Different induction of p55 and p7075 interleukin-2 receptor subunits in human cells

BIOCHEMICAL

Vol. 164, No. 2, 1989 October

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Pages 686-692

31, 1989

DIFFERENT INDUCTION OF p55 AND p70/75 INTERIEUKIN-2 RECEPTORSUBUNITS INHUMANCELLS Toshio Tanaka', 1

Third

Susumu Kishimotol

and Osamu Saiki2*

Department of Internal Medicine, Osaka University, Fukushima-ku, Osaka, 553, Japan 2

Department of Microbiology, mime University, Shigenobu, Ehime, 791-02, Japan

Received September 4, 1989 There are two interleukin-2 receptor(IL-2R) subunits(p55 and p70/75) on Induction of the expressions of these IL-2R subunits was human lymphocytes. examined by the protein kinase-C(PK-C) activator(phorbo1 myristate acetate, PMA) and the calcium ionophore, ionomycine(IM). IM induced predominantly p70/75 expression on human T and B cells as indicated by the results of chemical crosslinking studies and binding assays. In contrast, PMA induced p55 expression significantly. These results suggest that the calciumcalmodulin and PK-C pathways regulate p70/75 and p55 expressions differently, and indicate that these intracellular signal messengers could control the responsiveness to IL-2, changing the affinity and number of receptors in vivo. 0 1989 Academic

Press,

Inc.

Interleukin

2 (IL-2)

of growthand

differentiation

There are two IL-2

IL-2

with

an

is suggested

that

p70/75

affinity

whereas the intracellular

with

at high

in T and B lymphocytes.

regulation

(0.1-10

p70/75

glycoprotein Isolated

and ~55, alone,

concentrations

with

a broad spectrum

a 55-kilodalton

IL-2R subunits

conditions

p55 associates

activities

subunits,

Higkaffinity

through

lymphokine

on human lymphocytes.I-lo

intermediate

under physiological

to function

(IL-2R)

and ~70175,

a low affinity.*l' function

promoting

receptor

(~55, Tat antigen) binds

is a T cell-derived

binds

are necessary

u/ml),

although

(100-10000

IL-2 for

IG2 to

IL-2

is able

Inc. reserved.

686

It

IL-2R,1-10

remains unresolved.

*All correspondence should be addressed to 0. Saiki : Department of Ehime University School of Medicine, Shigenobu, Ehime, Microbiology, 791-02, Japan. 0006-291X/89 $1.50 Copyright 0 1989 by Academic Press, All rights of reproduction in any form

with

u/m1).6t8-10

to form high-affinity

of these expressions

~70175

Vol.

BIOCHEMICAL

164, No. 2, 1989

Here,

we report

predominantly lack protein

p55

the

calcium

p70/75 expression expression.

kinase

suggesting

that

that

5r10

AND BIOPHYSICAL

on human T cells

In contrast

phorbol

11

the calcium-calmodulin

, ionomycine(IM)

and

induces

SKW 6-4 B cells

myristate

predominantly

C (PK-C) activator,I*-l4

p70/75 and p55 expressions

ionophore

RESEARCH COMMUNICATIONS

induced

acetate

that

(PMA), a

p55 expression,

(CaM) and PK-C pathways12-l5

regulate

independently. MATERIALSAND METHODS

Preparation of cells Human tonsillar cells were obtained by gentle teasing of the specimens in heparinized Hanks' balanced salt solution (HBSS). Then T cells were separated by rosette formation with 2-aainoethylisothiouronium-bromide(AET)-treated sheep erythrocytes as reported previously.8 The T cell population contained about 98% OKT3-positive cells. These T cells and a human B lympho-blastoid line, SKW6-4, were culturedin RPM11640 (GIBCO, Grand, Island, NY) with 10% fetal calf serum (FCS, Sterile Systems, Logan, UT), 2mM L-glutamine, 1OOU penicillin, and 100 Ug streptomycin/ml in the presence of IM (Calbicchem, La Jolla, CA, 1 or 0.5 uM) or PMA(Siqma Chemical Co., St. Louis, MO, 10 or 1 rig/ml) for 3 days. Binding assay of radiolabeled IL-2 R-IL-2 was iodinated with Na125 I by the chloramine-T method and the specific activity of 1251-labeled IL-2 (l*' I-IIr2) was 1.5-2 x lo4 cpm/ng. Stimulated T cells were washed three times with HBSSand incubated for 4 hours at 37OC in the culture medium. After washing, 2 x lo6 T cells were incubated for 60 min at 4'C with serial dilutions of 125 I-II?2 in RPM11640, 25 mM Hepes, 0.02% NaN3 ay$i51% bovine serum albumin (BSA). For the detection of the binding of I-IL-2 to p70/75 IL-2 binding molecules, T cells were preincubated with H-31 monoclonal antibody (50 pg/ml) at 4'C for I hour, and then 1251-ILr2 was added. H-31 recognizes Tat antigen (~55) and blocks the proliferationof Tcells by IL-2 (17). The mixtures were then centrifuged, the supernatants were removed, and the precipitated cells were resuspended in 100 Ul of phosphate-buffered saline (PBS). The cell suspension was layered on 750 ii1 of RPM11640 containing 1M sucrose and 0.02% BSA and centrifuged at 12,000 rpm for 3 min at 4'C. The radioactivities of the resulting su rnatants and cell precipitates were then counted. The specific binding of 12!? I-IL2 to cells was calculated by subtracting the count of the sample containing excess unlabeled IL-2 (loo-fold), for competitive binding, from the observed counts. Gel-electrophoresis of IL-2 binding molecules "'I-IL-2 was crosslinked to IL-2 binding molecules as previously described (24) with minor modifications. Briefly, stimulated cells (10') were resuspended with 400 11 of RPM1 1640 containin 25 mM Hepes and 1% BSA (RPMIHEPES) and incubated with 200 pM or 10 nM l2 ? I-IL2 for 1 hour at @C. After incubation, cells were pelleted at 400 g and resuspended in 5 ml of PBS, pH8.3, containing 1 mM MgC12. Then, disuccinimidyl suberate (DSS) was added to the cell suspension at a final concentration of 0.3 mM, cells were tumbled at 4'? for 20 min and 5 ml of 10 mM Tris and 1 mM EDTA was added. Cells were pelletedat gand extracted in 50 ~1 of 300 mM NaCl, 50 mM tris and 0.5% NP40, pH7.4. Postnuclear lysates were heated at IOO°C in the presence of SDS and analyzed by SDS-PAGEunder the reducing condition. 687

Vol. 164, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESULTS Induction

of the expression

T cells

was examined

activator

PMA.12-l4

using

for

by SDS-PAGE analysis

IM stimulation,

three

and the intensities and 3), although that

to detect

(KD) (Fig.

correspond calculated

the

molecules

(10 nM), followed When T cells around

were

85 and 90

previously.5'16 around

Under

70, 85 and 90 KD,

dose-dependently(Fig.

1, lanes 2

of the 85 and 90 KD bands were stronger

In contrast, (lanes

to ~55, p70 and p75

PMA(lO, 1 rig/ml) 5 and 6). IL-2

4

Thebands

binding

to 15 KD IL-2.

180-KD appeared

65

IL2

migrated

4), as reported

IM (1, 0.5pM)

binding

proteins. bands

and the PK-C

with

of IG2

bands appearedthatmigrated

to be crosslinked

IMIl

were cultured

labeled

1, lanes land

the intensities

around

ioncarrier

of 1251-labeled

the radiolabeled

the 70 KDband

band migrating

T cells

of these bands increased

of the 70 KD one.

expressicnof

calcium

crosslinking

medium alone,

kilodaltons

p70/75 and p55 in human tonsillar

3 days and expressions

were examined by chemical

with

the mobile

Human tonsillar

or PMA (10, 1 rig/ml)

cultured

of subunits

strongly at70,

mclecules,

As reported

induced

respectively, a broad

although

3 21

Figure 1. Induction of the expressions of p55 and p70/75 IL-2R subunits in human T cells by calcium ionophore (IM) and PMA. Chemical crosslinking of stimulated tonsillar T cells with 1251-labeled IL-2 was carried out. The lanes showSIX-PAGE patterns of T cells stimulated with medium alone (lanes 1 and 41, IM(luM,lane 21, IM(0.5 nM, lane 31, PMA(lOng/ml, lane 51, and PMA(lng/ml,lane 6).

688

the

85, and 90 KD

previously,'

by each stimulation,

than

the

Vol.

164, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL

Table 1. IL-2R number and affinity stimulation

High affinity Kd(pM) sites/cell

medium IM IM(H-31)*

PMA

of tonsillar

T cells

Intermediate affinity Kd(pM) sites/cell

c 50 1,000 < 50 2,000

24 27

RESEARCH COMMUNICATIONS

Low affinity Kd(PM) sites/cell

425 8,500 9,000

1,000 1,350 2,000

16,700

17,500

T cells were cultured with PMA(10ng/mI) or IM(l PM) for 3 days and binding studies were performed. The data were analyzed by Scatchard plots. Dashes(-) indicate the absence of measurable receptors of the resmtive affinity class. IM(H-31)* shows that the binding studies were carried out in the presence of anti-p55(H-31, ref.17) antibcdy(50 Ug/ml).

constitution

of this

IM induces

p70/75

suggesting

that

band remains expression

calcium

to be clarified.

These

predominantly

ionophore

results

show that

and p55 is induced

and PK-C activator

induce

IL-2R

by PMA, expression

differently. Next,

we examined the effects

of IL-2 binding experiments alone,

was

IL-2

molecules

By IM stimulation,

significantly

the limits

increased,

with

the

to

leaving

absence of

measurable IL-2 binding

ones correspond high-affinity

in number by of the chemical

binding

sites

presence

of anti-p55(H-31)

isolated

to p55 and disrupted as reported

molecules

medium

an intermediate binding

sites

sites

below

binding

sites

intermediate-affinity

IL-2R.l-lo

those

H-31 reduced

with

with

binding

correspond

to p70/75

to ~55, and these two IL-2R subunits

consistent

intact,

were cultured cell

number

of representative

PMA induced low-affinity

increased

binding

the

and

the number of low-affinity

complexes were

of low-affinity

Results

When T cells

In contrast,

constitute

with

analysis.

bound to

The intermediate-affinity

and the low-affinity suggested

plot

affinity

the number of intermediate-affinity

of detection.

predominantly, sites.

by Scatchard

are shown in Table 1.

some

affinity.

sites

of IM and PMA on the

High-affinity

both stimulations. crosslinking.

(p55), Scatchard IL-2R antibody.17

the number of high affinity 689

was carried

H-31 prevented

high affinity previously.8

analysis

IGZR bindings, When cells sites

IL-2R

These results To exclude

are

the effects out in the IL-2

leaving

were stimulated

to an undetectable

are

binding p70/75 by IM,

IeveI(Table

Vol. 164, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure 2. Induction of p70/75 IL-2R subunit expression in SKW6-4 B cells by IM. SKW 6-4 cells were cultured with IM (lyM, lane 3 or 0.5UM, lane 4), PMA(lOng/ml, lane 2), or medium alone (lane 1) for 2 days. Then, chemical crosslinking studies of stimulated SKW6-4 cells (107) with 1251-IL-2 (10nM) were carried out. Arrows show the bands corresponding to plOl75 IL-2R ones.

1) *

In contrast,

increased.

the

number

These results

of

also

intermediate

suggest

that

affinity

sites

was rather

IM induces

p70/75

expression

significantly. In the present CD3 positive

cells

contaminating the possibility

study,

T cells

were

about 98% of the T cells.

B cells that

contaminating

cells.

on ~70175

expression

lymphoblastoid 6-4 cells

line,

expressed

at high

concentrations,

Chemical

crosslinking

dose-dependent

and

were separated

macrophages

may be

the observed results In order were

manner

(Fig.

negligible,

possibility,

examined

by

employing

In a previous

but lacked

2).

report,I'

the ability

IgM secretion

revealed

formation,

that

690

of

these

the effects

of IM

a monoclonal we showed that

to express

IM induced p70/75 PMA did

of

we cannot deny

by these cells

In contrast,

and

the effects

may be due to the effects this

induces studies

Although

to exclude

SKW 6-4.18 p70/75,

by E rosette

not

B SKW

~55, and IL-2, through expression induce

p70/75. in a ~70175

BIOCHEMICAL

Vol. 164, No. 2, 1989

AND BIOPHYSICAL

expression

significantly.

expression

in the absence of contaminating

the induction

These results

of p70/75 expression

show that cells,

RESEARCH COMMUNICATIONS

IM directly

induces ~70175

and PMA has little

effect

on

in SKW 6-4 cells. DISCUSSION

IM transports calmodulin

calcium

(CaM) branch

pathway).IIr15

suggest that pathways

the

calcium

turnover.

l*-l4

membranes and activates

message

PMA activates

system

PK-C directly

Based on these

~70175 and p55 expressions

are regulated

the

(calcium-CaM

without

provoking

evidence,

our results

by PK-C and calcium-CaM

differently.

Peripheral

blood

T cells

separated."16

and a factor

generally

from adult

show evidence

that

that

cells

differently

An increase transduction.

by

This

T cell

is

and suggest

that

the calcium
because

and/or

absence of ~55,

of IL-2 by itself.

The subunit intermediate

IL-2

of p55

is

to high

suggest

predominantly,

to function

that

is

evidence However,

at

in the presence the calcium-CaM

the affinity

to

that

we

expression

expressions

alone,

role

are

at

and

high

induces

cells6r8-10

high affinity

in

ILr2R complexes

an affinity

to

p70/75

p55 expression

and number of IL-2R in viva.

are

(0.1-10 u/ml). IL-2

Taken together,

regulates

the

to the signal

concentrations

of ~70/75*~.

in signal

IL-2,

p55 is related

convert

691

is

Here,

p70/75

an important

responding

regulates

it

stable.3-5

internalized8'*l

pathway

to induce

PK-C pathways.

physiological

suggested

are

forskolin

stimulations,

and p55

p70/75

by

and the PK-C pathway

manner, by changing

p70/75

may play

u/ml),

these

predominantly

through

is no

were reported

and

differentiation

and there

lines,

is relatively

induces

when cells

PMA, IL-l,

Under

expression

~70175

including

leukemia

ionophore

(100-10000

proliferation

for

agents,

of ~70175 expression

concentrations

necessary

p70/75

calcium

in human lymphoid

transduction

predominantly

p55 expression.3-5r1g-20

accepted

regulated

express

Avarietyof

derived

predominantly

results

of

Incontrast,

phosphatidylinositol

freshly

ions across biological

from these

expression

in a different

Vol. 164, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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