Effect of dexamethasone on the expression of interleukin-2 in a mouse T cell line

0020-7 I I X/89 $3.00 + 0.00 Copyright ‘cm1989 Pergamon Press plc

Int. J. Biochem. Vol. 21. No. 9, pp. 961-970, 1989 Printed in Great Britain. All rights reserved

EFFECT

OF DEXAMETHASONE ON THE EXPRESSION INTERLEUKIN-2 IN A MOUSE T CELL LINE BARBARA

A.

SORG,’ NANCY S. MAGNUSON~

and

OF

RAYMOND REEVES’

‘Department of Genetics and Cell Biology and ‘Department of Microbiology, Washington State University, Pullman, WA 99164, U.S.A. [Tel. (509) 335-36341

(Received 19 January 1989) Abstract-l. The effect of the synthetic glucocorticoid, dexamethasone (dex), on the production of interleukin-2 (IL-2) and IL-2 mRNA was examined in the mouse T cell line, LBRM-33.4A2. 2. Treatment of Concanavalin A (Con A)-stimulated LBRM-33 cells with low concentrations of dex (10 nM) inhibited the production of IL-2 activity by approx. 70%, with a corresponding decrease in IL-2 mRNA levels. 3. In contrast, much higher concentrations of dex (up to 10pM) inhibited the level of another Con A-inducible mRNA, c-myc mRNA, by only 30%, and did not affect j?-tubulin mRNA levels at all. 4. Thus it appears that inhibition by dex in stimulated LBRM-33 cells is specific for the expression of IL-2 mRNA. 5. Experiments with actinomycin D suggest that dex does not mediate its suppression of IL-2 mRNA accumulation by decreasing the stability of this message; rather, it appears that dex inhibits transcription of the IL-2 gene.

INTRODUCTION

scription of these genes (Knudsen et al., 1987; Culpepper and Lee, 1985). In other cell types, the site of glucocorticoid action occurs at various levels of RNA and protein synthesis (Belanger et al., 1981; Firestone et al., 1982; Ringold, 1983; Vannice ef al., 1984; Raghow et al., 1986). The present work was undertaken to determine the effects of the synthetic glucocorticoid, dex, on the production of IL-2 protein and mRNA in the mouse T lymphoma cell line, LBRM-33, clone 4A2. We also examined the effects of dex on c-myc and /I-tubulin mRNA accumulation. The constitutively expressed gene, /I-tubulin, was chosen to determine if the effects of dex were specific for IL-2 mRNA or if dex reduced expression of all mRNAs. The levels of c-myc mRNA were measured for several reasons. (1) This mRNA has been shown to be induced by mitogens in T lymphocytes (Persson et al., 1984; Reed et al., 1986). (2) It was of interest to test whether dex affected mRNAs that have short half-lives in cultured cell lines [c-myc (Dani et al., 1984) IL-2 (Shaw et aI., 1987)] to a greater extent than it affected longer-lived mRNAs (p-tubulin), thus allowing us to determine if dex has a more general effect on mRNA turnover. (3) Several reports have suggested that mRNAs which are transiently expressed during induction of the immune response, including IL-2 and c-myc, contain short AU-rich sequences in their 3’ untranslated regions (Caput et al., 1986; Shaw and Kamen, 1986; Reeves et al., 1987). It is proposed that these regions are recognized by a factor which regulates the turnover of these mRNAs. Several mRNAs containing the AU-rich sequences are also those which are suppressed by glucocorticoids (Caput et al., 1986) suggesting that these genes may be regulated by similar processes. Thus, we wanted to determine if IL-2 and c-myc were affected by dex via a mechanism acting at the same level of their gene expression.

Glucocorticoids elicit a wide range of effects on the immune system, resulting in an overall inhibition of the inflammatory and immune responses (Fauci, 1979). Some of these effects are mediated by the inhibitory action of glucocorticoids on the production of interleukin-1 (IL-l) (Snyder and Unanue, 1982; Knudson et al., 1987), IL-2 (Gillis et al., 1979; Crabtree et al., 1980; Arya et al., 1984; Kelso and Munck, 1984; Grabstein et al., 1986) and interleukin3 (IL-3) (Culpepper and Lee, 1985). IL-2 has several biological activities which promote the growth and differentiation of lymphocytes (Ruscetti et al., 1977; Gillis and Smith, 1977; Henney et al., 1981; Farrar et al., 1982; Cantrell and Smith, 1984). Several studies have shown glucocorticoid-mediated inhibition of IL-2 production in spleen cells and peripheral blood lymphocytes (Gillis et al., 1979; Crabtree et al., 1980; Arya et al., 1984; Grabstein et al., 1986); however, mixed lymphocyte cultures do not allow for the distinction between direct effects of glucocorticoids on IL-2 synthesis and indirect effects by signals from other cell types. The suppression of IL-2 by glucocorticoids has been studied in some T cell lines, and the extent to which IL-2 production is inhibited is dependent upon the particular clone used (Ayra et al., 1984; Culpepper and Lee, 1985). The mechanism by which glucocorticoid-mediated suppression of IL-2 production occurs is not known. Studies with IL-l and IL-3-producing cell lines suggest that glucocorticoids act by suppressing the tran-

Abbreviations: AD, actinomycin

D; Con A, Concanavalin A; dex, dexamethasone; DME, Dulbecco’s modified Eagle’s medium; IL-I, interleukin-I; IL-2, interleukin-2; IL-3, interleukin-3. 961

BARBARA A. SORG et al.

962 MATERIALS

AND METHODS

Cell lines The T cell lymphoma line, LBRM-33 cells (clone 4A2) (Gillis et al., 1980) was obtained from the American Type Culture Collection (Rockville, Md). Cells were grown in Dulbecco’s modified Eagle’s medium (DME) supplemented with nonessential amino acids, 2 mM glutamine, 10mM hepes, 1 mM oxalacetic acid, 0.2 U/ml insulin, 0.5 mM sodium pyruvate, 10% NCTC 135 (Sigma), 10% fetal calf serum. 165 U/ml penicillin, and 75 U/ml streptomycin (LBRM-33 medium). The IL-2 dependent mouse-cell iine, HT-2 (Watson, 1979), was maintained in RPM1 1640 supplemented with 10% fetal calf serum, 165 U/ml penicillin, 75 U/ml streptomycin (HT-2 medium) and 30 U/ml human recombinant IL-2 (Chiron Corporation, Emeryville, Calif.). IL -2 assay LBRM-33 cells (7 x 105/ml) were treated with 20pg/ml Con A in the presence or absence of various concentrations of dex (Sigma). Dex was dissolved in 95% ethanol. In all “untreated” or “control” cells refer to cells experiments, treated with equivalent concentrations of 95% ethanol as were used for dex-treated cells. At the end of the time periods, supernatants were harvested and IL-2 activity was determined in a modification of a standard microassay (Gillis et al., 1978). Serial dilutions of the IL-2-containing supernatants in HT-2 medium were incubated with I x lo4 HT-2 cells/well for 20 hr. Cultures were pulsed for 4 hr with I PCi of [‘Hlthymidine per well (New England Nuclear, 6.7 Ci/mmol), cells were harvested onto glass fiber filters, and [)H]thymidine incorporation was determined by a liquid scintillation counter. One unit of actitivity is defined as the amount of IL-2 that induced 50% of maximal [rH]thymidine incorporation of a 48 hr culture medium conditioned by Con A or PHA stimulation of LBRM-33 cells. Incorporation of [‘Hlthymidine and [-‘H]uridine Proliferation of LBRM-33 cells was measured by incubation of 2 x 10“ce11s/we11 in LBRM-33 medium in a 96-well microtitre tray with 20 pg/ml Con A or 5&100 pg/ml PHA. [‘HlThymidine (1 pCi/well) was added during the last 4 hr of a 24 hr incubation. Cells were harvested and radioactivity measurements were performed as described above for the IL-2 assay. Cell viability was determined by the Trypan Blue exclusion method. To measure total RNA synthesis, LBRM-33 cells were incubated (5 x 104cells/well) in LBRM-33 medium in the presence of PHA (ranging from l(rlOO~g/ml) and in the presence or absence of dex (1 x lO-8 M). Cells were incubated in the presence of 1 pCi/well [3H]uridine (New England Nuclear, 50 Ci/mmol) for the last 4 hr of the incubation period. Cells were frozen at -80°C and thawed twice and spotted onto glass fiber filters. RNA was precipitated by placing filters in ice-cold 10% trichloroacetic acid for 30min. Filters were washed with 10% trichloroacetic acid followed by 95% ethanol, dried and radioactivity was determined by liquid scintillation counting. RNA isolation LBRM-33 cells (7 x 105cells/ml) were incubated in the presence of Con A (20 pga/ml) for various times in the presence or absence of dex b;‘5 p’g/ml AD (Sigma). At the end of the time periods, total cellular RNA from 100 ml cells was isolated by’a modification of the procedure by Cathala et al. (1983). Cells were centrifuged and resuspended and homogenized in 5 ml of a 4 M guanidine isothiocyanate buffer. An equal volume of chloroform was added to the mixture and homogenization was continued. After brief centrifugation, the aqueous layer was removed and mixed with 5 vol 4 M lithium chloride for 16 hr at 4°C to precipitate the RNA. Following centrifugation at 13,800g for

15 min, samples were subjected to proteinase K (Sigma) digestion and RNA was extracted twice with an equal volume of phenol:chloroform (1:1, v/v) and precipitated with ethanol. Northern blot analysis The RNA samples were separated in a 1.2% agarose, 6% formaldehyde gel and transferred to a nylon membrane in 20 x standard saline citrate buffer. The blot was exposed to U.V. light for 3 min and treated as described by Sorg et a/. (1987). The 18s and 28s ribosomal RNAs served as RNA size standards. Quantitative estimates of mRNA levels were obtained by cutting out the bands and determining radioactivity by liquid scintillation counting. Probes IL-2 cDNA. A mouse IL-2 cDNA (M-pIL2.1) was a generous gift from Dr Verner Paetkau (University of Alberta, Edmonton; Shaw er al., 1987). c-myc cDNA. A mouse c-myc cDNA (pSVc-myc) was kindly provided by Dr Robert Weinberg (Massachusetts Institute of Technology) (Land et al., 1983). Tub&n cDNA. The chicken /3-tubulin cDNA (pT2) was a kind gift from Dr Donald Cleveland (University of California, San Francisco; Cleveland et al., 1980). Statistical analysis Differences between means were analysed test (Steele and Torrie, 1960).

by Student’s

t

RESULTS

Effect

of dex on IL-2production

Figure 1 shows the time course of induction for IL-2 in LBRM-33 cells incubated with Con A in the presence or absence of 1 x IO-* M dex. Preliminary concentrations > indicated that experiments 1 x IO-’ M dex inhibited IL-2 production to < 1% of control levels by 24 hr. Therefore, 1 x lo-@ M dex was chosen for all subsequent experiments. Figure 1 shows that IL-2 was induced rapidly to high levels by 20 hr, but in the presence of dex, inhibition of IL-2 accumulation occurred from the first time point of 4 hr and reached a maximal inhibition (18% of control levels) by 20 hr. The inhibition of [3H]thymidine uptake by HT-2 cells was not due to dex in the supernatants, and inhibition of HT-2 cell

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Fig. I. Time course of IL-2 production by LBRM-33 cells in the presence or absence of 1 x IO-’ M dex added simultaneously with 20 pg/ml Con A. At the indicated times after Con A stimulation, the supernatants were collected and assayed for IL-2 activity as described in Materials and Methods. Each value represents duplicate assays of duplicate cultures.

Effect of dex on IL-2

proliferation by supernatants of dex-treated cells could be overcome by the addition of exogenous human recombinant IL-2 to HT-2 cells. Measurement of IL-2, c-myc and p-tubulin mRNAs To investigate whether dex treatment caused a corresponding decrease in the levels of IL-2 mRNA as were observed for IL-2 activity (Fig. I), total RNA was isolated and Northern blot analyses were performed. Initial experiments showed that IL-2 mRNA levels were expressed as early as 2 hr after Con A stimulation, with peak levels occurring between 8-12 hr. For all subsequent studies, total RNA was isolated from cells incubated for 8 hr or less, a time during which cellular viability remained high (data not shown). Figure 2 shows Northern blots of total RNA isolated from cells incubated with Con A in the presence or absence of 1 x lo-‘M dex. When blots were hybridized with IL-2 cDNA, a major mRNA was present migrating at 1.Okb. An additional minor band migrating at 3.3 kb (arrow 1) was observed consistently in the blots, and may be a stable nuclear precursor of IL-2 mRNA. From Fig. 2 it is apparent that no IL-2 mRNA was present in uninduced cells (t = 0). Treatment of cells with dex severely inhibited IL-2 mRNA accumulating at both the 4 and 8 hr time points. The second panel of Fig. 2 shows that c-myc mRNA, migrating at 2.5 kb, was expressed in uninduced cells. The level increased approx. 2-fold in 4 and 8 hr stimulated cells. In dex-treated cells, no decrease in c-myc mRNA levels was apparent at 4 hr, but a small decrease was observed in 8 hr cultures. Figure 2 also shows a Northern blot of B-tubulin mRNAs. Two sizes of mRNA, migrating at 3.2 and 2.0 kb, were found in approximately equal abundance at all times tested. It was apparent that the levels of /I-tubulin mRNA were not affected by dex treatment. Quantitation of mRNA levels Quantitative estimates of the levels of IL-2, c-myc and /I-tubulin mRNA are shown in Table 1. The levels of IL-2 mRNA in dex-treated cells were reduced compared to their time-matched controls. For IL-2 mRNA levels, significant differences between dex-treated and control cells were found at 4 hr (38% of time-matched control) (P < 0.005) and at 8 hr Table I. Effect of dex on steady-state levels of IL-2, c-myc and /I-tubulin mRNAs in LBRM-33 cells’~b cDNA probe Treatment Untreated Con A, 4 hr Con A + dex, 4 hr Con A, 8 hr Con A + dex, 8 hr

IL-2 _c 32i3 12+5 100 30 + 8

c-myc

fI-tubulin

51*7 78 +9 101 * 22 100 67 k 4

137*31 117k6 106*14 100 84 f 9

“LBRM-33 cells were incubated in the presence or absence of I x 10-s M dex added simultaneously with 20 pg/ml Con A. Total RNA was isolated at the indicated times as described in Materials and Methods. Levels of mRNA were quantified by cutting out the bands and determining radioactivity by liquid scintillation counting. bEach value is expressed as a percentage of mRNA levels relative to their levels found in 8 hr cultures. Values represent mean * SD from three experiments. ‘Below detectable levels.

963

(30% of control) (P < 0.001). The reduction of IL-2 mRNA correlated with the level of suppression observed for IL-2 activity in the cell supernatants. The levels of c-myc mRNA were not significantly reduced by dex treatment at 4 hr, but by 8 hr, c-myc mRNA levels were decreased to 67% of control levels (P < 0.001). These levels were significantly different from the reduction in /I-tubulin levels at 8 hr (P < 0.05). Levels of I-tubulin mRNA remained unchanged by dex treatment at 4 hr, and a small but significant reduction (84% of control levels) was found at 8 hr (P < 0.05). The decline in the levels of fl-tubuhn mRNA between t&8 hr parallels the decrease observed for total RNA synthesis and cell viability during this period (data not shown). Dosage effects of dex Figure 3 shows a Northern blot of total RNA isolated from cells treated with dex concentrations ranging from 1 x lo-’ to 1 x lo-” M dex added simultaneously with Con A and hybridized with IL-2, c-myc or /I-tubulin cDNAs. The level of IL-2 mRNA was affected in a dose-dependent manner. IL-2 mRNA was reduced to negligible levels by concentrations > 1 x lo-‘M dex while c-myc was reduced by much less at these dosages. The third panel in Fig. 3 indicates that B-tubulin mRNA levels were not affected by dex treatment as high as 1 x 10m5M. These data suggest that dex-mediated inhibition is specific for IL-2 mRNA. AD treatment and mRNA half-life To determine whether the suppression of IL-2 mRNA by dex was mediated by changes in IL-2 mRNA half-life, AD was added to cultures to inhibit transcription at various times following Con A induction. Addition of AD at the same time as Con A did not allow expression of any detectable levels of IL-2 mRNA, indicating that transcription of mRNA molecules was required for the accumulation of IL-2 mRNA. Cells were also treated with AD 4 and 6 hr after induction by Con A, with similar results between the two sets of experiments. Figure 4 shows a Northern blot of IL-2 mRNA isolated from cells treated with AD 6 hr after Con A induction. After 1 hr incubation with AD, dex was added to half the cultures and incubation was continued for 30 min or 4 hr to determine if dex decreased the half-life of IL-2 mRNA. The Northern blot shows that the addition of dex did not decrease the half-life of IL-2 mRNA by 0.5 or 4 hr after dex treatment. Additionally, Fig. 4 shows that treatment with AD resulted in a rapid decrease in IL-2 mRNA levels compared to the 0.5 hr control level by 1.5 hr (0.5 hr lanes), and that little additional decrease occurred by 5 hr (4 hr lanes) after AD treatment. In separate experiments, IL-2 mRNA levels were decreased to approximately half of control levels within just 30 min after AD treatment of 4 hr stimulated cultures, indicating that there was an immediate decrease in IL-2 mRNA levels (data not shown). Also observed in the Northern blot in Fig. 4 was the disappearance of the 3.3 kb mRNA and the appearance of a 3.0 kb mRNA in AD-treated cultures (arrows 1 and 2, respectively), suggesting that some processing of an IL-2 nuclear precursor

BARBARA A. SORGet al.

964

may occur in these cells. The addition of dex did not affect this processing. Identical Northern blots (not shown) hydridized with fi-tubulin and c-myc cDNAs showed that /I-tubulin mRNA levels remained unaffected by the treatments described above, while c-myc mRNA levels were reduced to negligible levels after only 30min of incubation with AD. DISCUSSION

We examined the effects of dex treatment on the synthesis of IL-2 in a Con A-stimulated mouse T cell lymphoma line, LBRM-33, clone 4A2. Treatment of the cells with I x lOmEM dex resulted in a marked inhibition of IL-2 in the supernatant from the earliest time point throughout the time period of this study (8 hr), during which cell viability remained high. Analysis of total RNA on Northern blots indicated that no IL-2 mRNA was present in unstimulated cells. If AD was added at the same time as Con A, IL-2 mRNA did not accumulate in LBRM-33 cells, indicating that the appearance of IL-2 required synthesis of new RNA molecules. This is consistent with the results of Efrat and Kaempfer (1984), who showed that induction of IL-2 mRNA requires de nouo transcription. Northern blot analysis of IL-2 mRNA levels in Con A- plus 1 x IO-’ M dex-treated cells indicated that the dex-mediated suppression of IL-2 activity in the supernatants correlated with reduced levels of IL-2 mRNA accumulation in these cells. An approx. 70% reduction of IL-2 mRNA levels in dex-treated cells was present at both early (4 hr) and late (8 hr) time points. The dosedependence of IL-2 mRNA levels on dex indicated that the effects of dex on IL-2 may occur by a receptor-mediated process. The suppression of IL-2 mRNA levels by dex treatment could result from a block in one or more steps of IL-2 mRNA biosynthesis. We did not detect any accumulation of stable RNA processing intermediates in dex-treated cells, as determined by Northern blot analysis. The stability of mature IL-2 mRNA in the presence of dex was examined. When AD was added to LBRM-33 cells to block transcription, the addition of dex did not decrease the stability of IL-2 mRNA. These results suggest that transcription of the IL-2 gene may be inhibited in the presence of dex. Transcription of other genes has been shown to be blocked by glucocorticoids (Nakanishi et al., 1977). Glucocorticoid receptors bind to specific DNA sequences to regulate the expression of mouse mammary tumor virus (MMTV), (Payvar et al., 1983) metallothionein II (MT-II) (Karin et al., 1984) and growth hormone (GH) (Moore et al., 1985) genes. These canonical DNA sequences, termed glucocorticoid regulatory elements (GREs), are found in the 5’ flanking region of these genes. A region containing homologous sequences to the GRE is also present in the mouse IL-2 gene (Fuse et al., 1984), as shown below [last four sequences are taken from Slater et al. (1985)]. mIL-2 MMTV hGH

5’-AAAaTGTTCT-3’ 5’-AAAcTGTTCT-3’ 5’-AAtgTGTcCT-3’

hMT-II, CONSENSUS

5’-ActgTGTcCT-3’ 5’-AMWNTGTYCT-3’

This region in the mouse IL-2 gene is found - 327 to - 3 18 bp from the cap site, just 26 bp upstream from a unique DNA sequence in IL-2 that has been shown to be involved in the induction of IL-2 expression and other T cell-specific genes (Fujita et al., 1986). If dex does inhibit transcription of the IL-2 gene, then it does not act as rapidly as AD, since dex addition after stimulation (46 hr) results in only a slight inhibition (not shown) compared to that of AD-treated cells. The finding that substantial levels of c-myc mRNA were present in LBRM-33 cells prior to Con A stimulation is in agreement with others who report an increased expression of c-myc during proliferation of normal and neoplastic cells (Erikson et al., 1983; Thompson et al., 1985). Upon Con A stimulation, c-myc mRNA levels were increased approx. 2-fold. The effects of dex on c-myc mRNA did not mimic what was observed for IL-2 mRNA. Dex does not appear to act early after Con A stimulation because no differences were detected between dex-treated and control cells at 4 hr, and by 8 hr only a small decrease in c-myc mRNA levels was observed. Thus, dex does not appear to cause a preferential turnover of shortlived mRNAs. It seems unlikely that dex inhibits the transcription of c-myc mRNA, at least early after stimulation, since the addition of AD to stimulated cells caused c-myc mRNA levels to disappear within 30min (not shown), while dex treatment did not cause this rapid and severe reduction. Levels of c-myc mRNA were maximally inhibited by ~50% over a wide range of dex concentrations, from 1 x lo-’ to 1 x lo-* M dex. It is possible that c-myc mRNA levels would be reduced over a longer incubation time with dex, as has been observed in 24 hr cultures of dex-treated PMBC (Reed et al., 1985). p-Tubulin mRNA was constitutively expressed in unstimulated and Con A-stimulated cells, and on a qualitative level, their levels appeared to be unaffected by dex at all concentrations of dex tested. These studies suggest that inhibition by dex appears to be specific for IL-2 mRNA, and that dex suppresses accumulation of IL-2 mRNA levels by regulation at the transcriptional level. SUMMARY

We examined the effects of the synthetic glucocorticoid, dex, on the production of IL-2 and IL-2 mRNA in the mouse T cell line, LBRM-33.4A2. Treatment of Con A-stimulated cells with 10 nM dex inhibited the production of IL-2 activity by approx. 70% with a corresponding decrease in IL-2 mRNA levels. Accumulation of IL-2 mRNA following Con A stimulation required RNA synthesis, and its suppression by dex occurred early (within 4 hr) during the induction phase in a dose-dependent manner. AD experiments suggest that dex does not mediate its suppression of IL-2 mRNA by decreasing the stability of this message. The effect of dex on another Con A-inducible gene, c-myc, was compared with that of IL-2. Although c-myc mRNA was present in rapidlyproliferating, uninduced cells, the level of this mRNA increased approx. 2-fold upon Con A stimulation. In

HRS DEX

0 -

8

4 ‘--

I--

IL-

2

0 4 -n --+-+-

C-MYC

8

0

4 nn -+

-+

Tubulin

Fig. 2. Northern blots of IL-2, c-myc and /?-tubulin mRNAs isolated from cells in the presence or absence of 1 x 1Om8M dex added at the same time as each lane refer to the hours of incubation following treatment. Total RNA (20 cDNA probes for IL-2, c-myc and P-tubulin. Total RNA and Northern blots in Materials and Methods.

965

8

Con A-induced LBRM-33 Con A. The numbers above pg/lane) was analysed using were prepared as described

C-MYC

1

Tubulin

234567

Fig. 3. Effect of various dex concentrations on steady-state levels of IL-2. c-myc and b-tubulin mRNAs isolated from Con A-stimulated 8 hr cultures. Dex was either absent (lane 1) or added at the same time as Con A to a final concentration of 1 x 10m5 (lane 2), 1 x 10e6 (lane 3), 1 x lo-’ (lane 4), I x 10-s (lane 5), 1 x 10m9 (lane 6) or 1 x IO-” M (lane 7), and incubated for 8 hr. Total RNA (20 pg/lane) was analysed as described in the legend for Fig. 2.

966

HRS

DEX

0.5

-

-

4

+

-

-

i-.

c-

Fig. 4. Effect of dex on IL-2 mRNA stability in Con A- plus AD-treated LBRM-33 cells. AD (5 pg/ml) was added to the cultures as indicated above following 6 hr of Con A stimulation. Cells were incubated for 1 hr, then I x 10m8 M dex was added to half to the cultures, and total RNA was isolated 0.5 and 4 hr later. Total RNA (20 pg/lane) was analysed using the IL-2 cDNA probe as described in the legend for Fig. 2.

967

969

Effect of dex on IL-2

contrast to IL-2 mRNA, much higher concentrations of dex (up to IO PM) only partially inhibited the levels of c-myc mRNA (by 30%) and, unlike for IL-2 mRNA, the effects of dex on c-myc mRNA occurred much later (8 hr) after induction. The levels of a-tubulin mRNA remained the same in both unstimulated and stimulated cells, and were unaffected by dex concentrations as high as 10pM. These data suggest that inhibition by dex in stimulated lymphocytes is specific for IL-2 mRNA and that this inhibition is not mediated by a decrease in mRNA stability. Acknowledgements-The authors wish to thank Dr V. Paetkau. University of Alberta, for helpful discussions and for providing the mouse IL-2 probe. This work was supported by U.S. Public Health Service Award AI-07025 from the National Institutes of Health, USDA Grant 85CRCRI-1730 (to R.R. and N.S.M.) and NSF Grant DCB-8602622 (to R.R.). REFERENCES Arya S. K., Wong-Staal F. and Gallo R. C. (1984) Dexamethasone-mediated inhibition of human T cell growth factor and y-interferon messenger RNA. J. Immun. 133, 273. Belanger L., Frain M., Baril P., Gingras M.-C., Bartkowiak J. and Sala-Trepat J. (198 1) Glucocorticosteroid suppression of a-fetoprotein synthesis in developing rat liver. Evidence for selective gene repression at the transcriptional level. Biochemist& 20, 6665. Cantrell D. A. and Smith K. A. (1984) The interleukin-2 T-cell system: a new cell growth model. Science 224, 1312. Caput D., Beutler B., Hartog K., Thayer R., Brown-Shimer S. and Cerami A. (1986) Identification of a common nucleotide sequence in the 3’untranslated region of mRNA molecules specifying inflammatory mediators. Proc. natn. Acad. Sci. U.S.A. 83, 1670. Cathala G., Savouret J.-F., Mendex B., West B. L., Karin M., Maria1 J. A. and Baxter J. D. (1983) A method for isolation of intact, translationally active ribonucleic acid. DNA 2, 329. Cleveland D. W., Lopata M. A., MacDonald R. J., Cowan N. J., Rutter W. J. and Kirschner M. W. (1980) Number and evolutionary conservation of OL-and p-tubulin and cytoplasmic r!I- and y-actin genes using specific cloned cDNA probes. Cell 20, 95. Crabtree G. R., Gillis S., Smith K. A. and Munck A. (1980) Mechanisms of glucocorticoid-induced immunosuppression: inhibitory effects on expression of Fc receptors and production of T-cell growth factor. J. Steroid Biochem. 12, 445. Culpepper J. A. and Lee F. (1985) Regulation of IL-3 expression by glucocorticoids in cloned murine T lymphocytes. J. Immun. 135, 3191. Dani D., Blanchard J. M., Piechaczyk M., El Sabouty S., Marty L. and Jeanteur P. (1984) Extreme instability of myc mRNA in normal and transformed human cells. Proc. natn. Acad. Sci. U.S.A. 81, 7046. Efrat S. and Kaempfer R. (1984) Control of biologically active interleukin 2 messenger RNA formation in induced human lymphocytes. Proc. narn. Acad. Sci. U.S.A. 81, 2601. Erikson J., Ar-Rushdi A., Orwingda H. L., Nowell P. C. and Croce C. M. (1983) Transcriptional activation of the translocated c-myc on&gene in Burkitt lymphoma. Proc. nom. Acad. Sri. U.S.A. 80, 820. Farrar J. J., Benjamin W. R., Hilfiker M. L., Howard M., Farrar W. L. and Fuller-Farrar J. (1982) The biochemistry biology, and role of interleukin 2 in the induction of cytotoxic T cell and antibody-forming B cell responses. Immun. Rev. 63, 129.

Fauci

A.

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