Phospholipid-metabolism of a stimulated murine T cell clone

BIOCHEMICAL

Vol. 144, No. 3, 1987

AND BIOPHYSICAL

May 14,1987

RESEARCH COMMUNICATIONS Pages 1303-1312

PHOSPHOLIPID-METABOLISM OF A STIMULATEDMURINE T CELL CLONE Berthold

Behl,

Margarete

Goppelt-Striibe, Beate Schwinzer, and Klaus Resch

Karsten Wiebe,

Division of Molecular Pharmacology, Department of Pharmacology and Toxicology, Medical School Hannover, D-3000 Hannover 61, FRG Received March 4, 1987

SUMMARY:The release of IPa and the incorporation of arachidonic acid into phospholipids was measured in a stimulated murine alloantigen-specific, non-cytolytic T cell clone. While Concanavalin A provoked a sharp increase in IP3, Interleukin 2 had no effect on the production of IPa. An increased reacylation of phospholipids with arachidonic acid was seen within the first 4 hours after addition of Concanavalin A, while an effect upon Interleukin 2 was only observed after 8 hours of incubation with Interleukin 2. A similar retarded response to Interleukin 2 was observed in proliferation experiments. These retarded cell responses may be due to changed properties of IL 2 receptors induced by IL 2. 0 1987 Academic Press, Inc.

The proliferation

of T lymphocytes

different

ligands.

mitogenic

response by inducing

the concomittant to its

receptor

Antigens

expression

the synthesis

of functional

concanavalin

mitogens

of Interleukin

mitogenic

signal

Amongst these,

turnover

a

(1). as well

as

The

A (Con A) has been shown to induce numerous functional

Con A was shown to activate

and to induce a subsequent

4, 5). Furthermore

by

of IL 2

understood.

changes of the plasma membrane, and also of the phospholipid (2).

initiate

Binding

(or mitogen),

is not completely

activation

2 (IL 2) and

IL 2 receptors.

the immediate

transmission

the stepwise

substituting

mechanism of both the antigen

the IL 2 induced signal mitogen

or antigen

then constitutes

The molecular

requires

we showed that

increase

metabolism

the phosphoinositide

in cytosolic

changes in the deacylation

Ca++ and pH (3, / reacylation

ABBREVIATIONS: Con A, Concanavalin A; FCS, fetal calf serum: Hepes, 4-(2-hydroxyethylj-1-piperazineethanesulfonic acid; IL 2, Interleukin 2; IPz, inositolbisphosphate; IP3, inositolIPI, inositolmonophosphate; trisphosphate; PC, phosphatidylcholine; PI, phosphatidylinositol; Tris, tris (hydroxymethyl)-aminomethan. 0006-291X/87 $1.50 1303

Copyright 0 I987 by Academic Press, Inc. All rghts of reproduction in any form reserved.

Vol. 144, No. 3, 1987

vtere early

cycle

tion

BIOCHEMICAL

and continuing

by lectins

AND BIOPHYSICAL

events associated

cellular

receptor.

proteins

Farrar

Several

after

cytosol

demonstrating

hydrolysis

are some reports,

(10).

It

that

is however difficult activation

constitutively

tions,

as it

of early

effects

In the present metabolism

fatty

acid metabolism

like

metabolism

population

specific with

induced

stimuli

C from the

whether

this

On the contrary

mechanism of the IL 2

IL 2 receptors by lectins,

(11).

PI-breakdown

of unoccupied

receptors.

two different

aspects turnover

condi-

of the and the

There are no examinations

pathways in physiological

Our experiments

Con A or antigen

hand investigarequire

on both metabolic

T cell

T-cell-

and ion signals

the phosphoinositide

so far.

are not

anti

On the other

membrane phospholipids.

non-cytolytic

by IL 2 without

lymphocyte

phosphoinositide

to assess the molecular

of lymphocytes,

homogenous T-cell

(6, 7, 8). Recently

of diacylglycerol.

have high levels

of

of

IL 2 did not provoke an enhanced PI-turnover

They are

of Con A- and IL a-effects

upon activation

generation

of

phosphorylation

is not yet clear,

paper we investigated

lipid

proliferative

It

and by IL 2 itself

under which cells

activation

an early

of proteinkinase

could be shown that

expressed.

antibodies

alloantigen

stimula-

the binding

a mechanism of IL 2 mediated the translocation

and a concomittant

receptor

reported

is the consequence of an increased

there

induced

events following

of IL 2 to lymphocytes

to the plasma membrane (9).

translocation

tions

authors

binding

and Anderson proposed

stimulation,

with lymphocyte

(12,131.

Much less is known about the primary IL 2 to its

RESEARCH COMMUNICATIONS

clone.

were performed

These cells

could be investigated

by lectins.

on an

proliferate

and show a good proliferation

preactivation

and

rate

on

Thus both types of

with regard

to lipid

in these clone cells.

EXPERIMENTALPROCEDURES Culture of clone cells: The alloantigen-specific non-cytolytic murine T lymphocyte clone II-8 was generated from a C57B1/6 anti DBA/2 mixed lymphocyte culture as described elsewhere (14). Cells were cultured in multiwell plates (Falcon 3047) in medium 199 (Gibco) supplemented with 15 1304

Vol.

144,

No.

3, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

U/ml IL 2 and 10 % fetal, calf serum (FCS; Gibco). They were maintained by weekly passage and restimulation with lo6 irradiated stimulator cells per well. Clone cells were used for the investigations of lipid metabolism 6 to 7 days after the last restimulation. Proliferation assay: 106 clone cells were incubated in 5-ml polystyrene tubes (Falcon 2058) in 1 ml RPM1 1640 supplemented with 16 U/ml IL 2 from a Con A stimulated rat spleen supernatant (15) for 2 - 20 h at 37V. and 5 % COZ. Then the cells were washed twice with RPM1 1640 (Gibco), resuspended in 1 ml RPM1 1640 supplemented with 10 % FCS and transferred into microtiterwells (200 ~1; Nunc 167008). Proliferation was measured after 20 h by incorporation of alI-TdR for the following 4 h (0.5 uCi/well). Accumulation of inositolphosphates: Clone cells (IL 2 starved for 3 days) were washed 3 times with inositol-free medium (modified BMED; supplemented with 20 mM Tris, 24 mM sodiumbicarbonate, 200 mM glutamine; Flow Laboratories). Cells (2 x 106/ml) were incubated with 50 DCi/ml 3Hinositol (Amersham) for 18 h in inositol free medium plus 0.5 % bovine serum albumin. During this preincubation period the cells were gently stirred in a glass vial. The prelabelled cells were washed 3 times with a RPMI-salt-solution (120 mM NaCl, 5.2 mM KCl, 5.1 mM NazHPOs, 0.5 mM MgClz, 0.43 mM CaClz, 11 mM glucose, 10 mM Hepes pH 7.2). Samples of 0.5 ml (106cells/ml) were incubated for 30 min in a water bath at 37V. Thereafter they were stimulated with either 5 pg/ml Con A (Pharmacia) or 20 U/ml IL 2 (recombinant human IL 2, Boehringer, or IL 2 from a Con A induced rat spleen supernatant). The incubation was stopped at the times indicated by addition of 1.88 ml chloroform/methanol (1:2). After addition of 0.625 ml water and 0.625 ml chloroform the two-phases-system was mixed and centrifuged for phase separation. The upper phase was collected, while the lower phase was washed twice with a theoretical upper phase (upper phase from a mixture containing chloroform/methanol/l00 mM sodiumcyclohexan-1,2diamintetraacetate (16:8:5)). The collected upper phases were applied to AG l-X8 columns (200-400 mesh, formate form, BIO-RAD). Each column contained 500 ~1 of an aqueous slurry (ion exchanger gel/water (1:21). Inositolphosphates were separated by stepwise elution with 15 ml Hz0 for inositol; 10 ml 5mM disodiumtetraborate, 60 mM sodiumformate for glycerophosphorylinositol; 6 ml 100 mM formic acid, 200 mM ammonium formate for inositolmonophosphates (IPr 1; 10 ml 100 mM formic acid, 400 mM ammonium formate for inositolbisphosphates (IPz) and 7 ml 100 mM formic acid, 1 M ammonium formate for inositoltrisphosphates (IPa). Samples were dried at 95OC and radioactivity was determined by liquid scintillation counting. Incorporation of fatty acids into phospholipids: Cells from 2 to 4 plates were pooled, washed and resuspended at a final cell density of 1 x 10b cells/ml. They were incubated in polypropylen tubes in a total volume of 1 ml HEPESbuffered RPM1 containing 0.5 % FCS and 0.5 % albumin (essentially fatty acid free: Sigma). Con A (2.5 pg/mll, IL 2 from a Con A induced rat spleen supernatant (corresponding to 16 U/ml) or recombinant IL 2 (16 U/ml) were used as stimuli. Arachidonic acid (Amersham Buchler) was dried under a stream of nitrogen and resuspended by brief sonification in medium containing 0.5 % albumin. 0.1 pCi (1.75 nmol) were added at the times indicated. The reaction was stopped by the addition of 5 ml methanol. Lipids were extracted as described previously (16) and separated by thinlayer chromatography on silica sheets (F 1500; Schleicher and Schuell). The system chloroform/methanol/ triethylamine/isopropanol/H20 (30: 17:25:18:7) was used for the separation of unesterified fatty acid, neutral lipids and phospholipids and the system chloroform/ methanol/acetic acid/O.9 % NaCl (50:25:8:2.5) for the separation of phosphatidylinositol and phosphatidylserine, which were not separated with the first system. Labeled lipids were identified by comparison with known standards and the amount of radioactivity determined by liquid scintillation counting. 1305

Vol. 144, No. 3, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESULTS Accumulation

of

inositolphosphates:

inositolphosphates

in

and IL

2 was measured.

in

level

the

within

of

hydrolyse

added

to the

phosphates

(fig. the

were

seen Fatty

Licl

production

of

of

acid

to

Con A for

effect

2).

hours

with

the

cells

incubated

2 effect pulse from IL

were

with to

2 the

2 incubation.

manner,

saturation

range

level,

control

of

of

170 and

phosphatases, 5 mM LiCl

the

levels

lb,

lc).

of IL

inositolphosphates (fig.

Id,

le,

which

caused

was

inositol

2 had no

under

IL

only

enhanced IL

lipids,

the

If).

con-

There

the

high

SEM-

4 hours

the

on the

the

2 were

When the of

PE was less

effected.

acid cells

uptake were

labelled

(tablel).

Therefore

Cells

were

incubated

two hours

with

arachidonic

achieved

acylation

between 1306

8 to

during

the

cells

were acid

contrast,

the

first for

cells the

after

20

acid,

incubated kinetics

the

with

of

the

IL

IL

2 and

As can be seen 6 to

8 hours

in a concentration 16 U/ml.

IL

about

or without

acid.

was

hours

arachidonic

with

was observed the

to

In

with

while

incorporation

cells

arachidonic

preincubated

stimulated,

levels

primarily

incorporation

fatty

up by the

(PI),

and triacylglycerol.

2 stimulated being

was taken

phosphatidylinositol

and subsequently

control

for

acid

and neutral

however,

investigated.

labelled fig.

When, stimlus

Con A returned

the

C reaction,

la,

increase

enhancement

of

of Licl

Arachidonic

(PE)

had no significant fig.

the

inhibition

(fig.

IPr

in PC and PI (tablellwhile

(tablel,

the

presence

phospholipids

phosphatidylethanolamine

enhanced

the

11 2 on the

(PC),

with

a sharp

370 % of

in

to Con A

lc.

phosphatidylcholine

stimulated

in IP3

of Con A was tested

incorporation:

and was acylated

up to

phospholipase

without

influence

in fig.

the

than

the

effect

For

in

on the

Con A provoked

than

and lc). of

as a response

Con A. The Con A induced

Thus

higher

was a variable

la

was less

products

effect where

values

lb

of radiolabelled

cells

(IPs) of

and IPr

samples.

significant

in fig.

addition

in IPz

which

prelabelled

As shown

after

200 % respectively

ditions

3H-inositol

inositoltrisphosphates

3 minutes

radioactivity

The increase

Recombinant

of

dependent IL 2

2

Vol. 144, No. 3. 1987

BIOCHEMICAL

%

IP3

AND RIOPHYSICAL

RESEARCH COMMUNICATIONS

a)

LOO3cKlzooI"r loo-

~+--

o0

*zo

L

8

12

[mlnl %

b)

16

20

[mlnl

e)

‘P2

Loo 300 2cx 100

I

!

I

,

0

L

8

I.2

1

I

I6

20

0

I 0

I L

,

1

8

12

Cmlnl %

I

1

16 20 Cmd

I P1

f1

300 200

OJ

I 0

1 L

, 6

, 12

( 16

, 20

Lm!

L

100

e--t--+

o-Jr

I1 L

0

8

1 12

I 16

I 20

Cminl

Cmlnl

Fiaure 1. Effect of concanavalin A and Interleukin 2 on the release of inositolphosphates in alloantigenic, non-cytolytic T-lymphocyte clone cells; Clone cells were labelled with 3H-inositol for 18 hours. After washing the cells were incubated for 30 min and then stimulated with either 5 ug/ml Concanavalin A or 20 U/ml Interleukin 2. Experiments were performed with and without addition of 5 mM LiCl. Incubations were stopped at the indicated times by addition of chloroform/methanol. Watersoluble inositol-phosphates were extracted and the individual inositol-phosphates were separated by ion-exchange chromatography. The figures show the time course of the accumulation of radioactivity in the individual inositol-phosphates as percentage of the controls. Data represent the mean t/- SEM values of 3 independent experiments. (Open la - lc: symbols : without LiCl, closed symbols: with 5 mM LiCl, figure 5 pg/ml Concanavalin A, figure Id - If: 20 U/ml Interleukin 2).

was not

significantly

different

from

the

of

clone

IL

2 containing

spleen

cell

supernatant. IL

2 dependent

alloantigen-specific

proliferation non-cytolytic

clone 1307

11-8: II-8

The proliferation is

dependent

on the

of

the

presence

Vol. 144, No. 3, 1987

BIOCHEMICAL

AND BIOPHYSICAL Table

Incorporation

into

1

Stimulus

% Radioactivity 0

Phosphatidycholine

Con

A

IL

Phosphatidylinositol

2

Con

A

IL

of IL

2. 6 to 7 days

stimulator

cells

clone

the

cells

A weak proliferation cells

with

reached

5 to

IL

2 concentrations

of

arachidonic

proliferative

(more

incubated

with

different

times

16 V/ml (fig.

the than

on the

h

20

(n=5)

109

+/-

1

(n=21

109

+/-

3

(r-r=51

148

+/-

9

(n=9)

127

+/-lo

+/-

4

(n=2)

111

+/-

+/-

4

(n=51

(n=8) 4

(n=51

be obtainned

added

by stimulating

the

growth

rat

8

12

the

a comparison

exposure

cells

of

with

dependence

to IL

2. lo6

were

however,

by saturating

the

spleen

the

kinetics of

the

cells/ml

supernatant) washed

IL 2. clone

cells,

induced

we investigated

the

irradiated

by exogenously

proliferation

of

125

with

be stimulated

For

89

twice

were for

and

% zoo-

loo-

o0

Figure 2. Incorporation stimulated by Interleukin

L

h

5

12 U/ml).

Thereafter

24

+/-

maximal

period

-

137

IL 2 (Con A induced 3).

4

restimulation

also

incorporation

response

-

. The Con A-induced

20 % of

acid

last could

could

Con A (1 vg/ml)

only

2

after

RESEARCH COMMUNICATIONS

16

20 hr

of arachidonic acid into phosphatidylcholine 2; clone cells were incubated with 11 2

(16U/ml) for the times indicated. For the last two hours 0.1 oCi nmol/mll *4C-arachidonic acid was added. Thereafter lipids were extracted and separated by thinlayer chromatography. The presented are the percentage of radioactivity in phosphatidylcholine related the controls as mean +/S.E.M. of 2 to 3 independent experiments.

1308

(0.18 data to

Vol. 144, No. 3, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

zo-

15-

10

5-

0' r,,

I

I

0 24

I

I

1

6810

20 of lncubatlon

time

30 (hr)

Figure 3. The dependence of the proliferative response of the lymphocyte clone II 8 on the period of exposure to Interleukin 2; Clone cells were incubated with 16 U/ml Interleukin 2 for the indicated times. Cells were washed twice and incubated for 20 hours (2 x 10J cells/well) including the time of Interleukin 2 incubation. Proliferation was measured by incorporation of 3H-thymidine (0.5 vCi/well) during the following 4 hours. Values are means of triplicates. Standart deviations did not exceed 8% of the mean.

resuspended for

the

in medium following

to be present the

clone

for

without

IL

2. Proliferation

16 h by 3H-TdR

incorporation.

a minimum

of

time

4 to

was measured As shown

6 hours

to

in

induce

after

fig.3

24 h IL 2 had

proliferation

of

cells.

DISCUSSION In

the

noncytolytic

breakdown effect

also

phosphatases lymphocytes

The incorporation stimulated

(4)

II-8

the

and the

Con A was largely

hydrolysing

small.

clone

of phosphoinositides of

to bulk

T cell

increased were the

blocked increase

rate

by Con A within

of

release when

in

first

1309

the

Con A stimulated of

the

by LiCl

arachidonic the

mitogen

the

inositolphosphates.

The

inositolphosphate (see

level acid

fig.

la-lc).

As compared

of

inositolphosphates

into

phospholipids

hours(tablel),

and reverted

was was to

Vol.

144,

No.

nearly

BIOCHEMICAL

3, 1987

background

levels

after

compared to bulk cultures observed effect stimulation to e.g.

of mitogen (17).

stimulated

the supernate

under similar

In this

Clone II-8 markedly

Proliferation

was

with

this

of incubation

with recent

effect

In contrast

in PI phosphate

turnover

phospholipids

the increase

with IL 2, it

cannot be excluded

or PI turnover, As in

most

the proliferative

cellular

IL 2

however,

systems,

transient

after

coincided

of arachidonic

response.

A possible

the first

phosphate

of the phospholipid

event,

accumulation fatty

acid incorporation

acids. into

about 6 hours of exposure to

with the time required

by the lymphokine

incorporation

to IL 2 during

that

which becomes apparent

inositol

of arachidonic

detectably

This strikingly

of proliferation

the increased

in response

response could be observed after

of the turnover

was stimulated 2).

et al.

lymphocytes.

to the lack of increased

In our experiments,

by Mills

remote.

IL 2 caused an activation

responses

findings

of PI breakdown has been found to be a rapid seems

responded

even in the presence

hours of stimulation.

such a possibility

induction

as it

phosphates,

period

IL 2 (fig.

which were stimulated

of inositol

a prolonged

induction

of IL 2

there was no

the onset of the proliferative

several

no secretion

of

of proliferation

As however,

after

induction

induction

alterations

increasing

response compared

that

in clone cells

to IL 2 in human or murine IL 2 sensitive

a small

clone

amounted to about 20% as compared

This is consistent

(101, who could not find

as

the

T lymphocyte

these investigations,

of IL 2 on the release (fig.ld-f).

Again,

Con A.

was chosen for

to IL 2. Despite

induces

particular

Accordingly,

small.

could be detected

conditions

COMMUNICATIONS

lymphocytes

mouse

of excess exogenous IL 2, suggesting

endogenous IL 2 synthesis

of LiCl

RESEARCH

with Con A induced only a small proliferative

spleen lymphocytes.

effect

BIOPHYSICAL

about 20 hours of stimulation.

was moderate

to the addition

into

AND

(see fig.

for the

3) indicating

that

acid might be associated

with

reason for the lack of cellular

hours of stimulation 1310

with

IL 2

may

be

Vol. 144, No. 3, 1987

the acquirement initiate

BIOCHEMICAL

of new properties

cellular

acid metabolism

resonses

lectins

(12,131.

lymphocyte

i.e.

activities the fatty role

response.

In molecular

in a low affinity

fatty

terms,

state

such a

to those in a

within

the first

time to an altered content

of the fatty

lymphocytes

(LAT) (18).

of

of the enzyme

These changes were found to

membrane phospholipid

of polyunsaturated acid composition

fatty

fatty

acid composition,

acids

(19).

were shown to effect

Since the

membrane bound enzymes (20) the observed increase

acid incorporation

in the modulation

by

of

10 minutes

with Con A, due to the activation

acyltransferase

of other

of resting

plasma membranes the incorporation

acids was increased

stimulation

cycle have been shown to be

event in the stimulation

In isolated

an increased

alterations

to

of the phospholipid

in the deacylation-reacylation

fatty

lysophosphatide lead with

of receptors

and continuing

unsaturated

the activation

capable

state.

Alterations an early

RESEARCH COMMUNICATIONS

of the IL 2 receptors,

and the proliferative

change could be a shift high affinity

like

AND BIOPHYSICAL

as a response

of enzyme activities

in

to Con A and IL 2 may play a during

the initiation

of growth

in lymphocytes.

REFERENCES

1. Waldmann, T. A.(19861 Science 232, 727-732. 2. Resch, K., and Ferber E. (1986) In Molecular Analysis of Lymphocytes. (J.J. Marchalonis, ed.), Marcel Dekker, New York. 3. Imboden, J. B., and Stobo, J.D.(1985) J. Exp. Med. 161, 446-455. 4. Taylor, M. V., Metcalfe, J. C., Hesketh, T. R., Smith, G. A., and Moore, J. P. Nature (1984) 312, 462-465. 5. Hesketh, T. R., Moore, J. P., Morris, D. H., Taylor, M. V., Rogers, J. C., Nature (1985) 313, 481-484. d ., Smith, G. A., and Metcalfe, 6. Kohno, N., Kuwata, S., Namba, Y., and Hanaoka M., FEBS (1986) 198, 33-37. 7. Ishii, T., Sugamura, K., Nakamura, M., and Hinuma, Y., Biochem. Biophys. Res. Comm. (1986) 135, 487-494. 8. Gaulton, G. N., and Eardley, D. D., J. Immunol. (1986) 136, 24702477. 9. Farrar, W. L., and Anderson, W. B., Nature (1985) 315, 233. 10. Mills, G. B., Stewart, D. J., Mellors, A., and Gelfand, E-W., J. Immunol. (1986) 136, 3019-3024. 1311

8lOCHEMlCAL

Vol. 144, No. 3. 1987

AND B\OPHYSICAL

RESEARCH COMMUNICATIONS

11. Depper, J. M., Leonard, W. J., Drogula, C., Kronke, M., Waldmann, T. A., and Greene, P. C., Proc. Natl. Acad. Sci. USA (1985) 82, 4230-

4234.

Resch, K., and Ferber, E., Eur. J. Biochem. (1972) 27, 153. 13. Ferber, E., Reilly, C. E., and Resch, K., Biochim.- Biophys. Acta 12.

(1976) 14.

15. 16. 17. 18. 19. 20.

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Wei.8, J., Schwinzer, B., Kirchner, H., Gemsa, D., and Resch, K ., Immunobiol. (1986) 171, 234. Spiess, P. J., and Rosenberg, S. A., J. Immunol. Meth. (1981) 42, 213. Goppelt, M., and Resch, K., Anal. Biochem. (1984) 140, 152. Stoeck, M., Wildhagen, K., Szamel, M., Lovett, D., and Resch, K ., Immunobiol. (1985) 169, 239. Szamel, M., Schneider, S., and Resch, K., J. Biol. Chem. (1981) 256 9198-9204. Ferber, E., de Pasquale, G., and Resch, K., Biochem. Biophys. Acta. (1975) 398, 364. Szamel, M.. and Resch, K., J. Biol. Chem. (1981) 256, 11618-11623,

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