Induction, suppression and superinduction of lymphokine mRNA in T lymphocytes

Molecular Immu~al~gy, vol. 24, No. 5, pp. 409419, 1987 Printed in Great Britain.

0161.5890/87 $3.00 -t 0.00 Pergamon Journals Ltd

INDUCTION, SUPPRESSION AND SUPERINDUCTrON OF LYMPHOKINE mRNA IN T LYMPHOCYTES*

JENNIFER SHAW, KAREN MEEROVITCH, JOHN F. ELLIOTT, R. CHRIS BLEACKLEY and QRNER PAETKAU

Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

(Received 11 My 1986; Accepred 22 September 1986)

Abstract-The expression of several lymphokine genes is characterized by a common pattern of induction, suppression and superinduction. This pattern was studied at the level of cellular mRNA in the mouse T-~ympboma cell line EL4, the human T-leukemia line Jurkat and in normal human peripheral blood lymphocytes. Lymphokine mRNA was induced by stimulating the cells with the phorbot diester PMA (TPA), with or without T-lymphocyte mitogens. The induction of Interleukin-2, Interferon y and the Colony Stimulating Factor for granulocytes and macrophages was suppressed by Cyclosporin A at moderate concns. Furthermore, these mRNAs accumulated to extraordinarily high levels (superinduction) if the protein synthesis inhibitor cycloheximide was added during transcription. Superinduction was not due to an increased rate of transcription, CsA interrupted ongoing transcription of IL2 by a mechanism not dependent on the induction of a new protein. The co-ordinate regulation of these genes strongly suggests that common intracellular signals mediate their expression.

INTRODUCTION

Synthesis and secretion of lymphokines is highly regulated in normal T-helper Iymphocytes and in several human and murine T-lymphocyte cell lines. The phorbol diester 12-myristate-l3-acetate (PMA)f replaces the need for the macrophage factor Interleukin- 1 (ILI) in stimulating the production of Interleukin-2 (IL2) from normal T-lymphocytes (Farrar et al., 1980h). It is a co-factor in the induction of IL2 synthesis by cells of the human T-leukemia line Jurkat (Gillis and Watson, 1980), and it directly stimulates the production of lymphokines in the mouse T-lymphoma cell line EL4 (Farrar et al., 1980~). PMA induces EL4 cells to secrete a number of lymphokines, including IL2 (Farrar et al., 1980a), IL3 (Pearlstein et af., 1983), GM-CSF (Pearlstein et

ol., 1983; Bleackley et al., 1983) and both a growth factor (Howard et al., 1982) and a differentiation factor (Pure et al., 1982) for B lymphocytes.

In addition to being inducible, lymphokines are also affected by some immunosuppressive agents. Thus, many of the effects of Cyclosporin A (CsA) (Bore1 et at., 1976), an undecapeptide fungal metabo- lite with clinically useful immunosuppressive activity,

*This work was funded by the Medical Research Council of Canada, the National Cancer Institute of Canada, and the Alberta Heritage Foundation for Medical Research.

~Abbreviations: CHX, cycloheximide; CsA, CsD, Cyclo- sporin A (Cyclosporine), Cyclosporin D; DTT, dith- iothreitol; GM-CSF, Colony Stimulating Factor for granulocytes and macrophage; IFNy, Interferon y; ILI, IL2. Interleukin 1, Interleukin 2; MMTV, mouse mammary tumor virus; PBL, peripheral blood leukocytes; PMA, phorbol IZ-myristate 13-acetate (also TPA).

can be rationalized by its demonstrated ability to inhibit the induction of IL2 synthesis (Bunjes et al.,

1981). On the other hand, CsA has no effect on the proliferation of IL2-dependent T lymphocytes (Bunjes et al., 1981; Larsson, 1980; Orosz et al.,

1982). The expression of IL2 induced by PMA in the human T lymphoma cell line Jurkat is suppressed by CsA at the mRNA level (Elliott et al., 1984; Kronke et al., 1984; Wiskocil et al., 1985) and Interferon y (IFNy) is similarly affected (Wiskocil et aZ., 1985). IL2 mRNA activity, as measured by translation in Xenopus Iaevis oocytes, was also shown to be induced in EL4 cells by PMA and to be suppressed by CsA (Granelli-Piperno et al., 1984). Normal murine and human T lymphoblasts show the same pattern of induction by the mitogen Con A and suppression by CsA for IL2 and IFNy (Granelli-Piperno et al., 1984, 1986), and secretion of IFNy by human T lymphocytes is suppressed by CsA (Reem et al., 1983). Antigen-dependent induction of IL2 and CSF activities in T-lymphocyte hybridoma cells was stopped within l-2 hr of adding CsA (~aufmann et al., 1984). These effects of CsA may account in part for its inhibition of mixed leukocyte reactions and cytotoxic T-lymphocyte responses (Bunjes et al.,

1981). Elsewhere, we have shown that the effects of PMA

and CsA on the EL4 variant line EL4. E 1 are specific, being limited to the expression of only a few genes (Paetkau et aI., 1985; Elliott et al., unpublished). In this paper, we examine the effects of these agents on lymphokine and related mRNAs in mouse EL4. E 1 T cells, human Jurkat T cells, and human peripheral blood T lymphocytes (PBL).

409

410 JENNIFER SHAW et al.

Another aspect of l~phokin~ mRNA expression is superinduction, which is the accumulation of very high levels of an mRNA (or protein) in the presence of one or more inhibitors of macromolecule synthesis. Superinduction has previously been observed to occur with both murine IL1 (Mizel and Mizel, 1981) and human tonsil IL2 (Efrat and Kaempfer, 1984). In the present work, a number of lymphokine mRNAs have been found to be superinduced by the protein synthesis inhibitor cycloheximide (CHX).

MATERIALS AND .~ETHODS

Ceils and culture conditions

The culture medium and the conditions of growth of the mouse T-lymphoma line EL4. El and its stimulation with PMA to generate lymphokines are described elsewhere (Elliott et al., unpublished). The human T-leukemia cell line Jurkat was grown in the same medium. To induce the optimal synthesis and secretion of lymphokines from this line (Gillis and Watson, 1980) it was necessary to use both PMA (15 ng/ml) and the mitogen Con A (30 pgjml). Human PBL were isolated from normal venous blood and stimuIated in the same way as Jurkat cells.

Isolation and c~ara~teri~at~on of m&V.4

Total cellular RNA was isolated either by the method of Chirgwin et al. (1979), or by a rapid method as follows. PBS-washed cells (l-5 x lo6 total) were pelleted in Eppendorf tubes, resuspended in 0.1 ml of 10 mM Tris, pH 8.0-l mM EDTA (TE buffer) and lysed with 0.05% NP40 (final concn) at 4°C. Nuclei were then rapidly removed by centrifu- gation, and the cytoplasmic extracts were incubated for 15 min at 37°C in 0.4ml TE containing 5~lOO~g/ml Proteinase K (BRL) and 0.5% SDS. Fifty micrograms of yeast tRNA was added as car- rier, and the RNA was precipitated with ethanol and resuspended in 0.1% SDS-25 mM EDTA. In some

experiments, total cytoplasmic RNA was analyzed by the “cytodot” method (White and Bancroft, 1982).

Gels for Northern analysis of mRNA consisted 01 0.8% agarose (BioRad) in buffer containing 20 mM MOPS (3-[N-morpholinolpropane-sulfonic acid), pi-l 7.0-5 mM NaOAc-1 mM EDTA-1% formaldehyde and 1.5 pg/ml ethidium bromide. RNA samples (10-30 pg) were incubated at 55°C for 15 min in 20 /ll of the same buffer containing 6.5% formaldehyde and 50% deionized formamide, before being cooled to 4°C and loaded onto the gel. Gels were run for 3-4 hr at 100 V, soaked in 20 x SSC for 0.5 hr and transferred to BASS nitrocellulose filters (S~hleiclle~- and Schueli Ltd) which had been soaked for I hr in 2 x SSC. Nitrocellulose filters were baked in an 80 C vacuum oven for 1.5-2 hr.

Filters were prehybridized for 6-18 hr at 42 C in a solution containing either 20% (for oligonucleotide probes) or 50% (for full length cDNA probes) formamide, 50mM sodium phosphate, pH 6.S, 5 x Denhardts solution, 5 x SSC, 2.5 mM EDTA. 2 mM sodium pyrophosphate, 100 HIM ATP, lOO/~g/ml heat denatured salmon sperm DNA and lOO~gg!mI yeast tRNA (Maniatis ef ai., 1982). Hy- bridization to ‘2P-fabeled DNA probes was carried out in the same solution, for 18 hr at 42 C. Filters which had been hybridized with synthetic oligonucleotide probes were washed for 1.5 hr at 42 ‘C in 2 x SSC--0.1% SDS. Filters hybridized with cDNA probe were washed for 1 hr at 55.C in 2 x SSC--0. 1% SDS, followed by a wash for 0.5 hr at 55 C in 0.2 x SSC-0.1% SDS.

Probes

Probes used in this work are described in Table 1.

Oligonucleotide probes were synthesized on an Ap- plied Biosystems nucleotide synthesizer by Dr P. Barr, Chiron Corp. They were based on published sequences for human (Taniguchi et al., 1983) and mouse (Fuse et al., 1984) IL2. human IL2 receptor

Table I. Descriution of arobes

Probe Length

H-IL?-34 34 H-IFNy-30 30

M-GM-43.13 43

M-GM-43.14 M-GM-43.15 HpIL2.1

43 43

820

M-pIL2. I 960

H-pILZR.5 1150

M-pGM.1 900

M-p86T5 660

pHFyA-I 2100 Probe IO 750

Region encoded

I I amino acid residues at the C-terminus of human and mouse IL2 Ammo acids 21-30 of the primary translatmn product of human IFN-; Amlho acid residues 76-89 of primary translation product of mouse GM-CSF Same, amino acids 90-194 Same, amino acids 105-118 Coding region plus 100 n of 5’ and 260 II of 3’ untranslated region of human IL2 Coding region plus 130 n of 5’ and 320 n of 3’ untranslated region of mou*e IL2 Coding region plus 165 tl of 5’ and 155 n of 3’ untranslated region of human IL2 receptor Coding region plus 50 n of 5’ and entire 3’ untranslated region of mouse GM-CSF Amino acids 85-306 of mouse T-cell antigen receptor /? chain mRNA (Hedrick et a[., 1984) Human y-actin cDNA (Gunning et al., 1983) Constitutively expressed mRNA in EL4.EI cells, unaffected by PMA (Paetkau er al.. 1985)

Specific effects of PMA, CSA and CHX on lymphokine mRNA levels 411

(Leonard et al., 1984), mouse GM-CSF (Gough et al., 1985) and human IFNy (Gray et al., 1982).

A cloned cDNA for human IL2 receptor was isolated from a cDNA library derived from Jurkat cells which had been stimulated for 16 hr with PMA and Con A. The library (400,000 plaques in IgtlO) was produced in the same way as described for EL4.El cells (Elliott et al., unpublished). It was screened with two synthetic oligonucleotide probes specific for the 5’ and 3’ ends of the coding sequence of the receptor. A human IL2 cDNA clone (H-pIL2.1) was also isolated from the Jurkat cDNA library by screening with synthetic oligonucleotide probes specific for the carboxyl-terminus and the amino- terminal region. A library generated in lgtl0 from PMA-stimulated EL4.El cells was used to derive cDNA clones for mouse IL2 and GM-CSF. Two oligonucleotide probes were used to identify IL2 cDNA clones, and the GM-CSF cDNA clones were identified using a mixture of three oligonucleotides. We have previously described a cloned cDNA, probe 10, which detects an mRNA of unknown function which is ~onstitutiveIy expressed in EL4. El cells, and whose expression is unaffected by PMA (Paetkau et al., 1985).

Synthetic oligonucleotide probes were labeled with 32P using polynucleotide kinase, to a final sp. act. of 3-6 x lo* dpm/pg, and used at a concn of 3-10 ng/ml (0.9-3 x 106dpm/ml) in the hybridization solution. The cDNA probes were nick translated using DNA polymerase to a sp. act. of 2.7-4.8 x lO’dpm/pg, and used at 1.5-3 x lO”dpm/ml.

Isolation of nuclei and run-on transcription assay

Transcription in isolated nuclei, and hybridization of nascent RNA to cDNA probes bound to nitrocellulose filters was carried out essentially as described elsewhere (M&night and Palmiter, 1979; Kronke et al., 1984). EL4.El cells were washed once in buffer containing 1OmM Tris-HCl, pH 7.5, 2 mM MgCl,, 3 mM CaCl,, 3 mM DTT and 0.3 M sucrose, resuspended in the same buffer, and lysed by adding NP40 detergent to a final concn of 0.2%. They were kept for 10min at 4°C and then pelleted through a 2 M sucrose cushion (containing 10 mM Tri-HCl, pH 7.5, 2mM MgCl,, 3 mM CaCl, and 3 mM DTT) in a Beckman SW50.1 rotor at 20~000 rpm for 45 min at 4°C. Nuclei were resuspended in buffer containing 25mM Tris-HCl (pH 8.0), 25% glycerol, 125 mM KCl, 5.6 mM MgCl, and 2.5 mM DTT, and frozen at -70°C for up to 2 weeks before being used.

For run-on transcription, 5 x IO7 nuclei were incubated for 15 min at 26°C in a 200 gl reaction containing 500 PM each of ATP, CTP and GTP, 2 mM DTT, 20% glycerol, 1OOmM KCI, 20mM Tris-HCI (pH 8.0), 4.5 mM MgCl, and 0.1 mCi of z-~‘P-UTP (600 Ci/mmole; NEN). The reaction was stopped by adding yeast tRNA to lOO/~g/ml, and a mixture of DNase I and Proteinase K (final concn lOO~g/ml

each) which had been incubated for 15 min at 37°C in 10 mM CaCl, to selectively destroy RNase (Tullis and Rubin, 1980). SDS was added to a final concn of I%, and the RNA was isolated by one extraction with phenol-chloroform and one extraction with chloroform, followed by three precipitations with ethanol. Samples were dissolved in 0.1 ml of 0.1% SDS, added to 1.5 ml of the hybridization buffer, containing 30% formamide and 100 PIM UTP, and heated to 70°C for 10min before being added to filters.

Linearized plasmid DNA was denatured by incubation in 0.4N NaOH for 10min at room temp., cooled on ice and neutralized by adding an equal vol of 2 M NH,OAc, pH 5.5. DNA was filtered onto BA85 nitrocellulose using a Schleicher and Schuell manifold apparatus. Each spot contained 1 jig of probe DNA.

Samples containing 4-S x IO5 dpm were added to the DNA-containing filters. Hybridization was carried out for 3 days at 42°C. Filters were washed for 45 min in 2 x SSC-O.l% SDS at 55”C, and then incubated for 45 min at 37°C in 2 x SSC containing 10 I*g/mI RNase A. Proteinase K was added to a final concn of SOpg/ml, and incubation was continued for a further 45 min. Filters were washed again for 20 min in 2 x SSC-O.l% SDS and exposed to X-ray film.

RESULTS

IL2, GM-CSF, and IFNy mRNAs are selectivel,v induced by PMA and mitogens, and suppressed by CsA

Lymphokine genes are not constitutively expressed, but are induced by mitogens. In several cases induction has been found to be blocked by CsA. The results in Fig. l(A) show that in unstimulated cells of the mouse T lymphoma line EL4. El, no mRNA for IL2 or GM-CSF could be detected by hybridization with cloned cDNA probes. On exposure to PMA, both mRNAs increased to levels easily observed on Northern gel analysis. The induction of both IL2 and GM-CSF mRNAs was suppressed by moderate levels of CsA (100 ng/ml or less). By contrast, the overall levels of mRNA for the /? chain of the T-lymphocyte receptor were not increased by PMA nor were they diminished by CsA, although PMA did induce the production of a smaller form of the mRNA [Fig. l(B)]. The shorter mRNA probably corresponds to the truncated version lacking a V region, as reported earlier by Yoshikai et al. (1984). Other genes whose expression was not affected by PMA or CsA in EL4.El cells included actin and probe 10mRNA (Paetkau et al., 1985), which is ~onstitutively expressed in EL4.El cells (data not shown).

Human PBL were induced to accumulate IL2 mRNA by stimulation with Con A plus PMA, and this increase was blocked by CsA, as in the case of EL4.El IL2 and GM-CSF (Fig. 2). The mRNA for IFN? in human PBL showed the same pattern of

412 JEKNIFEK SHAW rt ~1

(A) EL4, I8 hr

OM-CSF fL2

Fig. 1. Induction, suppression of induction and superinduction of IL2 and GM-CSF mRNA in EL4. El cells. (A) EL4.El cells were exposed to PMA (20 ng/ml) in the absence or presence of CsA (100 ngjml) or CHX (20 pg/ml). PMA was present in all samples except the control (left hand lane in each set) from 0 time, and CHX CsA were added at the times indicated. Cells were harvested at 18 hr, and RNA was prepared by the rapid method and analyzed by agarose gel electrophoresis and Northern blotting. Cloned probes M-pGM. 1 or M-pIL2. I were used to detect mRNA for GM-CSF or IL2, respectively. Each track represents the total RNA from 2.5 x lo6 cells. Both mRNAs were undetectable in unstimulated cells, and induced by PMA. Induction was blocked by CsA added at 0 time for both cytokines. CHX added at 0 time blocked induction, but added at 14 hr (4 hr before harvest), it led to superinduction of both mRNAs. (B) EL4.El cells were stimulated as in panel A, and 10 fig per track of poly A+ RNA was loaded. The bands were detected using probe for the /I chain of the T-cell receptor (probe M-p86T5, Table I). The shorter transcript corresponds to the truncated mRNA. lacking a V region, reported earlier (Yoshikai (‘I

cl/., 1984).


PBL. 24 hr ..-... --.-.

IL2 \./ II.Z?-R

TIME: I

PMA CsA CHX

Fig. 2. Induction, suppression and superinduction of IL2 and IFN? mRNAs in human PBL. Human PBL were stimulated for 24 hr with PMA/Con A and the other agents as indicated. Total RNA prepared by the guanidinium isothiocyanateeCsC1 method (Chirgwin et al., 1979) was analyzed by gel electrophoresis as in Fig. I (20 fig per track). Replicate gels were probed for human IL2 (probe HpIL2. I), human IL2 receptor (probe H-pIL2R.5) human IFNy (probe H-II?+-30) or human actin (probe pHFy A-l) (probes described in Table 1). CHX was present where indicated at 20pg/ml, and CsA at l~ng~ml. Both IL2 and IFNg mRNAs were not expressed constitutively, but were inducible. They were both suppressed by CsA, and CHX at 0 time blocked induction, but not completely. Added at 20 hr, CHX led to superinduction of both mRNAs. IL2-R mRNA was induced, but not blocked by CsA, and was less sensitive to superinduction by CHX. Actin was largely unaffected by any of the conditions, except for

inhibition by long term exposure to CHX.

JENNIFEZR SHAW et (II

CONTROL

PMA+CsA (Oh)

PMA+CHX (Oh)

PMA

PMA+CHX (411)

Fig. 3. Rapid superinduction of IL2 mRNA by CHX. Jurkat cells were stimulated with PMA (I 5 ng/ml) and Con A (30 pg/ml) and total cellular RNA analyzed by gel electrophoresis and blotting with cloned probe H-IL2.1 to detect human Ii2 mRNA. Each track represents total RNA from 2.5 x IOh cells. CsA was present at 100 ng/ml and CHX at 20 pg/ml. CsA or CHX added initially completely blocked induction, but added at 4 hr, CHX superinduced IL2 mRNA levels in just 2 hr of further incubation. The

peak of IL2 mRNA levels in these cells generally occurs between 4 and 6 hr.

induction and suppression. The mRNA for human

IL2 receptor was not detectable in unstimulated PBL

(Fig. 2). Upon exposure to Con A plus PMA, two mRNA species were detectable by hybridization to probe specific for the human IL2 receptor, and these were of the sizes reported earlier for the predominant species of this mRNA (Leonard et al., 1984). In contrast to the complete suppression of IL2 and IFNy mRNA by CsA, there was only a slight inhibition of the expression of IL2 receptor mRNA. Actin mRNA was constitutively expressed, and the level was neither significantly increased by the inducing agents nor suppressed by CsA in the cultured PBL (Fig. 2).

The most rapid induction of IL2 mRNA was seen with the human T-leukemia cell line Jurkat (Fig. 3). This induction was maximal at 4-6 hr, and sub-

sequently declined (Shaw et al., unpublished). The intense signal of IL2 mRNA seen 4 hr after stimulation was completely sensitive to CsA. The T cell p chain mRNA was also measured for Jurkat cells, with the identical result as in Fig. l(B) for EL4. El cells- namely, there was neither significant induction nor suppression (data not shown).

IL2 and GM-CSF are co-ordinate1.l; suppressed by, cyclosporins

The simultaneous induction of several lymphokines under given conditions, and their suppression by CsA, suggests that a common intracellular mechanism regulates lymphokine gene expression. This suggestion is further supported by the responses of IL2 and GM-CSF mRNAs to suppression by two different cyclosporins, CsA and CsD, as shown in Fig. 4. Although the two immunosuppressive agents

differed somewhat in their efficacy, in each case the suppression of the two lymphokines followed, within experiments] error, the same dose-response curves.

Inhibition of protein synthesis blocks induction of lymphokine mRNA in two T-lymphocyte cell lines, but

not in PBL

The induction of both IL2 and GM-CSF mRNAs

in EL4 cells was blocked by the protein synthesis

inhibitor CHX when it was present from the outset

[Fig. l(A)]. This indicates that protein synthesis is

Fig. 4. Co-ordinate suppression of IL2 and GM-CSF mRNA expression by Cyclosporins. EL4. El cells were stimulated with PMA in the presence of various concen- trations of either CsA or CsD. The cytoplasmic contents of IL2 or GM-CSF mRNAs were determined at l2hr by cytodot analysis of serial 2-fold dilutions of RNA, and the autoradiograms were analyzed by densitometry. The unin- hibited response is given as 100% for both lymphokine mRNAs. The probes in this case were H-IL2-34 (for IL2) and the mixture of GM-CSF oligonucleotides (M-GM-

43.13. -14. -15).

Specific effects of PMA, CSA and CHX on lymphokine mRNA levels

required to induce the transcription of these genes. Similarly, no IL2 mRNA accumulated in Jurkat cells induced in the presence of CHX (Fig. 3). By contrast, CHX added at time 0 did not greatly alter the level of mRNA for IL2 or IFNy which accumulated by 24 hr in stimulated PBL (Fig. 2). The IL2 receptor, which was induced by mitogen plus PMA, was also not significantly affected. Curiously, actin mRNA, which was neither induced nor suppressed by CsA, did diminish over the 24 hr of the experiment if CHX was present.

Time of

harvest: IO h

125

25

The mRNAsfor IL2, GM-CSF and IFNy are superinduced bJ> CHX after transcription is initiated

“0 2 4 6 6 10

CHX added (hr)

If CHX was added after induction was well under way (6 hr before the cells were harvested in a 20 hr experiment), both IL2 and GM-CSF mRNAs accumulated to extraordinarily high levels in EL4.El cells, a phenomenon referred to as superinduction [Fig. l(A)]. Superinduction of both IL2 and IFNy mRNAs was also observed if CHX was added 4 hr before harvesting stimulated PBL at 24 hr (Fig. 2). Actin mRNA in PBL was not superinduced, but IL2 receptor mRNA was, albeit not as drastically as the two lymphokines. The most dramatic superinduction occurred with Jurkat cells, where the addition of CHX at 4 hr led, in 2 hr, to a massive increase in IL2 (Fig. 3).

Fig. 5. The effect of time of addition of CHX on superinduction of IL2 mRNA in EL4. El cells, Jurkat cells, and human PBL. The indicated cells were cultured in Costar 6-well Dlates for 10 hr. CHX (20 ueimf) was added at the times indicated on the abscissa; andcytddot analysis of IL2 mRNA was performed. The blots were probed and scanned as in Fig. 4. Data are expressed as relative densitometer

intensities.

seen, CHX did not completely block the accumulation of IL2 mRNA even when added at 0 time. However, the highest level of superinduction was achieved if CHX was added to 6 hr and 4 hr before harvest.

The latter effect is illustrated quantitively in Fig. 5. CHX was added to cells at various times, and cyto- dots were prepared at 10 hr. The results show again that both EL4.El and Jurkat cells could not be induced to accumulate IL2 mRNA in the presence of CHX from 0 time. However, CHX added either after 1 hr (for Jurkat cells) or 2 hr (for EL4. El) rapidly led to superinduction. Even when added just 2 hr before harvest (i.e. at 8 hr), CHX caused superinduction. PBL showed a slightly different pattern. As already

A summary of the inductive effects of PMA (with

or without mitogen, as required), and of suppression of induction by CsA, is given in Table 2. The synthesis of IL2, GM-CSF and IFNy mRNA was induced by PMA (plus Con A in the cases of Jurkat cells and PBL) and suppressed by CsA. The mRNA for IL2 receptor was also induced by PMA, but not affected by CsA, in Jurkat cells. PMA induces the appearance of the IL2 receptor (Tat) on these cells (Greene et al., 1984). Actin, and the control probe 10

415

Table 2. Expression and suppression of specific mRNA in T lymphocytes

Expression PMA

Product Cells Unstimulated (+ Con A) CsA Ref.

IL2 _____~ EL4, PBL _ + _ Figs I,2 Note I

Jurkat _ + _ Note 2 GM-CSF EL4 _ + _ Fig. I IFN? PBL, Jurkat - + _ Fig. 2

Note 3 IL2-R PBL, Jurkat _ + + Fig. 2

Note 4 TCR-8 EL4, Jurkat + + + Fig. l(B) MMTV EL4 f ++ i Note 5 Actin PBL, Jurkat + + + Fig. 2 Probe 10 EL4 + + + Note 6

The expression of a given mRNA is graded from - (undetectable by Northern gel analysis) to + , + and + + to indicate increasing levels relative to the control value for that mRNA.

Notes: 1. Granelli-Piperno ef al., 1984, 1986. 2. Elliott ef al., 1984; Kronke et al., 1984. 3. Wiskocil ef al., 1985. 4. Greene et al., 1984. 5. Kwon and Weissman (1984); Elliott et al., unpublished. 6. Paetkau er a/., 1985.


were unaffected by either PMA or CsA, as was the p chain of the T-lymphocyte antigen receptor. The transcription of Mouse Mammary Tumor Virus (MMTV) provirus in EL4. El cells was strongly

induced by PMA (Kwon and Weissmann, 1984). We have recently found that this transcription is also blocked by CsA (Elliot et al., unpublished).

CsA blocks IL2 mRNA synthesis by a mechanism

which does not depend on an induced protein

The rate of IL2 mRNA synthesis in intact EL4. El

CsA: CHX: Harvor 1 IL2:

Psubs: pucl.3

cells was assayed by nuclear run-on transcription. The nascent RNA in nuclei isolated from cells treated in various ways was radioactively labeled, and the products were hybridized to cDNA probes. As seen earlier for Jurkat cells (Kronke et al., 1984), CsA added at the time of induction of EL4. El cells with

PMA completely blocked the synthesis of IL2 mRNA (Fig. 6). In addition, however, CsA added at the time when mRNA synthesis was near its maximal rate (lo-16 hr) also interrupted transcription of IL2 Transcription was nearly completely halted within

CCon) - - 0 12 -_ .- 0

C: 12 12 163 12 . 1

iYe3 :: ::a3 -8 .IB .5

TIME:

CISA : - -

CHX : Harvcrrt: Ei to6

;g z - 11

IL2: 13 3.4

Probe: pUCl3

I L.2

PlO

Fig. 6. CsA interrupts IL2 mRNA synthesis in EL4. El cells. Nuclei were harvested from EL4. El cells following treatment of the cells in culture as indicated. CsA was added to 100 ng/ml, CHX to 20 pgiml and PMA to 15 ng/ml, final concn. The isolated nuclei were incubated under conditions for labeling nascent RNA for 15 min. The RNA samples were worked up and hybridized to filters carrying 3 different cloned probes: a negative control provided by the vector pUCl3 (top row); plasmid M-plL2.1 to detect IL2 transcripts; and probe 10, representing an mRNA of unknown function which is expressed at equal levels in stimulated and unstimulated EL4. El cells (Paetkau et al., 1985) as an internal standard. The transcription rate of IL2 is expressed relative to the signal given by probe 10, with autoradiograms being quantitated with a densitometer. The expression seen at 12 hr was given a value of 10. “CON’ is a control of unstimulated cells, harvested at 12 hr. The average rates of transcription are given as the fourth line

of numbers above each pair of duplicate samples.


4 hr of adding CsA. The interruption of transcription by CsA also occurred, at least to a large extent, if CHX was added 2 hr before CsA. Thus, CsA not only interferes with the induction of IL2 mRNA synthesis, it also interrupts on-going transcription, and this interruption does not depend on a CsA-induced protein. However, the CsA effect may depend on interaction with existing cellular proteins. Some evidence for this is given by the somewhat lesser inhibition by CsA when CHX was present (Fig. 6).

Superinductioft is not a result of an increased rate of transcription

Importantly for the mechanism to be proposed for superinduction (Discussion), CHX did not significantly alter the rate of transcription if added 2 or 6 hr before harvest (Fig. 6). It also did not change the transcription rate observed at 24 hr when it was added at 11 hr. As expected from the results in previous figures, CHX present from time 0 blocked transcription.

DISCUSSION

This work demonstrates a number of specific effects of the inducing agent PMA (with or without T-lymphocyte mitogen) and the immunosuppressive drug CsA on the expression of lymphokines in three kinds of T lymphocytes. Induction by PMA and suppression by CsA was previously demonstrated for IL2 and IFNy mRNA in Jurkat cells (Elliott et al., 1984; Kronke et al., 1984; Wiskocil et al., 1985), and in stimulated T lymphocytes in mixed leukocyte cultures (Granelli-Piperno et al., 1986). It had also been found, at the level of active proteins, that CsA blocked the production of IFNy (Granelli-Pipemo et al., 1984; Reem et al., 1983) and a Colony Stimu- lating Factor (Kaufmann et al., 1984), but not IL2 receptor (Kronke et al., 1984). The results shown here are consistent with these observations, and extend them to include GM-CSF mRNA in EL4. El cells. As summarized in Table 2, IL2 mRNA was induced in cells of the murine T lymphoma EL4. El by PMA, and in the human T-leukemia line Jurkat and in normal PBL by PMA plus Con A. In every case this induction was blocked by CsA at moderate levels (100 ng/ml or less). The dose-response curves for IL2 and GM-CSF were essentially identical for both CsA and CsD-mediated suppression, although the half- maximal concns were somewhat different for the two cyclosporins. This further argues for a common intracellular mechanism regulating suppression of the two lymphokines.

Our results also confirm the lack of effect of CsA on expression of the IL2 receptor in Jurkat cells (Kronke et al., 1984), by demonstrating a lack of inhibition at the mRNA level, and extend these effects to primary cultures of normal PBL (see Granelli-Piperno et al., 1986, for similar results on T lymphoblasts derived from PBL). The partial sup-

M.I.M.M. 24/SB

pression of IL2 receptor expression in PBL by CsA (Fig. 2) may be secondary to the block in IL2 production, since this lymphokine has been shown to up-regulate its receptor (Depper et al., 1985). Neither the constitutive expression of the T-cell antigen receptor /l chain mRNA, nor the level of actin mRNA were affected by PMA or CsA. The specificity of these effects has been studied extensively by bio- synthetically labeling the proteins synthesized by EL4. El cells in the absence or presence of PMA, and in the absence or presence of CsA (Elliott et al., unpublished), Fewer than five out of every 100 proteins synthesized by these cells were affected by adding either PMA or CsA or both, and a similar fraction of the mRNA pool was altered as well. Thus, neither the bulk of genetic expression nor the specifi- cally probed control mRNAs described here is changed by inductive or suppressive signals for lymphokine expression. Rather, the pattern of induction, suppression and also superinduction, is characteristic of a small subset of all expressed genes in T lymphocytes.

The mechanism of lymphokine mRNA induction in the T-cell lines EL4.El and Jurkat appears to differ from PBL T lymphocytes in that the protein synthesis inhibitor CHX blocks induction of transcription only in the T-cell lines. This could reflect altered states of differentiation of the cell lines relative to PBL. Earlier work (Kronke ef ai., 198.5) indicated that the rate of transcription of IL2 and IFNy in PBL was unaffected by CHX present from time 0. Our data do not directly contradict this result (Fig. 5). However, the lack of superinduction when CHX was present from very early times in PBL experiments is difficult to understand if the transcription rate was, indeed, unaffected by CHX added at time 0. There are experimental differences between the PBL experiments performed here and those of Kronke et al., including the use of an erythrocyte rosetting technique to purify T lymphocytes in their work. This may have either affected the state of activation of the cells even before mitogen was added, or removed regulatory cells which, in our experiments with whole populations of PBL, affected lymphokine transcription.

The mechanism of superinduction of lymphokine mRNA does not involve an altered rate of transcription. This can be seen from the nuclear run-on transcription results (Fig. 6). The highest level of superinduction at 10 hr in Fig. 5 was reached by adding CHX between 2 and 6 hr before harvest. The lower levels of IL2 mRNA accumulating if CHX was added earlier may have been due to a general inhibition of cellular function, including tran~ription. Alternatively, the induction of maximal transcription rate may have not yet been achieved at the earlier times. The half life of IL2 and GM-CSF mRNAs is relatively short in the cell lines studied here, of the order 1-2 hr (Shaw et al., unpublished), The mRNAs are stabilized by CHX, suggesting that the mechanism


of superinduction includes an inhibition of a degrad- ative mechanism of lymphokine mRNA. A candidate

for such a CHX-sensitive mechanism of mRNA degradation is the RNase L system (Slattery et al.,

1979; Lengyel, 1982), which selectively degrades inducible mRNAs in a number of other types of cells, including viral RNA (Newmark, 1985). The CHX- sensitive site in that case is the enzyme (2/-5’)(A), synthetase, which generates an obligatory co-factor of RNase L (Lengyel, 1982).

PMA induces a profound increase in MMTV RNA in EL4 cells (Kwon and Weissman, 1984). We have found that the primary effect of PMA is to induce the synthesis of a 13-14s transcript of MMTV in these cells, in contrast to the larger species found by Kwon and Weissman (1984). A surprising result (Elliott et

al., unpublished) is that the PMA-induced transcription of MMTV in EL4. El cells was blocked by CsA, although it did not interfere in a significant way with the constitutive expression of the 35s form. The similarity between lymphokines and MMTV expression includes the inhibitory effect of CHX added at the outset of induction, and its superinducing effect when added in the middle of the response (Table 2; Elliott et al., unpublished). This suggests that the induced, 13-14s MMTV transcripts are regulated by the same intracellular mechanisms as lymphokines in EL4. El cells.

Acknowledgements-The authors thank Dr Phil Barr, Chiron Corp., for the synthesis of the oligonucleotides described in this paper, and Dr F. Pasutto, University of Alberta, for cyclosporins. They are also grateful to Drs P. Gunning and M. Davis, both of Stanford University, for providing the human y-actin probe and the mouse T-cell receptor B-chain probe, respectively. They express their appreciation to Rosemary Garner for technical assistance.

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Induction, suppression and superinduction of lymphokine mRNA in T lymphocytes

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