Effect of interleukin-2 on the expression of cell cycle genes in human T lymphocytes

Vol.

133,

No. 2, 1985

December

17,

BIOCHEMICAL

AND

BIOPHYSICAL

COMMUNICATIONS

1985

Leszek

ON THE EXPRESSION OF CELL CYCLE GENES IN HUMAN T LYMPHOCYTES*

Kaczmarekl,

Bruno

Calabretta

and Renato

Department of Pathology and Fels Research Medical School, 3400 N. Broad Street, October

410-416

Pages

EFFECT OF INTERLEUKIN-2

Received

RESEARCH

25,

Institute, Philadelphia,

Baserga Temple University PA 19140

1985

We have studied the expression of seven cell cycle-dependent genes in phytohemablood mononuclear cells, in macrophage-degglutinin (PHA)-stimulated peripheral pleted 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 T cell

growth

in vitro

model

stimulated IL-2R

induced

gglutinin,

cytes, greatly cells

system

on the

PHA) while are purified the

mononuclear

ability

of T cells However,

restores

their the

included

is

to enter the

cycle

are

operationally

*This work Health.

was supported

defined

of either

here

by a grant

provided

(PBMC).

genes

system

(e.g.

of T cells If

GM 33694

1

that

after

PHA stimulation

IL-1

or IL-2 topurified (l-3),

(5).

$1.50

Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

410

T recently

The genes

IL-2R,

the H3

cDNA libraries

are preferentia.lly Institutes

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

is

We have

gene encoding

from the National

as

T lympho-

and B lympho-

as cell cycle genes from stimulated to proliferate.

as genes

the

phytohema-

activity.

in human T cells the

An

by mitogen

In this

to the mitogen c-myc,

gene and several genes identified from hamster or mouse fibroblasts

(l-4).

in a subpopulation

DNA synthesis

response

of cell

is

(IL-l)

between

(IL-2R)

of monocyte-macrophages

addition

the proto-oncogene

interaction

by the mitogen

induced

removal

proliferative

expression

cells

Interleukin-1

PBMC, after

on the receptors

proliferation

of T lymphocytes

production

diminished.

and its

T cell

blood surface

IL-2

from

investigated

genes

studying

of macrophage-released

investigated histone prepared

for

is dependent

IL-2)2

(Interleukin-2,

human peripheral

is

a consequence cytes

of T lymphocytes

factor

Cell

cycle

expressed of

in

Vol.

133,

No. 2, 1985

a specific

phase

from a Syrian from

of the cell

hamster

ts13

manner

expressed

cells

at very

extended

of cell

the

genes

tested

cycle

we have rather

studies

genes

were

and 2F1, were KC-l

expressed

PHA (5).

levels

It

in

isolated

and JE-3, a

Specifically,

in Go lymphocytes

on T-cell

lines

panel

pression

is

The term

gene expression

be noted

cycle

cell

cycle

they

and were

were induced

by PHA in puri-

communication

we have

of IL-2

we have

on cell

studied

the

cycle

on gene

with

expression

B-actin

(10)

in one of its

we have whose

ex-

and fibroblasts

accepted

to the mechanism(s)

(2,3,8,9)

on MDC,

In addition

including

lymphocytes

PMBC (MDC), reports

directly

T lymphocytes. here,

other

usages

by which

genes

expression

PMBC, macrophage-depleted

in both

prejudice

induced

at variance

genes

is used here

effect

purpose

or precultured

of cell

were

In this the

that

of IL-2

cycle-dependent

mRNA, without

(5).

subpopulations:

the effect

a larger

and IL-ZR

For this

should

analyzed

cell

2Fl

to investigate

in three

investigated

cytoplasmic

4Fl

COMMUNICATIONS

and two others,

genes

with

T lymphocytes

our

IL-Z.

than

genes,

(6),

these

only

human T lymphocytes.

and mc plus

All

low or detectable

(macrophage-depleted)

in purified

cDNA library (7).

RESEARCH

stimulus.

Among all fied

BIOPHYSICAL

Two of these

in human PBMC stimulated

by the mitogenic

further

AND

cycle.

a mouse 3T3 cDNA library

dependent all

BIOCHEMICAL

(11,12).

as levels

these

levels

of are

achieved. METHODS The procedures for PBMC isolation and cultures, RNA extraction, RNA electrophoresis, Northern blotting, ization were described in a previous paper (5).

purification nick-translation

of T cells, and hybrid-

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 from the same individual aliquot as MDC + IL-2,

were performed was cultured

in such a way that an aliquot of the cells as PBMC, a second aliquot as MIX, and a third

PROBES USED: The were from the following and pKC-1 (7), pIL-2R2 plasmid 7B6 has an insert cycle-dependent manner

probes used for nick-translation and hybridization (13) plasmids: pMC415 (c-myc) (l4), p4Fl and p2Fl (6), pJE-3 (the IL-2 receptor) (15), pHF Ba-1 (B-actin) (16). The corresponding to a gene that is not expressed in a cell (5). RESULTS

Response

of purified In all

entering period

the

T cells

experiments S phase

of incubation

60% of the level

lymphocytes

IL-2.

we monitored

of the cell of 72 hr.

Table 1. In unstimulated cultures incubated with in the

to

cycle

enter

under

The results

PBMC, only [3H]-thymidine

of PHA-stimulated

autoradiographically

S phase cells

different

culture

of these

analyses

an occasional for 72 hr. in

the first

replicating 411

the percentage conditions are

in

found in the with PHA

A significant

DNA was observed

during

summarized

labeled cell is After stimulation 72 hr.

of cells

after

decrease T lympho-

a

Vol.

133,

BIOCHEMICAL

No. 2, 1985

Effect

AND

RESEARCH

TABLE 1 on the Entry of Lymphocytes

of Interleukin-2 PMBC

MDC

(50-70)

% of labeled 9.7 (l-17)

61

BIOPHYSICAL

into

COMMUNICATIONS

S Phase

MDc+IL-2 cells 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).

that

PBMC contain

only

are

T lymphocytes

of exposure phase. cells

It

should

MDC is

that

DNA synthesis,

after

always

produced

PHA,cells

40%.

already

repeatedly We performed genes:

2F1,

in each

three

However,

cells.

(data

not

at 22 hr after sented

in Fig.1

shown). stimulation and 2.

to MDC

of the

cell

by anti

in MDC (5).

of Tat + cells

of the expression

S-actin,

All

since

Tat Addition

(18),

we have

as

omitted

For the other either

except

note the

deals

the same amount 7B6,

are

inducible

was confirmed with

confirmatory

cycle-

and one control

that

and this

cell

in

a comparison data

the be-

on the kinetics

PBMC. cellular RNA extracted from all PHA stimulation as well as from

of 4Fl and JE-3

at any of the with

genes,

short

of seven

and c-myc,

to ensure

kinetics,

this

The expression

Figure

IL-2R

of these

different

were prepared from total at 6,12, and 22 hr after

MDC or MDC+IL-2

IL-2

to

(8,9,10,19).

one was used

of PHA-stimulated

Northern blots types of culture

non-stimulated lation

lane. with

PBMC, MDC and MDC+IL-2,

in either

JE-3,

The last

albeit

tween

of IL-2

the

to

depleted

S phase

for

S

of accessory

gene expression.

in PBMC and 32-39%

analysis

4F1, KC-l,

of mRNA was blotted

of gene expression

72 hr

any response

partially

entering

in the percentage

blot

7B6.

experiments.

deprived

The addition

positive

After

T Lymphocytes.

by PHA in PBMC (5), present

of cells

cells

had entered

abolishes

of using

18).

in the literature

Northern

gene,

totally cells

of cells

41-50%

an increase

reported

not

in mind

all

to PHA.

and one can relate (5,

in the number

in stinarlated

cycle

in size in size

about

one keeps

in MDC virtually

to respond

The advantage

The percentage

to MDC induced

dependent

grow

PHA is

of IL-2

Gene expression

MDC are

(17).

if

Q 10% of the T lymphocytes

of accessory

or growth

40 hr after

while

able

only that

in size

an increase

to about

of T cells,

elimination

growth

is even more striking

potentially

be remembered

complete

either

non-cell

50-70%

and therefore

PHA, including

cycle

about

decrease

to PHA in MDC cultures

and that

antibody

This

genes

could

not

be detected

investigated times following PHA stimugenes the clearest results were obtained PHA or PHA+IL-2.

1 shows the pattern

412

These

of expression

results

are pre-

of c-myc

(2.4

kb

Vol.

133,

No. 2, 1985

BIOCHEMICAL

a

b

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

c

-

24

-

1.9 b

a

-

c

1.5

-

1.2

-

0.6

-

3.5

-

1.5

t

01

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. 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).

Fig.1.

Fig.%.

mRNA band), after the (a),

@-actin

stimulation pattern

of PBMC (lane

are from

films

were

in arbitrary

and 7B6 (0.6kb)

and MDC+IL-2

gene at 22 hr after

The IL-2R

genes,

but

after

gene probe

genes

were

laser

(c).

at 22 hr

Figure

2 shows

PRA stimulation recognizes

of PBMC

two messages

Since

would

this

be two.

pression

of the

IL-2R

pression

of 2Fl

does not

over

denistomer

(5),

in arbitrary

-

conditions

In unstimulated Based on the data

the levels after

to MLX causes found IL-2

of gene

413

units.

of Fig.1

of results

of course,

expression

we have

a slight

These

varied,

and not

arranged

PBMC the levels

in MDC cultures. addition

to appropriate

and the levels

of expression

is a comparison

of IL-Z change

hybridization

levels

of each in different

the addition

blot

evaluated

The absolute

a comparison.

units

Northern

a soft

2.

(5).

so as to facilitate we can say that

(l.Zkb),

HOC (b),

using

cycle

in Table

gene to gene

different

(c).

obtained

scanned

of cell

summarized

a),

KC-l

(15).

The x-ray gene probes

(1.5kb),

of IL-2R

and MLlC+IL-2

and 1.5kb

expression

2Fl

of expression

MDC (b)

3.5kb

(1.9kb)

Table

2

of expression

and 2, and Table increase

in the ex-

The level

to MDC; however,

of ex-

as already

2,

Vol.

133,

BIOCHEMICAL

No. 2, 1985

Expression

AND

BIOPHYSICAL

RESEARCH

TABLE 2 Cycle Genes in T Lymphocytes under Different

of Cell

levels

COMMUNICATIONS

Culture

Condit‘ions

of expression

GENE

PBMC

MDC

KC-l c -myc 2Fl B-actin IL-2R

8 23 21 12 40

2 2 13 2 20

MDc+IL-2 4 18 12 4 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

are not

detectable

is markedly

induced

by PHA in MDC (5).

in MDC, either

before

induced

by IL-2,

and a good

should

be noted

that

8-actin.

It

expressed

in MDC+IL-2

with

As already

or after

at a much lower

the addition

increase the

is

also

exception

level

mentioned

than

4Fl

and JE-3

of IL-2.

observed

of c-myc

c-myc

with

KC-l

the other

in PHA-stimulated

and

genes

are

PBMC.

DISCUSSION The role recent

of IL-2

years.

It

mitogen

stimulation

earlier

times)

In PBMC IL-2

is

interaction

a necessary

event

IL-2

did

not

one carefully

produced

response

completely by IL-2

the percentage agreement ion IL-2 here

that

to enter

theyused

cultures

positively very

our approach

this

restoration partial.

Using of other IL-2

It

worth

the

is also S phase

showing

is

noting cell

that cycle

in MDC (5,18).

only

a slight

when compared

of purified (100 units

S phase. both

roughly is

if and

proliferative

the percentage is

of

Moreover,

recombinant

T cell

This

increase

of exin our

also

of IL-2R

of cells similar

to

in good gene express-

to MDC. of its

concentrations slightly

with

(l-3).

However, response

restores

at

The removal

concentrations

authors

appears

Addition

(1,3).

entering

never

of the

IL-2R

the expression high

higher

of cells

in

hr after

progression

IL-l.

of T cells.

response

12-24

(which

cycle

of proliferative

seems that

expressing

our results,

treated

influences

However,

(1,2).

of cells

with

in IL-2

it

receptor cell

response

reinstates

the results

IL-2,

IL-2

studied

about

of macrophage-released

the percentage

reexamines

naturally stimulated

was only increase

the

levels

in T lymphocyte

the proliferative dependent

human T lymphocytes

has been extensively

with

is an effect

to T lymphocytes the

proliferation

in PBMC at the highest

and its

diminishes

IL-2

experiments IL-2/ml)

in T cell

produced

production

of macrophage ogenous

is

Other authors have shown that own receptor (8,9,19,20,21).

of IL-Z.

different

414

from

that

We would like to re-emphasize used by other investi-

Vol. 133, No. 2, 1985 gators

BIOCHEMICAL

who generally

added

IL-2

AND BIOPHYSICAL

to T-cell

lines,

or precuftured

to MDC. This (9,211 , while we have added IL-2 directly the slight differences. There is no question, though, literature, tein

that

IL-2

upregulates

the

cycle

tested

RESEARCH COMMUNICATIONS

expression

T lymphocytes

may account from the receptor

of its

for

some of

data in the at both the pro-

and the RNA levels. Among the cell

seems particularly in good

influenced

agreement

with

this

gene

is usually

with

their

receptors

during

the

actin

and IL-2R)

(4F1,

JE-3

phase

of stimulated

these

in cell

factors

as suggested

from

induced

we have shown transition

respond

other

after

of the

c-myc protooncogene

by Reed et a1.(22).

cell

types

that

the interaction

to IL-2

do not.

cycle

that

of seven

of Go lympocytes

This

the expression of growth

with

progression.

to different

that

different

must be kept

in mind

increased

and in

is of

factors

cycle-dependent four

RNA levels,

More

the

important,

cycle

in the genes

in any attempt

complex

may respond

to dissect

cell

that

results

in8-

others the G1

these

genes

suggest

that

mitogenic to different cycle

genes c-myc,

three

in mapping

fuction

these

(KC-l, while

may be helpful

identifying

components cell

cell

to S phase,

The differences

lymphocytes

respond

The possibility

the expression

(23,24,25).

and 2Fl)

genes

by IL-2,

information markedly

In conclusion duced

may have

genes

stimulus. growth

progression

in T-lymphocytes.

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