Article
Reference
Cyclosporin A prevents induction of the interleukin 2 receptor gene in cultured murine thymocytes
GAUCHAT, Jean-François, KHANDJIAN, Edouard W., WEIL, Roger
Abstract
Blast formation and mitotic activation of G0-arrested mouse thymocytes were triggered by the addition of concanavalin A plus interleukin 2 (IL-2) to the culture medium. When added alone, Con A induces within 6 hr a complex reprogramming ("priming") that comprises the activation of the IL-2 receptor gene. The primed thymocytes are competent to interact with IL-2 and to respond to its growth-promoting effect, which corresponds to blast formation and mitotic activation. Cyclosporin A, an immunosuppressive cyclic peptide of fungal origin, prevents in T lymphocytes the activation of a set(s) of genes encoding lymphokines and the IL-2 receptor but does not affect their expression once they have been activated. The biomedical implications of these observations are discussed.
GAUCHAT, Jean-François, KHANDJIAN, Edouard W., WEIL, Roger. Cyclosporin A prevents induction of the interleukin 2 receptor gene in cultured murine thymocytes. Proceedings of the National Academy of Sciences, 1986, vol. 83, no. 17, p. 6430-6434
DOI : 10.1073/pnas.83.17.6430 PMID : 2428033
Available at:
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1 / 1
Proc.Natl.Acad.Sci. USA
Vol. 83,pp.6430-6434, September1986 Cell Biology
Cyclosporin A prevents induction of the interleukin 2 receptor gene in cultured murine thymocytes
(control of cellproliferation/immunosuppression/blastformation/mitoticactivation/chemotherapyofmammaliangeneexpression)
JEAN-FRANiOIS GAUCHAT, EDWARD W. KHANDJIAN, AND ROGERWEIL
Department of MolecularBiology, University of Geneva,1211Geneva, Switzerland CommunicatedbyV.Prelog, April16, 1986
ABSTRACT Blast formation and mitotic activation of GO-arrestedmousethymocytesweretriggered by the addition of concanavalin A plus interleukin 2 (IL-2) to the culture medium. When added alone, Con A induces within 6 hr a complexreprogramming ("priming") thatcomprisesthe ac- tivation of the IL-2 receptor gene. Theprimed thymocytes are competent to interact withIL-2 andtorespondtoitsgrowth- promoting effect, which corresponds to blast formation and mitotic activation. Cyclosporin A, an immunosuppressive cy- clicpeptide of fungal origin, preventsin T lymphocytes the activation ofaset(s) of genes encoding lymphokines and the IL-2 receptor but does not affect their expression once they have been activated. The biomedical implications of these observations arediscussed.
Cyclosporin A (CsA), acyclicundecapeptide (Mr 1203) of fungal origin, is a powerful immunosuppressive drug of considerableclinicalimportance. CsA prevents blast forma- tion and mitoticactivation ofT lymphocytes derived from differentmammalianspecies (1-3).Itinhibitstheproduction ofinterleukin 2 (IL-2) and ofother lymphokines (4-8) and prevents theactivationbyphorbol 12-myristate13-acetateof the IL-2 gene in ahuman T-cell line (Jurkat; ref. 9) and a murineT-cellline(EL-4;ref. 10). ItwassuggestedthatCsA exertsitsimmunosuppressiveactionby interactingwithorby preventing the appearance of receptors forlymphokines or growth factors (11-13); however, whentested inthe Jurkat T-cellline,CsA had noinhibitoryeffectontheexpression of the IL-2 receptors, as judged by indirect immunofluores- cence(9).Itwasreported (14)thatCsAdirectlysuppresses the response ofT-lymphocyte-independent B lymphocytes toanti-immunoglobulin antibodies. Althoughseveralhypoth- eseshave beenproposed, nocoherentpictureof the mech- anismof actionofCsA hasasyetemerged(15, 16).
Lectin-inducedproliferation of peripheralTlymphocytes requirestheproductionand releasebythestimulated cellsof IL-2and its interaction with the IL-2 receptors(17)onthecell surface. Tosimplifytheexperimental approach, weused in the presentworkG0-arrestedmousethymocytes,which upon stimulation withlectinproducelittle if anyIL-2;thymocytes aretherefore unable to undergo blastformation and mitotic activation unless exogenous IL-2 is added to the culture medium(18). We report here the results from a study on the sequence of molecular events during mitogen-induced thymocyte activation and on the inhibitory effect of CsA.
MATERIALS ANDMETHODS
Thymocytes isolated fromfive1-month-old femaleBALB/c mice (obtained from IFFA-Credo, France; ref. 19) were mixed and distributed inaliquots of2or25 ml(about2 x
106
cells perml) in modifiedRPMI medium(20) supplemented with10%fetalbovine serum (GIBCO).Afterpreincubation for20 hrat370C,morethan80% ofthecellswereviable,as determined by the use of fluorescein diacetate. IL-2 was obtained fromthe supernatantsof rat spleen cells (agift from S. Cammisuli, Sandoz Pharmaceutical, Basel;ref. 21) or a mouse cell line (EL-4; ref. 22) by gel filtration of the concentrated supernatants (21, 22). The preparations were used at a concentrationthat led to maximal mitotic activa- tion.Tostimulate the thymocyte cultures, either Con A alone (2,ug/ml; Pharmacia)orConAplus IL-2 was added to the medium at "time zero" (i.e., 20 hrafterexplantation). CsA (Sandoz Pharmaceutical) was solubilized (3) and added at time zero tothe cultures at afinalconcentrationof0.3,ug/ml, unless indicated otherwise. Duplicate 2-ml cultures were labeledby incubationwith [3H]uridine(5 ,uCi, 25 Ci/mmol;
1Ci=37GBq)or[35S]methionine(10,Ci,1000Ci/mmol)for 1hr orwith[3H]thymidine(5
ACi,
45Ci/mmol, Amersham) for4hr(19);acid-precipitable radioactivity wasdetermined andaliquots of[3H]thymidine-labeled
cells were processed forautoradiography(19, 23). Forcolorimetricdeterminations ofDNA,RNA, andproteincontent(24), about108cellswere used perexperimental point.IL-2receptorswerevisualized onthe cell surfaceby indirect immunofluorescence(allsteps performed on ice): 2 x 106cells were washedwith culture medium supplemented with 10 mM methyl a-D-mannoside and wereincubated for1 hrwitharatmonoclonalantibody (diluted 1:10)directedagainstthe mouse IL-2 receptor(PC61;ref. 25) and then for 1 hr with fluorescein-conjugated goat anti-rat serum (Nordic, Lausanne, Switzerland). For each experimental point,twoaliquots(4000cells) wereexamined by phase-contrast and ultraviolet microscopy (LeitzPralux 22EB).
To measure synthesis of the IL-2 receptor protein, thymocyte cultures
(101
cells) were metabolically labeled with[35S]methionine
(200 ,Ci/ml) for 15 min at 15 hr.Membraneproteinswereextracted (26), and the IL-2 recep- tor was isolated by immunoaffinity chromatography using PC61 antibody(25)and subjected toNaDodSO4/PAGEand revealedbyautoradiography (7 days exposure).
Tomeasuresteady-state levels of IL-2 mRNA, total RNA wasextractedfrom
108
thymocytes with phenol at 65°C (27) and contaminating DNA was removed by treatment with pancreaticDNaseI(BoehringerMannheim). RNA (5 ,ug)was subjectedtoelectrophoresisin1%agarose gelscontaining 6%formaldehyde (28), electrophoretically transferred to Gene- Screen nylon membranes (New England Nuclear-DuPont), and fixed by UV irradiation (29). The membranes were stained withmethylene blue to localize andquantitatethe28S and 18S rRNA and then hybridized with a
32P-labeled
nick-translated probe (pmIL-2R-1; ref. 30) specific for the IL-2receptor mRNA(s); radioactivity was revealed by ex-
Abbreviations: CsA, cyclosporin A; IL-2, interleukin 2; hnRNA, heterogeneous nuclearRNA; kb, kilobase(s).
6430 Thepublicationcostsof thisarticleweredefrayedinpartbypagecharge payment. Thisarticle must therefore beherebymarked"advertisement"
inaccordancewith 18 U.S.C. §1734solelytoindicate thisfact.
Proc. Natl. Acad. Sci. USA 83 (1986) 6431 posureof the membranestoKodak XAR-5films for 2-15 hr
at -70'C in the presence ofanintensifying screen.
RESULTS
DNA Synthesis and Thymoblast Formation. Unstimulated thymocyte cultures consistmainly of thymocytes arrested in phaseGoof the mitotic cycle (A. Grieder, personal commu- nication); only 0.8-1.5% of the cells synthesize DNA, as judged by autoradiography(Fig. lA). Throughout the exper- iments the cell number remainedvirtually stable. To induce blast formation and mitotic activation the cultures were stimulated with Con Aplus IL-2, added at time zero; the percentageof DNA-synthesizing cellsbegantoincrease by 20-25 hr(3-5%)and reached a maximum by 50-60hr,when 35±5%ofthe cellswereengagedin DNAsynthesis.Aslight increase in thecytoplasmic rim could be observed by phase- contrast microscopy in 5-10% of the cells around 15 hr, whereas by 40 hr and later30-40%oof thecells exhibited the morphologyofthymoblasts. Con A alone (i.e., without added IL-2) induced DNA synthesis in only 2-5% of the cells, whereas IL-2 alone was, as expected (18), without effect.
Inunstimulatedcultures, CsA (0.3pg/ml)had noeffecton residual DNAsynthesis,evenafter exposure for up to 100 hr.
InConA-plus-IL-2-stimulated cultures, CsA, added at time zero, reduced the maximum number ofDNA-synthesizing cells to 2-3% (Fig. 1A); thesecorresponded tothymocytes which escaped the inhibitory effect of CsA and underwent blastformationandmitoticactivation (seebelow).WhenCsA was added at 8 hror later(afterCon A plus IL-2) it nolonger inhibited blastformationand DNAsynthesisinducedby Con Aplus IL-2(data not shown). In cultures stimulated with Con Aalone, CsA reduced the number ofDNA-synthesizingcells tothat observed in unstimulated cultures(datanotshown).
RNA Synthesis. Determined colorimetrically, the RNA content ofunstimulated thymocyte cultures was very low (0.5-1,ugof RNA per106 cells; Fig.1B),being less than10%o of the RNA content of G0-arrested mouse kidney tissue culture cells(24); in contrast, the DNA content (6.5
,g
per106 thymocytes) corresponded to that expected for quiescent, diploidmousecells (24). In unstimulated thymocytecultures, therateofincorporation of[3H]uridineinto RNA was low, as determinedby1-hr labeling (Fig.2Inset); itmainly reflected residual heterogeneous nuclear RNA (hnRNA) synthesis (Fig. 2), whereas synthesis of 5S RNA, tRNA, 45S pre- rRNA, 18S rRNA, and 28S rRNA was barely detectable, evenafterlabelingfor 3 hr(datanotshown). ActinomycinD (0.05 ,ug/ml) had no detectable effect on residual hnRNA synthesis (19).40
._
u 30
s:
@ 20 ae 10
5=l 14S0L-
3 Qj
2
T.
1 -t :
0 20 40 60 0 20 40 60 80 100 Time, hr
FIG.1. (A) Percentage of DNA-synthesizing cells,determinedby autoradiography, in cultures ofunstimulated thymocytes (A---A)orof thymocytes stimulated with ConAplusIL-2 in theabsence(o-o)or presence(*-*)ofCsA.Cellswereincubatedwith[3H]thymidinefor 4hr; times plottedrepresentmidpoints of labeling periods. (B)RNA contentofthymocytecultures(symbolsasinA),determined colori- metrically.SinceCsA hadnodetectableeffectonDNAsynthesisand RNAcontentof unstimulatedcultures, the resultswereomitted.
E I
03 6 9 12
TimehrX 28S
18S
100 80 60 40 20 0
Gel slice
FIG. 2. Analysisof[3H]uridine-labeledcellular RNAby electro- phoresis in tubular 2% polyacrylamide/0.5% agarose gels (31).
Thymocyte cultures (108 cells) were labeled with [3H]uridine (200
,uCi/ml)from 5 to 6hr,total RNA was extracted with phenol,and aliquots(10pLgofRNA) were subjected toelectrophoresis.Gels were scanned at 260 nm (data not shown) and radioactivity wasmeasured in 1-mmslices. The positions of 18S and 28S rRNA are indicated by arrows.Cultures were unstimulated (---) or were stimulated withCon Aplus IL-2 in the absence (-) or presence (-) of CsA. (Inset) Incorporation of[3H]uridineinto cellular RNA during 1-hrperiods by unstimulated thymocytes (A---A) or by thymocytesstimulated with Con A and IL-2 inthe absence (o-o) or presence(-o*)of CsA. In unstimulatedcultures, CsA decreased incorporation of[3H]uridine byabout5%withoutdetectably affectingthemigrationpatternofthe RNA(data not shown).
Inculturesstimulated with Con A plus IL-2, RNA content began to increase around 12hr,reaching amaximum plateau by60-80hr,when the cultures contained 4-5 ,ugof RNA per 106cells(Fig. 1). Rate of[3H]uridineincorporationincreased around 3 hr and reached a maximum plateau by 50-60 hr (Fig.
2 Insetand ref. 19). Increased incorporation of[3H]uridine was resistant toactinomycin D (0.05,g/ml)until about 9 hr and reflected "early" stimulation of hnRNAsynthesis(Fig.
2 and ref. 19). A rather sudden and massive increase in nucleolar and nucleoplasmic transcription (45S pre-rRNA, 32S rRNA, 28S rRNA, 18S rRNA, hnRNA, 5S RNA, and tRNA) began by 10-11 hr (19). Judged from the high sensitivity of transcription toward actinomycin D (0.05 ,ug/ml)and from the migration pattern of phenol-extracted RNAduring gelelectrophoresis
(A260
andradioactivity), the bulkof RNA synthesized by 10-11 hr (and later) was rRNA (19). Direct evidence fortheactivation oftheribosomalgenes was obtained by measuring steady-state levels of 45S pre- rRNA(ref. 19andunpublishedresults). Con A alone ledto early stimulation of hnRNAsynthesis indistinguishablefrom that induced by Con A plus IL-2 (Fig. 2), whereas the subsequent stimulation of overall RNA synthesis did not occur(datanotshown).Inunstimulatedcultures,CsA(testedupto80hr)remained without effect on RNAcontent and decreased incorporation of[3H]uridine by onlyabout5%(datanotshown).Incultures stimulated with ConAplus IL-2 orwithCon Aalone,CsA had no detectable effectontheearly stimulation of hnRNA synthesis,until 6-7hr(Fig.2); CsAprevented, however,the subsequent stimulation of overall RNA synthesis and the increase in total RNA (Figs. 1B and 2 Inset). The small increase inRNA content (3-5%; Fig. 1) in the presence of CsAreflects residual blastformation. WhenCsAwasadded to Con A-plus-IL-2-stimulated cultures by 8 hr orlater, it exertednodetectable effectonsynthesisandaccumulationof RNA(data not shown).
B Cell
Biology:
GauchatetAProc. Natl.Acad. Sci. USA83 (1986)
4 er ei, Z o x 2
e 0
cn u0.
WIcUli
0 40 80 0 40 80
Time, hr
40 ',I.K
30 °
W
20 X
10 .S
0
FIG. 3. (A)Incorporationof[35S]methionineintoprotein during 1-hrperiods by unstimulated thymocytes (A---A)orby thymocytes stimulated with Con AplusIL-2 in theabsence(o-o)orpresence
(l-o*) ofCsA. (B) Protein content of cultures (symbolsasinA), determined colorimetrically. Inunstimulatedcultures, CsA hadno
detectable effect on the incorporation of [3-S]methionine and on
proteincontent(datanotshown).
Protein Synthesis. Unstimulated cultures contained about 15ggof proteinper106 thymocytes(Fig. 3), corresponding toless than10% of theproteincontentof G0-arrestedmouse
kidney cells(24).After addition of Con Aplus IL-2, protein content increased by 20-25 hr and reached a maximum around 80 hr(Fig. 3).
Unstimulatedcultures and cultures stimulated withCon A plus IL-2 and incubated in theabsenceorpresenceof CsA
werelabeled for 1hrwith[35S]methionineatdifferent times between 3 and70hr,andacid-precipitable radioactivitywas
determined (Fig. 3A and ref. 19); aliquotsof allpreparations
wereanalyzed by electrophoresis inone(NaDodSO4/PAGE)
ortwo dimensions (32), and theproteins were revealedby stainingthegelswithCoomassie blue andby autoradiography
orfluorography.From the results(19), weconcluded that in unstimulated cultures, residualincorporation of[35S]methi- onine reflected overall protein synthesis, similar to that observed in G0-arrestedmousekidneycells(24), thoughata
much lowerrate(19).Incultures stimulated with Con Aplus IL-2,theapparent rateofproteinsynthesisincreasedaround 6hr-i.e., about 3 hr after the onsetofstimulated hnRNA synthesis-reachingamaximumby60-80 hr(Fig. 3A).The stimulation of [35S]methionine incorporation that was in- duced by Con A and IL-2 reflected, at all times tested, increased overallprotein synthesis (19).
In unstimulatedcultures,CsA hadnodetectable effecton
proteincontent, ontheincorporation of[35S]methionine, or
on the protein patterns (ref. 19 and unpublished data). In cultures stimulated with Con A and IL-2, CsA virtually preventedtheincreaseinproteincontent(Fig. 3B). However, stimulationof[35S]methionine incorporation became notice- able around 6hr,reachedamaximumaround 15 hr(Fig. 3A), andthen decreasedtothelowlevel ofunstimulatedcultures;
whentested between 10 and 12 hr, incorporation of [35S]me- thionine was 30-50%lower than in parallel cultures stimu- lated in the absence of CsA.Theproteinpatternsofcultures stimulated with Con AplusIL-2in thepresenceof CsAwere
indistinguishable from those observed in parallel cultures incubatedwithoutCsA, withtheexception that radioactivity
wasmarkedly lower(unpublished results).
Expressionof the IL-2 ReceptorGene. TheIL-2 receptors on the cell surface were visualized by indirect immuno- fluorescence using a monoclonal antibody (PC61; ref. 25) directed against the mouse IL-2 receptor. In unstimulated cultures and also in Con A-plus-IL-2-stimulated cultures, tested at 0.5 and 3 hr, less than 0.5% of the thymocytes exhibitedanimmunofluorescencereaction,whereasby6hr afterstimulation with Con Aplus IL-2, 15-20%of thecells showedontheirsurfacenumerous, ratherregularly distrib- uted fluorescent dots (Table 1); the relative number of immunofluorescent cells then increased, reaching a maxi-
Table 1. Cell surfaceexpression of IL-2receptor
%immunofluorescent cells
Cultures 6 hr 9hr 20hr 40hr
Unstimulated <0.5 <0.5 0 0
Stimulated
NoCsA 15 22 19 (6*) 32*
Plus.CsA (O hr) 6 4 3(1*) 2*
PlusCsA (8 hr) ND ND ND 28*
Stimulated cultureswere exposedtoConA plusIL-2 from time
zero.ND, notdetermined.
*Strongimmunofluorescence reaction.
mumplateau of 35 ± 5% by 20-25 hr (tested until 60 hr). A
very strongincrease in the intensity of theimmunofluores-
cence reaction, which thencovered the entire cell surface,
was noted on thymoblasts around 20 hr and later. Acti- nomycin D (1 ,ug/ml)orcycloheximide (25 ,ug/ml) addedat 3 hr or 4hr, respectively, prevented the appearance of the IL-2receptors, determinedat7 hr;incontrast,exposureof ConA-plus-IL-2-stimulated cultures tocycloheximidefrom 36-40 hrhadnodetectable effectonthe immunofluorescence reaction. After addition ofCon A alone, the timecourseofthe
appearance of fluorescent dots was indistinguishable until about 20hr,whenthepercentageof fluorescent cells rapidly decreased, corresponding by 40-60 hrtoonly 2-3% strongly positive thymoblasts. In cultures stimulated with Con A plus IL-2, CsA reduced the percentage of immunofluorescent cellsto3-5% whentestedatdifferenttimes between6and20 hr, and by 40-60 hronly 1-3% of the cells (thymoblasts) exhibitedastrongimmunofluorescence reaction (Table 1).In contrast,whenCsAwasaddedat8 hrorlater,the timecourse of cell surfaceIL-2receptorexpression, asjudgedfrom the immunofluorescence reaction(Table 1 and unpublished da- ta),wasindistinguishable from that observed intheabsence of CsA. In cultures incubated with Con Aalone,CsA reduced themaximumpercentage ofpositive cellstoabout3%,and by 40 hr and later, less than 1% immunofluorescent cells could be detected.
B
A 2 3
kDa 205-
116- 97-
12 3 45 6 7
kb
66- 28S-
45-3 185-
w-3.5 2.2 1.5
29- W
12-
FIG. 4. (A)Synthesis of theIL-2receptor.Cultureswerelabeled for15minwith[35S]methionine,and theIL-2receptorwasisolated byimmunochromatographyandsubjectedtoNaDodSO4/PAGEand autoradiography (7 days exposure). Lane 1: unstimulated thymo- cytes. Lanes2 and3: thymocytes stimulatedwithConAplusIL-2 for 15 hr in the absence or presence of CsA, respectively. (B) Steady-statelevelsof IL-2receptormRNA(s), measuredbyhybrid- ization with nick-translated pmIL-2R-1 plasmid. Autoradiography
wasfor15hr,withtheexception oflane 7. Lane1:unstimulated cells.
Lanes2-4:cellsstimulatedwith Con AplusIL-2 for8, 15,and 30 hr.
Lanes5and 6:cells stimulated withCon AplusIL-2 inthepresence ofCsA for 15and30 hr. Lane7:same aslane4,butautoradiography
wasforonly2 hr.In cultureswhereCsAhad been added8hr after ConAplus IL-2, themRNA patterns wereindistinguishable from those shown in lanes 1-4.
-A B -
-~ ~~ X ~~~~~~~---
- --- 11X1 ---Ala-- Am--In
6432 Cell
Biology:
Gauchatetal.Proc. Natl. Acad. Sci. USA 83 (1986) 6433
PRIMING
"early"stimulation of
hnRNA
-vra,
synthesis synthesis
expression of the IL-2 receptor gene
PHASE 1
BLA$T FORMATION AND MITOTIC ACTIVATION massive stimulation
IL-2 of nucleolar.and + nucleoplasmic
transcription and of overall protein synthesis
J-S-phase-mitosis
PHASE 2
FIG. 5. Sequence of the major molecular events during mitogen-induced blast formation and mitotic activation of GO-arrested mouse thymocytes.
Tostudybiosynthesis of the IL-2receptor, cultureswere incubated with [35S]methionine for 15 minat 15hr,and the receptor wasisolated by immunochromatography and sub- jected to NaDodSO4/PAGE. Autoradiography of the gels (Fig. 4A) revealed that isolates from Con A-plus-IL-2- stimulated cultures contained radioactive polypeptides of apparent molecular mass ranging from 30 to 65 kDa; the broad upperband (50-65 kDa) corresponds to the surface- labeled IL-2 receptor (25), whereas the group of faster moving bandsmostlikelycorrespondstotheunglycosylated (30) and underglycosylated forms of the IL-2 receptor. In contrast, noradioactivebands couldbedetectedinextracts from unstimulated culturesorfrom culturesstimulatedwith Con Aplus IL-2butincubatedin thepresenceofCsA.
Steady-state levels of IL-2receptormRNA(s)weredeter- minedin total RNA extracted withphenol 8, 15, and 30 hr after stimulation by Con A plus IL-2 in the absence or presence of CsA and from unstimulated parallel cultures.
Aliquots ofRNA were subjectedto electrophoresis, trans- ferred toGeneScreen membranes, fixed by UV irradiation (29), and then hybridized with 32P-labeled nick-translated pmIL-2R-1, a probe specific for the mouse IL-2 receptor mRNA(s) (30).Autoradiography revealed, in samples taken 8 hr after the addition of Con Aplus IL-2,radioactivebands atthepositions expected formouseIL-2receptormRNA(s) (3.5, 2.2,and 1.5kilobases(kb)andtraceamounts at4.5kb;
Fig. 4B);themRNA(s) markedly increased around 15-20hr, coinciding in time with the strong increase in the im- munofluorescencereaction for the IL-2receptor(see above).
Inunstimulatedculturesandalsoin culturesincubatedwith Con AplusIL-2 inthepresenceofCsA,onlytrace amounts of3.5-kb IL-2 mRNA could be detected (Fig. 4), possibly reflecting the small percentage of immunofluorescent thymocytes observed in unstimulated cultures. When CsA
was added at 8 hr or later, steady-state levels of IL-2 mRNA(s)wereindistinguishable from those foundinparallel cultures stimulated with Con A and IL-2 but incubated without CsA (datanot shown).
DISCUSSION
Inunstimulated, G0-arrested mouse thymocytes, transcrip- tionisverylow and virtuallyrestrictedtohnRNA,whereas residual translation reflects overallprotein synthesis. Addi- tion ofConAplusIL-2 leadsin 35 + 5%of thethymocytes toblastformationandmitoticactivation.Thesequenceofthe major molecular events reported here is shown in Fig. 5.
"Priming" (phase 1) is induced by Con A alone; "early,"
low-levelstimulationof hnRNA andprotein synthesis begins around 3 and 6 hr, respectively, and is paralleled by the expression of the IL-2 receptor gene; phase 1 apparently reflects a complex, Con A-induced reprogramming that renders thethymocytescompetent tointeract with IL-2 and to respond to its growth-promoting effect, referred to as
phase2. Thisphase beginsaround 10hr;itcomprisesblast formation, mitotic activation, and, by 15-20 hr, amarked increase in the expression ofthe IL-2 receptorgene. The
sequenceof theeventsinphase 2 issimilartothatobserved during mitotic activation triggered inquiescent mammalian cells by oncogenic DNA viruses andserum growthfactors (23, 24, 33).
Inunstimulatedthymocytes, CsA (0.3 ,g/ml) has virtually
no effect on residual DNA, RNA, and protein synthesis.
When added at time zero (i.e., together with Con A plus IL-2), CsApreventstheexpression of the IL-2receptorgene
andphase 2, whereastheearly stimulation (until 6-7hr)of hnRNAandprotein synthesis isnotdetectably inhibited. If CsA isaddedat8 hrorlater, after priming, itnolongerexerts
anyinhibitory effects; this is in accordance with the obser- vation that CsA acts early in T-lymphocyte activation (3).
Prevention ofIL-2receptor gene activation seems to be a
major, thoughmostlikelynotthe only, effect by whichCsA blocks mitogen-inducedblastformation and mitotic activa- tion. Inquiescent mouse 3T3 fibroblastand mousekidney cell cultures, CsA (1 ,ug/ml) inhibits neither cellular RNA synthesis northe virus- or serum-induced mitotic reaction (19); this tendstoexclude the possibility that CsAexertsits immunosuppressive effect by inhibiting mammalian RNA polymerase II (34)orby blocking the transition ofmammalian cells fromaquiescent to aproliferative state.
Theexperimental observationsnowavailable indicatethat CsApreventsin Tlymphocytes the activation ofaset(s) of
genesencodinglymphokines and the IL-2receptorbutthatit doesnotaffect theexpression of thesegenes oncethey have beenactivated; theysuggestthatprevention of theactivation ofspecificgenesinTlymphocytes playsapivotal rolein the immunosuppressive action of CsA. Wemayexpectthat the understanding of themolecular mode ofaction ofCsA will provideasteptoward thechemotherapy of mammaliangene
expression, leadingtothedevelopment of drugsthatmodu- late the activity of specificgenes.
WearegratefultoDr. S.Cammisuli (Basel) for the EL-4 cell line;
Drs. H. R.MacDonald and M.Nabholz(Lausanne, Switzerland)for themonoclonal antibody PC61; Dr. M. Nabholz forperformingthe radioimmunoassayshowninFig. 4; Prof.T.Honjo (Kyoto, Japan) for thepmIL-2R-1 plasmid;and Drs.S.Cammisuli,B.Ryffel,and H.
Stahelin(Sandoz A.G., Basel, Switzerland) for useful discussions andsuggestions. We gratefully acknowledge the critical readingof the manuscript by Drs. R. Lamers and T. Staehelin (Max Planck InstitutfurImmunobiologie, Freiburg, F.R.G.) and Prof. J.-C.Jaton (UniversityofGeneva). We thankMrs. Y.Eprechtand Mr.0.Jenni forpreparing the figures. This workwassupported byGrant3.072.81 from theSwiss National Science Foundationandbygrantsfrom the SandozResearch Foundationandthe Swiss CancerSociety.
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