenesis (20, 26, 29).

Equilibration of exogenous 45Ca2+ in resting cells was

achieved rapidly and did not change over the 48 h in culture.

300

200

ï 100Q.

10

Minutes

20

Fig. 2. CsA spares inositol triphosphale formation. Lymphocytes, prelabeledwith 'H-myi>¡nos¡iol.were suspended in balanced salt solution containing 0.1%glucose, 25 HIM4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid, and 5% fetalbovine serum. Con A stimulation was terminated after 0, 10, and 20 min byacidification. Soluble inositol phosphates were extracted and chromatographicallyseparated on AGI-X8 (formate form). Data for inositol triphosphate content isexpressed relative to that of unstimulated cells. D, without CsA; •¿�,with 20 ngCsA.

Table 1 cAMP content during Con A stimulation (pmol/million cells)Lymphocytes were stimulated with 10 fig Con A for 0, 10, and 20 min at 37'C,

both with and without 20 ng CsA. Cells were washed to remove serum, homogenized with trichloroacetic acid and extracted with water-saturated ether. Theaqueous sample was evaporated to dryness, dissolved in assay buffer, and acety-lated. cAMP content was quantitated by commercial radioimmunoassay. Recovery of cAMP after extraction exceeded 90%.

O min 10 min 20 min

Con ACsA1.6

±0.06"

1.5 ±0.061.6±0.02

1.5 ±0.031.7±0.09

1.7 ±0.04

Although the value of 180 DM is similar to resting cytosolicmeasurements with fluorescent indicators, it must be emphasized that this procedure does not indicate intracellular localization. Addition of Con A caused DNA stimulation index of100 ±10. Con A caused the resting equilibrium to rise from180 ±27 to 300 ±18 nM after l h (Fig. 3). Calcium contentpeaked between 4 and 6 h at 360 ±9 nM and remained relativelyconstant at 300 ±15 nM until 18 h in culture. Between 24 and26 h, when Con A is no longer required for activation, cellcalcium content returned to the resting concentration of 180 ±36 HM.After 27 h in culture, a second episode of calcium uptakebegan which peaked at 36 h at 380 ±45 nM and returned toresting concentration by 42 h.

The calcium equilibrium of resting cells exposed to CsAremained at 180 ±27 for the duration of culture. Cocultivationof Con A and 20 ng CsA reduced DNA stimulation index to 2±2 and elevated calcium content to 460 ±20 nM after 1 h (Fig.3). Cell calcium content slowly decreased and reached 360 ±15 HMafter 4 h of cultivation. Calcium content remained stableat 300 ±20 nM until 18 h of cultivation, then rapidly droppedto resting cell levels. After 27 h in culture, a second episode ofcalcium uptake was initiated. Calcium concentration from 180nM peaked at 36 h at 380 ±30 HMand returned to 180 nM by42 h.

The effect of CsA concentration on isotopie calcium contentwas determined after 1 h of Con A stimulation (Table 2).Inclusion of 20,1, or 0.05 ng CsA reduced the DNA stimulation

480

420

360

300

240

18015 20 25 30

Hours ¡nCulture

Fig. 3. CsA elevates calcium balance during first hours of lectin stimulation.Lymphocytes were equilibrated with 1 >iCi45Ca2*in complete medium for 30 minat 37'C. Con A (D) was added and cells were incubated for up to 48 h. At varioustimes during the incubation, the amount of cell-associated radioactivity wasdetermined. Unbound 4'Ca2* was removed by rapid washing, the cell pellet waslysed with 0.5 ml water, freeze-thawed, then diluted with scintillation cocktail.CsA (•)was included in the incubation mixture at a concentration of 20 ng. CsAdid not affect the 180 nM <5Ca2*content of resting cells.

Table 2 Effect of CsA concentration on calcium levelLymphocytes were equilibrated with 1 nCi 4!Ca2* in complete medium for 30

min prior to the addition of Con A. Cells treated with Con A or Con A and CsAwere harvested after 1 h and the amount of cell-associated 45Ca2*was determined.

The absolute cell calcium concentration was extrapolated from the free, unlabeledcalcium present in medium. Proliferation was measured by 5-iodo-2'-deoxyuri-

dine (IdUrd) incorporation after a total of 48 h of cultivation. IdUrd incorporationin unstimulated cells equaled 104 ±20 cpm. "Calcium content in resting cells

was 180 ±27 with or without CsA.

Con AWith 20 ng CsAWith 1 ng CsAWith 0.05 ng CsACalcium

(nM)300±19°

460 ±7340 ±14306 ±1IdUrd

incorporation(cpm)26,700

±700253 ±25

10,941 ±12526,000 ±400

" Mean ±SD. ' Mean ±SD.

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ELECTROLYTE BALANCE IN RESTING LYMPHOCYTES ALTERED BY CsA

index from 129 to 2, 53, or 120, respectively. CsA induced aconcentration-dependent increase in isotopie calcium levels.CsA at 20, 1, or 0.05 ng raised the cell calcium concentrationfrom 180 to 460, 340, and 300 nM, respectively. Calciumcontent of Con A-stimulated cells was 300 nM.

CsC is not used clinically because of its potent hepato- andnephro-toxicity (2). Substitution of an equimolar concentrationof CsC for CsA caused a 2- to 6-fold increase in calcium contentrelative to that of CsA-treated cells after l h of cultivation withCon A. Similar to CsA treatment, calcium content normalizedafter approximately 4 h of cultivation. Addition of CsA or CsCafter 24 h of stimulation with Con A, when the cells are in Gìand are mitotically unaffected by cyclosporins, did not affectthe second episode of calcium accumulation.

Calcium Influx. The inappropriate calcium level produced byCsA had to result from increased calcium entry or decreasedcalcium exit from the cell. The effect of CsA on calcium influxwas compared to that induced by two concentrations of Con A.A mitogenic dose of Con A caused 69,053 cpm of 45Ca2+toenter per min (Table 3). A 5-fold increase in Con A concentration was not mitogenic and doubled the rate of calcium entry.Addition of 1 or 20 ng CsA inhibited DNA stimulation indicesby approximately 70 and 100%, respectively. The calcium influxrate in CsA-treated cells was the same as seen in cells treatedwith mitogenic concentrations of Con A.

Calcium Efflux. CsA did not affect the rate of calcium effluxfrom RBC. Because RBC use Ca2+ ATPase to efflux calcium,

the effect of CsA on calcium translocation via ATPase wasmeasured. RBC, with and without CsA, were loaded with 45Ca2+at 37°Cfor 30 min in the presence of serum as an energy source.

RBC were diluted into warm medium (which lacked isotopiecalcium) and the change in cell-associated radioactivity wasmeasured with time. CsA did not affect calcium loading ofRBC. RBC-associated 45Ca2+dropped by 90 and 95% after 2

and 5 min, respectively (Table 4). CsA did not inhibit calciumefflux from the RBC.

Effect of CsA on Sodium Equilibrium. The sodium content ofcells in culture for a maximum of 4 h was determined asdescribed for analysis of calcium equilibrium. Sodium contentin resting lymphocytes was stable for 4 h in culture (Table 5).

Table 3 Comparative effects of Con A and CsA on rate of calcium influxLymphocytes were equilibrated in complete medium for 30 min prior to Con

A addition. Various concentrations of Con A and CsA were added to the mixtureand incubation continued for 60 min. Two min prior to collection, 1 /iCi 45Ca2*

was added. Cells were pelleted in a microfuge, the supernatant was removed byvacuum, and the cell-associated radioactivity was determined. The effects of CsAand Con A on DNA synthesis were measured by 5-iodo-2'-deoxyuridine (IdUrd)incorporation after 48 h in culture. Idi 'rd incorporation in unstimulated cells

equaled 104 ±10 cpm.

ConAlOiig

50„¿�gCon

A 10 Mg,CsA l ngCon A 10,ig, CsA 20 ng4SCa2*

(cpm)69,053

±191138,200±5,45870,003

±20069, 132 ±85IdUrd

incorporation(cpm)14,167

±300400 ±153,145

±75424 ±10

Table 4 Calcium efflux by RBCEffect of CsA on calcium translocation via Ca2*ATPase was examined by

measuring calcium efflux in RBC. RBC, with and without 20 ng CsA, were loadedwith "Ca2* for 30 min in the presence of serum as an energy source. RBC werediluted into warm medium which lacked 45Ca2*.Cells were collected after 0, 2,and 5 min and the drop in cell-associated radioactivity was measured.

O min 2 min 5 min

Table 5 Effect of CsA on sodium contentLymphocytes were equilibrated with 0.6 (iCi 22Na*in complete medium for 30

min at 37'C prior to Con A addition. Con A and CsA (20 ng) were added into

the culture mixture for 1 or 4 h. Cells were washed, pelleted, and the amount ofradioactivity associated with the cells was determined. DNA synthesis was measured by 5-iodo-2'-deoxyuridine (IdUrd) incorporation after 48 h in culture. IdUrd

incorporation in unstimulated cells was 150 ±50 cpm.

Sodium(cpm)Resting

WithCsACon

AWith CsAAfter

1h20,

134 ±45328,560 ±88829,900

±16538,147 ±80After

4h20,134±

1,33324,770 ±85823,869

±3123,816 ±324IdUrd

incorporation(cpm)150

±20155 ±1519,500

±100450 ±50

RBCRBC, CsA23,189±

50023,189 ±3502,434

±1002,142 ±851,897

±901,539 ±110

CsA elevated the 22Na+equilibrium of resting cells from 20,134to 28,560 cpm. The differential between normal and CsA-treated resting cells was maintained for 4 h, although it wassignificantly reduced. Cells stimulated with Con A or Con Aplus CsA showed a similar differential elevation in 22Na+con

tent by l h which declined to similar concentration by 4 h ofcultivation.

DISCUSSION

Defective transport of an essential ion was implicated as thecausative effect of immunosuppression by CsA (1, 5, 7,15, 16).We speculated that CsA changed calcium availability and prevented the generation of appropriate calcium-dependent activation signals. The hypothesis was examined by measuring theeffect of CsA on the induction of intra- and extracellular calcium mobilization in T-lymphocytes during Con A stimulation.

CsA did not inhibit the generation of initiating activationsignals. This was approached indirectly by measuring the effectof CsA on the regulatory arms of Ca¡2+generation: Con A

recognition, and inositol triphosphate and cAMP formation.Maximal T cell activation requires prolonged, continuous binding of Con A to surface receptors and sustained endocytosis ofthe lectin-receptor complex (4, 5, 18-22). The first minutes oflectin binding induce inositol triphosphate formation whichtriggers the explosive and transient release of Ca,2' from an

internal source. The failure of CsA to prevent Con A bindingwas consistent with its inability to alter chemotactic recruitmentof lymphocytes in vivo (2). Because CsA did not impair Con Abinding, a reduction in inositol triphosphate generation wasneither expected nor found. Nor did CsA downregulate theinositol triphosphate-Car* cascade by activation of adenylate

cyclase. The occurrence of early activational events in thepresence of a potent mitogenic inhibitor shows clearly thatalthough initiating signals are essential for activation, they areinsufficient to commit the cell to the lengthy transition fromGo to Gìand from there to mitosis.

CsA caused an inappropriately elevated calcium promotionalsignal which prevented the Go to d transition. We have recentlyshown that two episodes of sustained extracellular calciumuptake perpetuate ongoing activation signals in T-lymphocytesand are responsible for driving the Go cell through mitosis (20,26). The first episode is regulated by Con A interaction andmust occur within particular threshold concentrations. Con Aconcentrations that are too low or too high are reflected in theCa«2*signal and prohibit cell cycle progression. The second

episode of sustained calcium entry appears regulated by growthfactor interaction (19, 20, 26, 30). CsA treatment produced aCa,,: ' signal remarkably similar to that seen with high, nonmi-

togenic concentrations of Con A. Both agents raised cell calcium content to nonpermissive levels for the first hours of lectin

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ELECTROLYTE BALANCE IN RESTING LYMPHOCYTES ALTERED BY CsA

stimulation. This suggests that excessive calcium content incells using C'a,,; ' as a promotional signal may be a common

mechanism for modulating proliferation without compromisingcell viability. Analogous results seen in calcium-dependentgrowth factor modulation of epithelial cells lend support to thisidea. The chemical basis for calcium modulation of biologicalevents can be explained, in part, by the alteration in cofactoravailability. Nonoptimal cofactor concentrations regulate essential enzyme activity as well as gene transcription (16, 29, 31,32) and may have similar quantitative effects on calcium-activated process such as calmodulin and G-protein activity.Whether this causes qualitative changes in the identity of regulatory products is a tantalizing possibility.

The relative normalization of Ca„2+in CsA-treated cells after

4 h in culture is coincident with mRNA synthesis (18, 23). Thisimplies that the increased rate of calcium efflux is due tosynthesis of new Ca2+ ATPase as well as potential kinetic

changes in enzyme activity (31). Although the mechanism ofcalcium entry is the same, growth factors probably regulate thesecond episode of extracellular calcium mobilization in the T-cell (11, 20). The inability of CsA to affect the second episodeof calcium accumulation is consistent with its biological sparingof cells in G, (2, 4, 5).

Whether Con A-regulated calcium entry is controlled bylectin binding or endocytosis, the failure of CsA to interferewith either event showed that the calcium excess resulted froma decrease in the rate of efflux. Direct confirmation was obtained by showing that CsA had no effect on the calcium influxrates. We turned our attention to the effects of CsA on effluxmechanisms. Cell calcium equilibriums are maintained byCa2+ATPase activity except when an abnormal elevation in

intracellular calcium occurs and enlists the assistance of theNa+-Ca2+ antiportal. The antiport aids calcium removal al

though the amount of calcium translocated via the antiport issmall relative to Ca2+ATPase. Because CsA spared enzymatic

translocation of calcium, it was clear that the drug reducedcalcium efflux via antiportal exchange. The means by whichthat occurs was clarified by the demonstration that CsA allowssodium to rush into the resting cell (Fig. 4). Because of theabnormal elevation in intracellular sodium, sodium antiport

Ce**ATPase

i*Na»-K*ATPase

Na'excess

Ca** excess

Fig. 4. Speculative mechanism of CsA immunosuppression. Con A interactionwith the resting lymphocyte produces a radical change in electrolyte balance.Calcium influx from the extracellular space is increased by Con A and establishesa new calcium balance in the cell. Sodium entry from the extracellular space issimultaneously increased with similar adjustments in sodium equilibrium. CsAescalates sodium entry in the Go cell. This reduces the rate of calcium efflux viathe Ca2*-Na* antiport and causes an inappropriately elevated calcium content

which does not permit progression through the cell cycle.

exchange across the plasma membrane becomes an unfavoredequilibrium. This is consistent with the observations of Danieland Ivés(15). They showed that the lack of cytosolic alkalini-zation in CsA-treated cells was caused by the inactivation ofNa+/H+ antiport. We hypothesize that Ca2+/Na+ antiport ex

change is similarly unfavored in the face of high intracellularsodium concentration. Because the Ca2+/Na'f antiport is im

potent in the face of a rising calcium burden, cell calciumcontent soars to nonpermissive levels. This results in an inappropriate Cao2+which prevents transition to Gìand results in

clinical immunosuppression. How CsA allows sodium to floodthe resting cells is unknown. It is clear that the mechanism isrestricted to Go which accounts for the insensitivity of cyclingcells to the drug. The consequences of elevated sodium onintracellular events are not known.

Why CsA-treated cells, which do not undergo blast transformation continue with biologically "downstream" events(mRNA synthesis, interleukin 2 expression) is puzzling (2-5,8, 9, 33). It suggests that cellular response to stimulation ispreprogrammed and that the program must go to completioneven when activation is inappropriate. This may render themetabolically active (but not activated) lymphocyte sensitive toproliferative modulation at various times and may keep cellresponse elastic and attentive to changing host needs. Thishypothesis would explain preliminary observations showingsuboptimally stimulated lymphocytes respond to proliferativerescue by subsequent exogenous addition of growth factors (11).

REFERENCES

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