effect of interleukin-2 on the expression of cell cycle genes in human t lymphocytes

Vol. 133, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 17, 1985 Pages 410-416

EFFECT OF INTERLEUKIN-2 ON THE EXPRESSION OF CELL CYCLE GENES IN HUMAN T LYMPHOCYTES*

Leszek Kaczmarekl, Bruno Calabretta and Renato Baserga

Department of Pathology and Fels Research Institute, Temple University Medical School, 3400 N. Broad Street, Philadelphia, PA 19140

Received October 25, 1985

We have studied the expression of seven cell cycle-dependent genes in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells, in macrophage-depleted cultures and in macrophage-depleted cultures plus Interleukin-2 (IL-2). The expression of all seven genes is increased in PHA stimulated peripheral cells. Only two (2Fl and the IL-2 receptor) are increased in PHA-stimulated macrophage depleted cultures. Addition of IL-2 to these cultures increased the RNA levels of four genes (KC-l, c-myc, S-actin and IL-2R), but has no effect on three others (4F1, 2F1, and JE-3). The results indicate that the expression of these cell cycle genes is regulated by different components of the mitogenic stimulus. 0 1985 Academic Press, Inc.

The proliferation of T lymphocytes is dependent on the interaction between

T cell growth factor (Interleukin-2, IL-2)2 and its receptors (IL-2R) (l-4). An

in vitro model system for studying T cell proliferation is provided by mitogen

stimulated human peripheral blood mononuclear cells (PBMC). In this system the

IL-2R is induced on the surface of T lymphocytes by the mitogen (e.g. phytohema-

gglutinin, PHA) while IL-2 production is induced in a subpopulation of T cells as

a consequence of macrophage-released Interleukin-1 (IL-l) activity. I f T lympho-

cytes are purified from PBMC, after removal of monocyte-macrophages and B lympho-

cytes, the ability of T cells to enter DNA synthesis after PHA stimulation is

greatly diminished. However, the addition of either IL-1 or IL-2 topurified T

cells restores their proliferative response to the mitogen (l-3), We have recently

investigated the expression of cell cycle genes in human T cells (5). The genes

investigated included the proto-oncogene c-myc, the gene encoding IL-2R, the H3

histone gene and several genes identified as cell cycle genes from cDNA libraries

prepared from hamster or mouse fibroblasts stimulated to proliferate. Cell cycle

genes are operationally defined here as genes that are preferentia.lly expressed in

*This work was supported by a grant GM 33694 from the National Institutes of Health.

1 To whom correspondence should be addressed.

2 Abbreviations: kb-kilobase (thousand of nucleotides). IL-2-Interleukin-2, IL-ZR-Interleukin 2 receptor, IL-1-Interleukin 1, MDC-Macrophage-depleted culture, PMBC-Peripheral blood mononuclear cells, PHA-Phytohemagglutinin.

0006-291X/85 $1.50 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. 410


a specific phase of the cell cycle. Two of these genes, 4Fl and 2F1, were isolated

from a Syrian hamster ts13 cells cDNA library (6), and two others, KC-l and JE-3,

from a mouse 3T3 cDNA library (7). All these genes were expressed in a cell cycle

dependent manner in human PBMC stimulated with PHA (5). Specifically, they were

all expressed at very low or detectable levels in Go lymphocytes and were induced

by the mitogenic stimulus.

Among all the genes tested only 2Fl and IL-ZR were induced by PHA in puri-

fied (macrophage-depleted) T lymphocytes (5). In this communication we have

further extended our studies to investigate the effect of IL-2 on cell cycle genes

in purified human T lymphocytes. For this purpose we have studied the expression

of cell cycle genes in three subpopulations: PMBC, macrophage-depleted PMBC (MDC),

and mc plus IL-Z. It should be noted that at variance with other reports (2,3,8,9)

we have investigated the effect of IL-2 on gene expression directly on MDC,

rather than on T-cell lines or precultured T lymphocytes. In addition we have

analyzed a larger panel of cell cycle genes here, including B-actin whose ex-

pression is cell cycle-dependent in both lymphocytes (10) and fibroblasts (11,12).

The term gene expression is used here in one of its accepted usages as levels of

cytoplasmic mRNA, without prejudice to the mechanism(s) by which these levels are

achieved.

METHODS

The procedures for PBMC isolation and cultures, purification of T cells, RNA extraction, RNA electrophoresis, Northern blotting, nick-translation and hybridization were described in a previous paper (5).

The macrophage-depleted cultures were incubated with PHA, either alone (MDC), or in the presence of human recombinant IL-Z (a kind gift from Cetus Corp.), at a final concentration of 20 units/ml (MDC + IL-Z).

All experiments were performed in such a way that an aliquot of the cells from the same individual was cultured as PBMC, a second aliquot as MIX, and a third aliquot as MDC + IL-2,

PROBES USED: The probes used for nick-translation and hybridization (13) were from the following plasmids: pMC415 (c-myc) (l4), p4Fl and p2Fl (6), pJE-3 and pKC-1 (7), pIL-2R2 (the IL-2 receptor) (15), pHF Ba-1 (B-actin) (16). The plasmid 7B6 has an insert corresponding to a gene that is not expressed in a cell cycle-dependent manner (5).

RESULTS

Response of purified T cells to IL-2.

In all experiments we monitored autoradiographically the percentage of cells

entering the S phase of the cell cycle under different culture conditions during a

period of incubation of 72 hr. The results of these analyses are summarized in

Table 1. In unstimulated PBMC, only an occasional labeled cell is found in the

cultures incubated with [3H]-thymidine for 72 hr. After stimulation with PHA

60% of the lymphocytes enter S phase in the first 72 hr. A significant decrease

in the level of PHA-stimulated cells replicating DNA was observed after T lympho-

411


TABLE 1

Effect of Interleukin-2 on the Entry of Lymphocytes into S Phase

PMBC MDC MDc+IL-2

61 (50-70)

% of labeled cells 9.7 (l-17) 39 (24-45)

All cultures were labeled for 72 hr with [3H]-thymidine (0.2 uCi/ml). PBMC = peripheral blood mononuclear cells stimulated with PHA (10 pg/ml) MDC = macrophage-depeleted PBMC stimulated with PHA. MLX+IL-2 = MDC stimulated with PHA and 20 units/ml of IL-2. In unstimulated PBMC, the percentage of labeled cells is 1%. Averages of seven experiments (range in parenthesis).

cyte purification (MDC). This decrease is even more striking if one keeps in mind

that PBMC contain only about 50-70% of T cells, while in MDC virtually all cells

are T lymphocytes and therefore potentially able to respond to PHA. After 72 hr

of exposure to PHA in MDC cultures only Q 10% of the T lymphocytes had entered S

phase. It should be remembered that MDC are not totally deprived of accessory

cells and that complete elimination of accessory cells abolishes any response to

PHA, including growth in size (17). The advantage of using partially depleted

MDC is that after PHA,cells grow in size and one can relate gene expression. to

either DNA synthesis, or growth in size (5, 18). The addition of IL-2 to MDC

always produced an increase in the number of cells entering S phase of the cell

cycle to about 40%. The percentage of cells positive for the IL-2 by anti Tat

antibody 40 hr after PHA is about 41-50% in PBMC and 32-39% in MDC (5). Addition

of IL-2 to MDC induced an increase in the percentage of Tat + cells (18), as

already repeatedly reported in the literature (8,9,10,19).

Gene expression in stinarlated T Lymphocytes.

We performed Northern blot analysis of the expression of seven cell cycle-

dependent genes: 2F1, 4F1, KC-l, JE-3, S-actin, IL-2R and c-myc, and one control

non-cell cycle gene, 7B6. The last one was used to ensure that the same amount

of mRNA was blotted in each lane. All of these genes, except 7B6, are inducible

by PHA in PBMC (5), albeit with different kinetics, and this was confirmed in the

present experiments. However, since this short note deals with a comparison be-

tween PBMC, MDC and MDC+IL-2, we have omitted the confirmatory data on the kinetics

of gene expression of PHA-stimulated PBMC.

Northern blots were prepared from total cellular RNA extracted from all

three types of culture at 6,12, and 22 hr after PHA stimulation as well as from

non-stimulated cells. The expression of 4Fl and JE-3 genes could not be detected

in either MDC or MDC+IL-2 at any of the investigated times following PHA stimu-

lation (data not shown). For the other genes the clearest results were obtained

at 22 hr after stimulation with either PHA or PHA+IL-2. These results are pre-

sented in Fig.1 and 2. Figure 1 shows the pattern of expression of c-myc (2.4 kb

412


a b c

- 24

- 1.9 a b c

- 1.5

- 3.5

- 1.5 - 1.2

t

0 1 - 0.6

Fig.1. Composite autoradiogram of Northern blots of total RNA from cultures of lymphocytes. Lane a: peripheral blood mononuclear cells 22 hr after stimulation with PHA; lane b: Macrophage-depleted cultures 22 hr after stimulation with PRA; lane c: Macrophage-depleted cultures stimulated for 22 hr with PRA and IL-2 (20 units/ml). The following probes were used: c-myc (2.4), B-actin (1.9), 2Fl (1.5), KC-1 (1.2). 7B6 (0.6). The numbersinparenthesis are the size in kilobases of the respective RNA's.

Fig.%. Autoradiogramof Northern blots of total RNA from cultures of lymphocytes. Lanes and conditions are the same as in Fig.1, except that the probe used was the IL-2 receptor (15).

mRNA band), @-actin (1.9kb) 2Fl (1.5kb), KC-l (l.Zkb), and 7B6 (0.6kb) at 22 hr

after stimulation of PBMC (lane a), HOC (b), and MDC+IL-2 (c). Figure 2 shows

the pattern of expression of IL-2R gene at 22 hr after PRA stimulation of PBMC

(a), MDC (b) and MLlC+IL-2 (c). The IL-2R gene probe recognizes two messages -

3.5kb and 1.5kb (15).

The x-ray films obtained after Northern blot hybridization to appropriate

gene probes were scanned using a soft laser denistomer (5), and the levels of

expression of cell cycle genes were evaluated in arbitrary units. These results

are summarized in Table 2. The absolute levels of expression varied, of course,

from gene to gene (5). Since this is a comparison of gene expression and not

different genes, but of each in different conditions we have arranged Table 2

so as to facilitate a comparison. In unstimulated PBMC the levels of expression

in arbitrary units would be two. Based on the data of Fig.1 and 2, and Table 2,

we can say that the addition of IL-Z to MLX causes a slight increase in the ex-

pression of the IL-2R over the levels found in MDC cultures. The level of ex-

pression of 2Fl does not change after IL-2 addition to MDC; however, as already

413


TABLE 2

Expression of Cell Cycle Genes in T Lymphocytes under Different Culture Condit‘ions

levels of expression

GENE PBMC MDC MDc+IL-2

KC-l 8 2 4 c -myc 23 2 18 2Fl 21 13 12 B-actin 12 2 4 IL-2R 40 20 25

The results are based on densitometer readings of the intensity of appropriate mRNA bands in autoradiograms of Northern blots hybridized with the respective probes, as described in Methods and Materials. The blots were prepared from total cellular RNA isolated from cells after 22 hr of culture. PBMC, MDC and MDC+IL-2 are the same as in Table 1.

reported, 2Fl is induced by PHA in MDC (5). As already mentioned 4Fl and JE-3

are not detectable in MDC, either before or after the addition of IL-2. c-myc

is markedly induced by IL-2, and a good increase is also observed with KC-l and

8-actin. It should be noted that with the exception of c-myc the other genes are

expressed in MDC+IL-2 at a much lower level than in PHA-stimulated PBMC.

DISCUSSION

The role of IL-2 in T cell proliferation has been extensively studied in

recent years. It is produced in PBMC at the highest levels about 12-24 hr after

mitogen stimulation and its interaction with the IL-2 receptor (which appears at

earlier times) is a necessary event in T lymphocyte cell cycle progression (l-3).

In PBMC IL-2 production is an effect of macrophage-released IL-l. The removal

of macrophage diminishes the proliferative response of T cells. Addition of ex-

ogenous IL-2 to T lymphocytes reinstates this response (1,3). However, in our

experiments the IL-2 dependent restoration of proliferative response of purified

human T lymphocytes was only partial. Using higher concentrations (100 units of

IL-2/ml) did not increase the percentage of cells entering S phase. Moreover, if

one carefully reexamines the results of other authors with both recombinant and

naturally produced IL-2, it seems that IL-2 never restores T cell proliferative

response completely (1,2). It is also worth noting that the percentage of cells

stimulated by IL-2 to enter the S phase of the cell cycle is roughly similar to

the percentage of cells expressing IL-2R in MDC (5,18). This is also in good

agreement with our results, showing only a slight increase of IL-2R gene express-

ion in IL-2 treated cultures when compared to MDC. Other authors have shown that

IL-2 influences positively the expression of its own receptor (8,9,19,20,21).

However, theyused very high concentrations of IL-Z. We would like to re-emphasize

here that our approach is slightly different from that used by other investi-

414


gators who generally added IL-2 to T-cell lines, or precuftured T lymphocytes

(9,211 , while we have added IL-2 directly to MDC. This may account for some of

the slight differences. There is no question, though, from the data in the

literature, that IL-2 upregulates the expression of its receptor at both the pro-

tein and the RNA levels.

Among the cell cycle genes tested the expression of the c-myc protooncogene

seems particularly influenced by IL-2, as suggested by Reed et a1.(22). This is

in good agreement with information from other cell types that the expression of

this gene is usually markedly induced after the interaction of growth factors

with their receptors (23,24,25).

In conclusion we have shown that of seven cell cycle-dependent genes in-

duced during the transition of Go lympocytes to S phase, four (KC-l, c-myc, 8-

actin and IL-2R) respond to IL-2 with increased RNA levels, while three others

(4F1, JE-3 and 2Fl) do not. The differences may be helpful in mapping the G1

phase of stimulated lymphocytes and in identifying the fuction that these genes

may have in cell cycle progression. More important, these results suggest that

these genes respond to different components in the complex mitogenic stimulus.

The possibility that different cell cycle genes may respond to different growth

factors must be kept in mind in any attempt to dissect cell cycle progression

in T-lymphocytes.

1.

2. 3.

4. 5.

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7. 8. 9. 10.

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12. 13. 14.

15.

16.

17.

REFERENCES

Maizel, A., Mehta, S.R., Hauft, S., Franzini, D., Lachman, L.B., and Ford, R.J. (1981) J. Immunol. 27:1058-1064 Cantrell, S.A., and Smith, K.A. (1984) Science 224:1312-1316. Bettens, F., Kristensen, F., Walter, C., Bonnard, C.D., and deWeck, A.L. (1984) Cell. Immunol. 86:337-346. Ruscetti, F.W., and Gallo, R.C. (1981) Blood 57:379-396. Kaczmarek, L., Calabretta, B., and Baserga, R. (1985) Proc. Natl. Acad. Scu. USA 82:5375-5379. Hirschhorn, R.R., Aller, P., Yuan, Z-A., Gibson, C.W., and Baserga, R. (1984) Proc. Natl. Acad. Sci. USA 81:6004-6008. Cochran, B.H., Reffel, A.C., and Stiles, C.D. (1983) Cell 33:939-947. Smith, K.A., and Cantrell, D.A. (1985) Proc. Natl. Acad. Sci. USA 82:864-868. Malek, T.R., and Ashwell, J.C. (1985) J. Exp. Med. 161:1575-1580. McCairns, E.,Fahey, D., Muscat, G.E.O., Murray, M., and Rowe, P.B. (1984) Molec. Cell Biol. 4:1754-1760. Campisi,J., Gray, H.E., Pardee, A.B., Dean, M., and Sonenshein, G.E. (1984) Cell 26:241-247. Greenberg, M-E., and Ziff, E.B. (1984) Nature 311:433-438. Thomas, P.S. (1983) Methods Enzymol. 100:255-266. Dalla Favera. R., Gelmann, E.P., Martinotti, S., Franchini, G., Papas, T.S., Gallo, R., and Wong-Staal, F. (1982) Proc.Natl. Acad. Sci. USA 70:6497-6501. Leonard, W.J., Deppel, J.M., Crabtree, G.R., Rudikoff, S., Pumphrey, J., Robb, R.J., Kronke, M., Svetlik, P.B., Peffer, N.J., Waldman, T.A., and Greene, W.C. (1984) Nature 311:626-631. Gunning, P., Ponte, P., Okayama, H., Engle, J., Blau, H., and Kedes, L. (1983) Mol. Cell Biol. 3:787-795. Williams, J.M., Ransil, B.J., Shapiro, H.M., and Strom, T.B. (1984) J. Immunol. 133:2986-2995.

415


18. 19. 20.

21.

22.

23.

24.

25.

26.

Mercer, W.E., and Baserga, R. (1985) Exp. Cell Res. in press. Reem, J.H., and Yeh, N.H. (1984) Science 255:429-430. Welte, K., Andreef, M., Platzen, E., Holloway, K., Rubin, B.Y., Moore, M.A.S., and Mertelsman, R. (1984) J. Exp. Med. 160:1390-1403. Depper, J.M. Leonard, W.J., Drogula, C., Kronke, M.. Waldmann, T.A., and Green, W.C. (1985) Proc. Natl. Acad. Sci. USA 82:4230-4234. Reed, J.C., Nowell, P.C., and Hoover, R.J. (1985) Proc. Natl. Acad. Sci. USA 82:4221-4224. Kelly, K., Cochran, B.H., Stiles, C.D., and Leder, P. (1983) Cell 35: 603-610. Muller, R., Bravo, R.,Burckhardt, J., and Cur-ran, T. (1984) Nature 312: 716-720. Bravo, R., Burckhardt, J., Curran, T., and Muller, R. (1985) EMBO J. 4: 1193-1197. Efrat, S., and Kaempfer, R. (1984). Proc. Natl. Acad. Sci. USA 81:2601-2605.

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effect of interleukin-2 on the expression of cell cycle genes in human t lymphocytes

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