Vol. 266, No. 16, Issue of June 5, pp. 10552-10561,1991
Printed in U.S.A.
THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
8 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Kinase C Regulates Both Production andSecretion of
Interleukin 2”
(Received for publication, June 4, 1990)
Jaime F. ModianoS, Rebecca Kolp, Roberta J. Lamb, and Peter C. Nowellg
From the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104
Inhibiting protein kinase C (PKC) activity abrogated
interleukin 2 (IL2) production by mitogen-stimulated
human Tlymphocytes. This effect was due partially to
a 60%decrease in IL2 gene expression. However, when
PKC inhibitors were added after IL2 gene transcription had already proceeded for 3-4 h, the IL2 in the
culture supernatants was stillreduced by 30-SO%, and
intracellular IL2 was increased byup to 50%. The
inhibition of PKC affected the expression of IL2 receptors by these cells differently; it had little effect on
gene expression or on the membrane-bound form of
the receptor, but it decreased soluble receptors in the
supernatants by SO-80%. These data indicate that in
addition to its previously defined role in gene expression, PKC can also regulate extracellular secretion of
proteins critical for T cell proliferation.
Activation of T cells by ligands that bind to the T cell
antigen receptor complex (Ti-CD3) stimulates hydrolysis of
phosphoinositides by phospholipase C within seconds to minutes (Imboden and Stobo, 1985; Imboden et d., 1987). This
in turnresults in mobilization of intracellular ionized calcium
(Ca2+,reviewed in Gardner, 1989) and activation of protein
kinase C (PKC)’ (Farrarand Ruscetti, 1986; Mire et al., 1986;
and Ne1 et al., 1987). Protein kinase C and Ca2+contribute to
a continuing cascade of biochemical events that lead to the
expression of genes associated with cellular growth including
interleukin (IL) 2 and IL2 receptors (reviewed in Waldmann,
1986, Smith, 1988; and Crabtree, 1989). The IL2 receptor 01
chain is further modified post-transcriptionally into a membrane bound form (p55/Tac; Uchiyama et al., 1981; Waldmann, 1986) that forms part of the high affinity IL2 receptor
complex or a secreted form (p45, Rubin et al., 1985; Robb and
Kutny, 1987), whose function is unclear. The subsequent
interaction of soluble IL2 with high affinity surface IL2
receptors provides the signal that drives lymphocyte proliferation (reviewed in Smith, 1988).
The importance of Ca2+and PKC as “second messengers”
is underscored by the fact that agonists which stimulate
mobilization of intracellular Ca2+or activation of PKC can
induce 1L2 production, IL2 receptor expression, and proliferation of T cells directly (Truneh et al., 1985; Farrarand
Ruscetti, 1986; Modiano et al., 1988), bypassing the requirement for binding of ligand to the Ti-CD3 complex. Specifically, the importance of PKC in IL2 production by human T
cells has been inferred from the observations that agonists of
PKC (including phorbol estersor sn-1,2-diC8) induce IL2
gene expression and production of biologically active IL2
(Kern et al., 1986; Isakov et al., 1987; Subramaniam et al.,
1988). Furthermore, H7 (an inhibitor of PKC), but not dibutyryl CAMP,dibutyryl cGMP, prostaglandin Ez, or polyamine
inhibitors, was shown to diminish both IL2 mRNA expression
and IL2 activity from human tonsilar T cells stimulated by
PHA + TPA (Yamamoto et al., 1986).
The specific role of PKC(s) in IL2 production is still unclear. The transcription of the IL2 gene is dependent on
signals mediated through the T cell antigen receptor, but is
not optimal without an additional accessory signal that can
be delivered by agonists of PKC (Kern et al., 1986; Crabtree,
1989). A phorbol response element (AP-l/Fos binding site) is
present in the IL2 promoter, andthis element can drive
transcription following the activation of PKC; however, the
deletion of this element from the promoter only inhibits the
transcriptional activity of the promoter partially (Serfling et
al., 1989; Muegge et al., 1989; Crabtree, 1989).
Once transcription of IL2 occurs, the protein appears to be
only slightly modified post-transcriptionally (Robb et al.,
1981; Robb et al., 1984; Taniguchi et al., 1986; Smith, 1988;
Kung et al., 1989). There are no N-glycosylation sites on the
IL2 protein (Taniguchi et al., 1983),it is variably O-glycosylated (Robb et al., 1984) or sialyated (Robb et al., 1981), and
the biological activity of unglycosylated, recombinant IL2
produced in Escherichia coli is indistinguishable from that
produced in mammalian cells (Taniguchi et al., 1986). Recently, human recombinant IL2 produced in E. coli or in Cos
cells was shown to be phosphorylated by PKC at the serine
on position 6 (Kunget al., 1989),but thisphosphorylation did
not affect the ability of the soluble protein to promote T cell
proliferation.
Our aim was to examine further therole of PKC in mitogeninduced activation of human lymphocytes. Our results indicate that the chemical inhibition of PKC diminished both
IL2 gene expression and IL2 secretion, as well as thesecretion
of soluble IL2 receptors in human T cells stimulated by PHA
or by PHA in combination with phorbol esters. It had little
effect, however, on the expression of the IL2 receptor 01 gene
or of IL2 receptors on the cell membrane.
* This work wassupported by National Institutes of Health Grants
CA-15822 and CA-42232. The costs of publication of this article were
defrayed in part hy the payment of page charges. This article must
therefore he hereby marked “aduertisement” in accordance with 18
U.S.C. Section 1734 solelyto indicate this fact.
$ Present address: Dept. of Pathology, Colorado State University,
College of Veterinary Medicine, Fort Collins, CO 80523.
To whom correspondence should be addressed. Tel.: 215-8981899 or 215-898-8061.
The abbreviaitons used are: PKC, protein kinase C; IL, interleukin; PHA, phytohemagglutinin, TPA, 12-0-tetradecanoylphorhol
EXPERIMENTALPROCEDURES
13-acetate; PBL, peripheralblood lymphocytes; HEPES, 4-(2-hyCells-Peripheral blood lymphocytes (PBL) were separated by
droxyethy1)-1-piperazineethanesulfonicacid; EGTA, [ethylenebisFicoll-Hypaque (Histopaque 1.077, Sigma) density gradient centrif(oxyethylenenitrilo)]tetraaceticacid SDS, sodium dodecyl sulfate.
‘
10552
PKC Regulates IL2 Secretion
ugation from heparinized whole bloodor from leukopheresis residues.
Whole blood wasobtained by venipuncture from healthy adult human
volunteers, and leukopheresis residues were obtained from the Philadelphia Red Cross. PBL populations were enriched for T lymphocytes by the removal of adherent cells as described (Modiano et al.,
1988; Kern et al., 1986).
The parental 532, H33HJ-JA1 (H33), and CTLL2 cell lines were
obtained from the ATCC (Rockville, MD). Subclones 532-7 and 53235 were a gift from MarianNakada(Department
of Pathology,
University of Pennsylvania, Philadelphia, PA); 532-68 and 532-68.3
were a gift from Malek Kamoun (Department of Pathology, University of Pennsylvania). The responses described in the results were
not significantly different among the various 532 subclones. All the
cell lines were maintainedin continuous culturein RPMI 1640
supplemented with 10% (v:v) heat-inactivated fetal calf serum, 2 mM
L-glutamine, 2 mM sodium pyruvate, 10 mM HEPES, and 55 p M 2mercaptoethanol. CTLL2 cultures were also supplemented with virusfree condidtioned media from MLA-144 cells as a source of interleukin
2 (IL2). The MLA-144 cell line was maintained in continuous culture
in HL1 serum-free media (Ventrex Laboratories, Portland, ME).
All of the cells were extensively washed in Hanks' balanced salt
solution to remove any tracesof serum before experimental conditions
were established, and the cells were then incubated in a 5% COZ
atmosphere at 37 "C in HL1 serum-free media, supplemented with 2
mM L-glutamine, 2 mM sodium pyruvate, and 10 mM HEPES for the
duration of the experiments.
Chemicals and Reagents-Staurosporine was obtained from Calbiochem and was diluted to 2 mM in dimethyl sulfoxide and kept as a
stock at -20 "C. This stock was further diluted in HL1 media to a
concentration of 10 p~ and stored at 4 "C for up to 8 weeks. Staurosporine was routinely used at a final concentration of 5 or 10 nM in
the experiments described, unless otherwise indicated. H7 (I-(&
isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride)
and
hydroHa1004 (N-(2-guanadinoethyl)-5-isoquinolinesulfonamide
chloride) were obtained from Seikagaku America (Rockville, MD)
and were used at a concentrationof 5 or 10 pM, respectively, according
to the manufacturer's instructions. Phytohemagglutinin (PHA-P)
and TPA were purchased from Sigma and used at 5 pg/ml and 20
nM, respectively. Human recombinant IL2 was a gift from Cetus
Corp. (Emeryville, CA). Tissue culture reagents were obtained from
various manufacturers through the University of Pennsylvania Cell
Center. All other chemicals were purchased from Sigma, unless otherwise noted.
Proliferation Assays-The proliferative response of PBL to mitogens was determined by the uptake of [3H]thymidine into DNA as
described (Modiano et al., 1988).
Assays for ZL2 Actiuity-IL2-containing supernatants were prepared from cultures of PBL, H33, 532, or MLA-144 cells incubated
for 6-24 h as noted under "Results." All the supernatantswere cleared
of cellular material by centrifugation and by filtration through 0.2pm filters (Nalgene, Rochester, NY). Gibbon sarcoma virus was
removedfrom the MLA-144 supernatants by ultracentrifugation
(100,000 X g for 2 h at 4 "C), followed by filtration. The IL2 activity
was assayed using a CTLL2 bioassay as described (Baker et al., 1979).
One unit/ml (units/ml) of IL2 was defined as the reciprocal of the
dilution of a test supernatant which induced half-maximal proliferation of the CTLL2 cells, where maximal proliferation was assessed
by stimulatin the cells with saturating concentrations (>lo0 pM) of
human rIL2 or of MLA-144 derived IL2. The CTLL2 clone used for
these experiments did not proliferate in response to PHA-Por TPA,
alone or in combination, or in response to IL4. The response of the
CTLL2 cells to IL2 was also neither enhanced by these agents nor
inhibited by staurosporine or H7.
The absolute amount of IL2 produced by mitogen-stimulated PBL
varied from donor to donor and ranged from 12.5 units/ml to 250
units/ml. The responses from the cell lines were more uniform: H33
cells stimulated by PHA + TPA for 16-24 h produced between 17
and 32 units/ml of IL2; 532 cells produced between 25 and 60 units/
ml of IL2 after stimulation by mitogens for 6 h and 200-400 units/
ml of IL2 when they were stimulated for 10-24 h.
Flow Cytometry-The expression of T cell surface antigens and
IL2 receptor a was analyzed by indirect immunofluorescence as
described (Modiano et al., 1988). The OKT3 (anti-CD3, unmodified
or conjugated to phycoerythrin) and O K T l l (anti-CD2) monoclonal
antibodies were purchased from OrthoPharmaceuticals (Raritan,
NJ). The anti-Leu-4 (anti-CD3) monoclonal antibody was obtained
from Beckton Dickinson (Mountain View, CA). Fluorescein isothiocyanate-conjugated goat-anti mouse antibodies were from Tag0 (Bur-
10553
lingame, CAI. Theanti-Tac (anti-CD25/p55 IL2 receptor) monoclonal antibody was a gift from T. Waldmann (National Cancer
Institute, Bethesda, MD). The samples were analyzed on a FACSAnalyzer (Becton Dickinson).
Purification of Protein Kinase C-PKC was partially purified from
lysates of0.5-1 X 10' PBL, 532, or H33 cells by sequential anion
exchange chromatography and gel filtration. Cells were lysed in buffer
A (330 mM sucrose, 20mM Tris, pH 7.3,2 mM EDTA, 0.5 mM EGTA,
0.1% Nonidet P-40, 1 mM N-ethylmaleimide, 0.03 p~ aprotinin, 0.5
p~ leupeptin), and the nonextractable material cleared by centrifugation (15,000 X g at 4 "C for 5 min). The cleared lysates were then
loaded onto 1.5-ml DE52 columns (Bio-Rad), eluted with a linear
gradient of KC1 (0-350 mM), and 0.5-ml fractions tested for activity.
Active fractions were pooled and dialysed extensively in buffer B (20
mM Tris, pH 8.0,2 mM EDTA, 0.5 mM EGTA, 0.5 mM dithiothreitol,
1 mM N-ethylmaleimide, 0.03 p M aprotinin, and 0.5 p M leupeptin).
The samples were then loaded on 5 ml G50-150 Sephadex columns
(Whatman) and eluted with buffer B. Active fractions were pooled
and used to assay the inhibition by staurosporine.
Protein Kinase C Activity Assays-The specific activity of PKC
was determined by the specific phosphorylation of histone I as described (Kikkawa et al., 1983). One unit/pg of PKC activity was
defined as the incorporation of 1 pmol of phosphate from [32P]ATP
into the substrate over 30 min at 30 "C/pg of protein in the assay
fraction. Assays for autophosphorylation were essentially identical to
the activity assays, but no exogenous substrate (histone I) was added.
The reaction was stopped after 30 min by the addition of sodium
dodecyl sulfate (SDS)-sample buffer, the samples were boiled for 5
min a t 100 "C, and the proteins separated over 7.5% polyacrylamide
gels by SDS-polyacrylamide gel electrophoresis as described (Laemmli, 1970). The gels were autoradiographed on XAR-5 film (Kodak)
at -70 "C, and autoradiograms quantitated by laser densitometry
using an Ultroscan XL (Pharmacia LKB Biotechnology Inc.).
Tyrosine Phosphorylation Assays-The activity of cellular tyrosine
kinases was determined by analyzing the relative phosphorylation of
tyrosine phosphoproteins with an anti-phosphotyrosine antibody
(Boehringer Mannheim). This antibody was used both for immunoprecipitation and immunoblotting studies.
Northern Blotting-Cells were routinely cultured for 3-6 h in the
presence or abscence of mitogens and with or without staurosporine.
Total RNAwas
isolated by the guanadine isothiocyanate/CsCl
method as described (Kern et al., 1986). Cytosolic RNA was isolated
by the method of Wilkinson (1988). Ten to 20pgof
RNAwere
separated electrophoretically over 1%agarose, 5% formaldehyde denaturing gels and transferred to Nylon 66 membranes (Genescreen
Plus, Du Pont-New England Nuclear) by capillary blotting. Steady
state levels of mRNA expression were assessed using a human IL2
cDNA (Holbrook et al., 1984) or a human IL2 receptor cDNA (Leonard et al., 1984). A cDNA encoding the human &microglobulin gene
(Suggs et al., 1981) was used to ensure that the amounts ofRNA
present in each sample were approximately equivalent. The cDNA
probes were labeled by random primer extension and hybridized to
the immobilized RNA as described (Kern et al., 1986). Blots were
autoradiographed on XAR-5 film and autoradiograms quantitated
densitometrically as described above.
WesternZmmunoblotting-PBL or either of the cell lines were
stimulated for 3-6 h as described above. Cell-free supernatants were
separated from the cell pellets by centrifugation, and thepellets were
lysed in buffer C (300 mM NaCI, 50 mM Tris, pH 7.6, 0.5% Triton X100, 1 mM N-ethylmaleimide, 0.03 p M aprotinin, 0.5 p M leupeptin).
The protein content in the supernatants and in the cell lysates was
determined by the bicinchoninic acid method (Pierce Chemical Co.).
The protein concentrations were routinely between 1.5-2.5 and 2-8
mg/ml in the culture supernatants and the cell lysates, respectively.
There was minimal variability from sample to sample. Each sample
was concentrated as necessary using a microfiltration unit (UltrafreeMC, Millipore, Bedford, MA). Subsequently, 800-1000 pg of supernatant protein or 20-100pgof
cellular protein were separated by
SDS-polyacrylamide gel electrophoresis over 15% polyacrylamide
mini-gels and transferred to nylon re-enforced-nitrocellulose membranes(Nitroscreen-West, Du Pont-New England Nuclear). The
blots were probed with either a rabbit anti-human recombinant IL2
antisera (Collaborative Research Inc., Bedford, MA) or a goat antihuman recombinant IL2 antisera (R & D, Minneapolis, MN) followed
by secondary anti-rabbit or anti-goat antibodies conjugated to alkaline phosphatase (Pierce). The alkaline phosphatase reaction was
carried out as described by the manufacturer. Quantitation of the
blots was performed by laser densitometry.
10554
PKC Regulates IL2 Secretion
Reverse Transcriptase Assays-Reverse transcriptase activity in
supernatants from MLA-144 cells were measured as described (Simonian et al., 1990). A murine Gross-murine leukemia viruswas used
as a positive control, and therewas no significantdifference between
the amount of phosphate incorporated into polyadenosine primers
from ['"PITTP by the reverse transcriptase from the murine versus
the ape viruses.
AUTOPHOSPHORYLATION
A
B
C
RESULTS
Staurosporine Inhibits Proliferationof Human PBL-When
staurosporine, a relatively specific inhibitor of PKC, was
added simultaneously with the mitogens to cultures of PBL
stimulated by PHA or by anti-CD3 antibodies, proliferation
was decreased in a dose-dependent fashion(Fig. 1A) as measured by incorporation of ["Hlthymidine intoDNA. This effect
was largely reversible if exogenous human recombinant IL2
was added at the onsetof culture and proliferation measured
after 48 or 72 h (Fig. 1B).
Staurosporine Inhibits PKC in Human Lymphocytes-We
also analyzed the effects of staurosporine on the in vitro
activity of PKC purified from human T cells, and on PKC
function in intact cells, to determine whether it inhibited
PKC from human lymphocytes in a similar fashion to PKC
100
-
A
Histone Phosphorylotion
0
c
2
-03
.-E
75-
'
&
f 50.-
.
X
0
E
25-
bp
04
.
0
,
.
2
,
'
4
,
.
,
6
[Staurosporine] (nM)
'"1
B
.
0
,
.
10
1
FIG.2. Staurosporine inhibits PKC activity in vitro. Protein
kinase C was purifiedfrom unstimulated H33 cells as described. PKC
activity was assessed by autophosphorylation (top panel)or by phosphorylation of histone I (bottom panel) in the presence of calcium,
magnesium, phosphatydylserine, and diolein (A); calcium,magnesium, phosphatydylserine, diolein, and staurosporine (10 nM) ( R ) ;or
EDTA (C). The molecular mass of autophosphorylated PKC was 82
kDa. The relative levels of autophosphorylation were quantitated by
laser densitometry. Similar results were obtained in one (autophosphorylation) and two (PKC activity) additional experiments.
from nervous tissues (Tamaoki et al., 1986; Nakadate et al.,
1988). Fig. 2 shows that PKCpurified from H33 cells became
autophosphorylated as an 82-kDa band in the
presence of
Ca2+and phospholipids (lane A , upper panel). Staurosporine,
a t 10 nM, inhibitedthisautophosphorylation
by approximately 80% (lane B, upper panel). Similarly, PKC activity as
measured by histone phosphorylation was inhibited approximately 50% by staurosporine (Fig. 2, lower panel). PKC was
notactivewhenits
cofactors were absent from the assay
mixture (i.e. no phospholipids were added and EGTA was
unstlrnulated
PHA
PHA+Stauro
PHA+Stauro+lU
used as a calcium chelator, lane C ) . A similar inhibition by
FIG. 1. Staurosporine (Stauro) inhibits proliferation of staurosporine of histone phosphorylation by PKC from huPHA-stimulated PBL. Unstimulated PBL or PBL stimulated by
man PBLwas observed (not shown).
PHA (5 pg/ml) were cultured for 48-120 h. ["HIThymidine uptake
As another, albeit indirect, approach to this
effect of stauwas measured for the last 8 h of culture. The relative responses were
not significantly different among the various time points tested, but rosporine on PKC in human lymphocytes, we explored the
fact that stimulationof T cells by agents that activate PKC
the absolute magnitude of the peak responses among the different
donors ranged from 18,000 to 81,000 cpm. ["HIThymidine uptake by induces the phosphorylation and subsequent modulation of
unstimulated cells was 3-876 of the maximal stimulation. A, stauros- CD3 complex molecules on the cell surface (Cantrell et al.,
porine was added to PBL stimulatedby PHA at the onsetof culture
1985). Fig. 3A shows that stimulation of H33 cells by TPA +
as indicated. The data shown are the means
f S.E. of measured after
72 h of culture of five experiments done. B, PBL were cultured as PHA for 30 min decreased the amount of CD3 on the cell
indicated for 72 h. Staurosporine was used a t 5 nM and IL2 a t 2 nM. surface by 50-60% as determinedby indirect immunofluorescence. Staurosporinealone(at10
nM) did not affect the
The results shown are the means f S.D. of two experiments done.
PKC Regulates IL2 Secretion
200
10555
1
P
FIG. 3. Staurosporine (Stauro) inhibits PKC activity in situ. H33 cells were incubated for 30-60 min at
4 "C (unstimulated) or at 37 "C in the presence of PHA (5pg/ml), TPA (20 nM), staurosporine (10 nM), a-Leu-4
(an a-CD3 antibody), orcombinations thereof. Samples were analyzed using a FACS analyzer. A , cells were stained
with OKT3 (an a-CD3 antibody which binds an epitope distinct from a-Leu-4)directly conjugated to phycoerythrin.
FL2 indicates relative red fluorescence; the y axis represents cell numbers; MFC, mean fluorescence channel. B ,
cells were first stained with a goat a-mouse Ig antibody directly conjugated to fluorescein and then followed with
OKT3 directly conjugated to phycoerythrin. FLl indicates relative green fluorescence and represents cell surface
expression of the Leu-4 epitope of CD3; FL2 represents relative red fluorescence and represents cell surface
expression of the OKT3 epitope of CD3. One of four experiments done is shown.
expression of surface CD3 on these cells. But when stauro- human T cell lines, which appear to use the same signaling
sporine was used in conjunction with TPA PHA, therewas pathways as normal PBL (Imboden and Stobo, 1985; Imboden
only a 20-30% loss of surface CD3 after a 30 min stimulation, et al., 1987)' but yield larger amounts of IL2 following stimsuggesting there was an inhibitory effect on the PKC-depend- ulation by mitogens.
Consistent with previous results from our laboratory and
ent modulation of the CD3 complex. Staurosporine also inhibited to a similar degree the modulation of surface CD3 others, IL2 was detectable in culture supernatants from PBL
from PBL stimulated by PHA or from 532 cells stimulated or either cell line 3-4 h following the mitogenic stimulation
by PHA plus TPA. As a control, we stimulated T cells for 30 as measured by the CTLL2 murine indicator line bioassay
min with soluble anti-CD3 antibodies, which appear to induce (Fig. 4). This response increased linearly for 10 to 15 h when
modulation of the CD3 complexby capping and are not strong it plateaued, presumably reflecting a steady state between
activators of PKC (Ledbetteret al., 1986; Manger et al., 1987). production and utilization of IL2. The addition of TPA enUnder these circumstances, staurosporine had no effect on hanced the production of IL2 by PBL stimulatedby PHA and
the internalization of CD3 on H33 cells (Fig. 3b) or on PBL was required for IL2 production by the T cell lines, which are
devoid of accessory cell function (Fig. 4). This synergism was
or 532 cells.
To determine whether staurosporine was acting as a non- not observed when the 4a-analog of TPA, which does not
specific inhibitor of protein kinases, we examined its effect activate PKC (Isakov and Altman, 1987),was used instead of
on another group of kinases which are specific tyrosine phos- the 4P-analog which does (not shown).
Either staurosporine or H7 reduced the IL2 levels in superphotransferases. We assessed the accumulation of tyrosine
phosphoproteins in mitogen-stimulated PBL or 532 cells by natants from 532 cells stimulated by PHA plus TPA up to
immunoprecipitation or by immunoblot analysis using an 100% (Fig. 6). IL2 production by H33 cells stimulated with
anti-phosphotyrosine monoclonal antibody and found that PHA plus TPA was also markedly inhibited by staurosporine
(Fig. 4). The inhibitory effect of staurosporine on IL2 producstaurosporine had no significant effect (not shown).
tion by these two cell lines was dose dependent (Fig. 4B).
Taken together, these observations suggest that stauroStaurosporine also showed a dose-dependent inhibition of IL2
sporine is an effective inhibitor of PKC purified from human
production by PBL stimulated by PHA alone or in combinaT cells as well as of PKC in situ, and at concentrations tion with TPA (Fig. 4). The observed decrease in the levels
between 5 and10 nM, staurosporine does notinhibit the of IL2 in these culture supernatants was directly related to
phosphorylation of major tyrosine phosphoproteins that may an inhibitory effect on IL2 production, rather than a consealso be important signaling molecules in T cells.
quence of the inhibitory effects of staurosporine on cellular
Inhibitors of PKC Decrease IL2 Production by Human T proliferation. The former effect was observable between 3 and
Cells-To begin to dissect the mechanism by whichPKC may 20 h after stimulation of PBL by mitogens, at which time
be important in regulating proliferation of normal human T staurosporine affected neither viability nor cell numbers. In
cells, we determined how staurosporine or another inhibitor contrast, the latter effect was not apparent for >36 h when
of PKC, H7, affected IL2 production and IL2 receptor expres- PBL entered the S phase of the cell cycle.
sion in PBL stimulated by PHA. We also analyzed the effect
R. Kolp and J. F. Modiano, unpublished observations.
of these inhibitors on IL2 production by the 532 and H33
+
PKC Regulates IL2 Secretion
10556
- +
-
PHA + T P A
Stourosporine(nM) 0
100
0--[7
0
10
20
PBL
H33
J32
30
-I- f
10 5
10 5 0
FIG.5. Staurosporine inhibits IL2 mRNA accumulation in
mitogen-stimulated human T cells. 532 cells were stimulated by
PHA ( 5 pglml), TPA (20 nM), or staurosporine as indicated for 3 h.
Cytosolic RNA was isolated from cell lysates, and expression of IL2
mRNA was determined by Northernblotting witha humanIL2
cDNA. Exposure of the autoradiogram was 72 h. The band densities
were quantitated densitometrically, normalized for loading, and the
amount detected in the cells stimulated by PHA + TPA was arbitrarilly considered to be 100%. The relative levels found in the rest
of the experimental conditionswere: unstimulated cells, 0%; staurosporine alone, 0%; PHA + TPA + staurosporine a t 5 nM, 48%, a t 10
nM 46%, and at 50 nM, 58%. Similar results were obtained in two
additional experiments.
0.6
0-0
A-A
0
40
50
[Sfaurosporins] (nM)
FIG.4. Staurosporine (Stauro) inhibits IL2 production by
human T cells. A, PBL, 532 cells, or H33 cells were stimulated by
I’HA (5 pg/ml), TPA (20 nM), or staurosporine (5 or 10 nM) for 1522 h as indicated. B, PBL were stimulated by PHA, and 532 or H33
cells were stimulated by PHA + TPA for 16-20 h with the indicated
concentrations of staurosporine. IL2 productionwas measured using
a murine IL2-dependent indicator cell line (CTLLZ). The range for
the absolute amount of IL2 (in units/ml) produced by the different
cell typesis defined under“Experimental Procedures.” The data
shown represent the means of 3-11 experiments for the different
conditions were the absolute amount of IL2 produced after stimulation by PHA + TPA in A and in B for the H33 (triangles) and 532
(squares) cell lines or by PHA in B for PBL (circles) was considered
t o be 100%.
Staurosporine Inhibits IL2 Gene Expression in Human T
Cells-The decrease in IL2 production induced by staurosporine could be through a transcriptional ora post-transcriptional effect. We used Northern blot analysis to determine
how staurosporine affected IL2 gene expression in mitogen
stimulated human T cells.
IL2 mRNA was not detected in unstimulated532 cells and
was initially seen in cells stimulated by PHA plus TPA after
2-3 h, with a peak at 4-5 h. Staurosporine, at a dose of 5-50
nM, inhibited the accumulation of IL2 mRNA in 532 cells
stimulated for 3-6 h (Fig. 5). Afterbeingnormalized for
loading differences, the levels of IL2 mRNA ranged between
48 and 58% of control in stimulated cellswhich received
staurosporine at concentrations between 5 and 50 nM. Thus
it appeared that unlike the IL2 activity present in culture
supernatants, the inhibitionof IL2 mRNA accumulation was
not dose dependent.
Staurosporine Inhibits Secretion of IL2 byHuman T CellsThe fact that staurosporine only reduced partially the accumulation of IL2 mRNA in human T cells presented an ap-
04
w
0
1
1
3
4
5
6
7
8
Time Inhibitor Added
0-0
Staurosporine
A-AH7
.“.Ha1004
FIG.6. PKC inhibitors decrease IL2 secretion by human T
cells. 532 cells were stimulated by PHA (5 pglrnl) + TPA (20 nM)
)
at
and staurosporine (10 nM), H7 (5 p ~ ) or, Ha1004 (10 p ~ added
the indicated time points. IL2 in the culture supernatants was then
measured after 22 h. For this experiment, the IL2 positive control
(no inhibitors present) was equivalent to -470 units/ml and is indicated as 100%; unstimulated cells produced <1 unit/ml. These data
are representativeof three (H7 andHa1004) or more (staurosporine)
experiments done.
parent paradox, since IL2 activity in culture supernatants
couldbecompletely
abrogated by either of the two PKC
inhibitors studied. This discrepancy might be explained by
changes in eitherIL2 mRNA stability, IL2 protein synthesis,
secretion of IL2, or any combinationof the three. Theobservations that PKC appears to have only a partial role in the
transcription of the IL2 gene (Crabtree, 1989) and that IL2
can be phosphorylated directly by PKC (Kung et al., 1989)
led us to investigate the latter
two possibilities We examined
how staurosporine or H7affected IL2 production by 532 cells
after maximal IL2 gene expression had already occurred (3-6
h following mitogenic stimulation).
Fig. 6 shows that both agents
reduced IL2 activity in culture
supernatants by -80 and 50%, respectively, when added 4 h
after the initial stimulation by mitogen. In contrast to this
effect seen with the inhibitorsof PKC, Ha1004 enhanced IL2
production in mitogen-stimulated 532 cells by >20% when it
PKC Regulates IL2 Secretion
was added at the onset of culture and had no effect when
added 6 h later. This compound is chemically similar to H7,
butitpreferentiallyinhibits
cyclic nucleotide-dependent
kinases rather than PKC.
Staurosporinealso reducedIL2 productioninmitogenstimulated PBL or H33 cells by >50% when it was added to
the cells 4-8 h after the onsetof culture (data not shown).
To determine whether these effects of staurosporine reflected an inhibition of IL2 synthesis versus secretion, we
assessed directly the levels of IL2 protein in culture supernatants and whole cell lysates from stimulated T cells by
Western immunoblotting. Fig. 7 shows that when staurosporine was added to the cells concurrently with the mitogens,
there was a decrease of 65-80% in the amount of IL2 protein
found in the culture supernatants at 6 h (top panel, lane3 ) ,
as well as 40-50% decrease in the IL2 found within the cells
(bottom panel, lane3 ) as compared with the stimulated controls (lane 2 ) ; and these changes were consistent with the
concurrent -50% decrease in IL2 gene expression(Fig. 8).
However, when staurosporine was added to the cells 4 h
after themitogens, and the culture continued
for an additional
2 h, the IL2 in the supernatants again was reduced by approximately 40-50% (Fig. 7, top panel, lane 4 ) , but there was
a concurrent increase of 30-40% in the amount of IL2 found
inside thecells (Fig. 7, bottom panel,lane 4 ) as compared with
the stimulated controls (lane 2 ) . The increase in the level of
intracellular IL2 did not reflect a generalized accumulation of
cytosolic proteins since the levels of the a and @ isoforms of
PKC were unchanged in these cells regardless of the stimuli
used (not shown).Also, in these experiments, the changes in
the amounts of IL2 protein, in both the supernatants and in
the cell lysates, correlatedwell with the changesin IL2activity
detected by the CTLL2bioassay (Fig. 8). Similar resultswere
obtained when H7,butnot
Ha1004, was used as A PKC
inhibitor.
Oneadditional piece of evidence suggesting that PKCPHA+TPA
Stouro (time)
-
+
-
+
+
Oh
4h
MW
10557
0 hours
4 hour#
Time of Staurosporine Addition
FIG. 8. Staurosporine inhibits the secretion of biologically
active IL2 from 532 cells. .J32 cells were stimulated t)y I'HA ( 5
pg/ml) + ' I T A (20 nM) andstaurosporineaddedattheonset
of
culture (0 h) or after 4 h. After 6 h. the cells were separated from the
cell-free supernatants. RNAor protein were isolated fromcell lysates.
Expression of I L 2 mHNA WAS determined hv Northern thtting as in
in cell
Fig. 6. IL2 activityinculturesupernatants(cell-free)and
lysates (cell-associated) was determined as in Fig. 4. The cell lysates
were diluted hv >100-fold to prevent lysis of the CTI,I,2 cells hy the
detergent,andthecell-associatedactivitywasnormalizedtothe
concentration of proteinpresentinitiallvineachsample.Similar
results were obtained in two (mRNA) nnd three (activity) experiments.
2800 h
24%
-
g2100U
-._
y1750-
-"
2 1400.
0
I-
=
1050-
7001
,
S50
0
,
0
&:,..,.::&:::B::::U
,
.
,
2
I
,
4
\
\
,
6
1
1
8
13500
f>:-lO
IO
log2 dilution
IL2( % of
control) 0
PHA+ TPA
Slouro(time)
-
100
+
-
20
57
+
+ rlL2
Oh4h-
IL2(% of
control) 0 I00
FIG. 9. Staurosporine (Stauro) inhibitR IL2 secretion but
not virusbuddingin M L A - 1 4 4 cells. M L A - 1 4 4 cells werecultured
in the presence (rlosrd symboh) or absence (own s y m b o k ) of stflurosporine for 16 h. The concentrations of 112 present in the virus56 units/ml and 340 units/
free supernatants from these cultures were
ml, respectively, as determined using the C'I'IL? cell hioassav (solid
lines). Reverse transcriptase activity was measuredin serial dilutions
from the culture supernatants (davhrd linrs). T h e data shown are
representative of four experiments done.
52
152
FIG.7. Staurosporine (Stauro) inhibits the secretionof IL2
protein by 532 cells. .I32 cells were stimulated as indicated for a
total of 6 h, at which time culture supernatants (top panel) or cell
lysates (bottom panel) were harvested and analyzed for the presence
of IL2 protein by Western immunohlotting. PHA was used a t 5 pm/
ml, TPA at 20 nM, and staurosporine at 10 nM. 800 pg of protein
from the culture supernatants and 35 pg of cell-lysate protein were
loaded onto each lane in the gels. T h e hlots were quantitated densitometrically and the value obtained for lane 2 (positive control) was
of
considered as 100"; in each case. These data are representative
four experiments done.
mediated processesmaybeinvolved
in IL2 secretion was
obtained from the gibbon ape T cell line MLA-144, which
produces IL2 constitutively.In the caseof this cell line, which
carries infective gibbon sarcoma virus, IL2 gene expression
may be driven by the viral promoter (Rabin et al., 1981). The
addition of staurosporine toMLA-144 cells for up to 20 h did
notmeasurably affect IL2 mRNA levels. Underthesame
conditions, however, there was up to an 85% decrease in the
amount of IL2 in culture supernatants (Fig. 9). This effect of
staurosporine onIL2 production appeared to be specific, since
budding of viral particles from the MLA-144 cells, as measured by reverse transcriptase activity in the culture supernatants, was not affected (Fig. 9).
Staurosporine Inhibits Secretion of Soluble IL2 Hrceptors,
but Not Expression of IL2 Receptor mRNA or of Mcmhrane-
PKC Regulates IL2 Secretion
10558
A
PHA+TPA Stouro(time) -
+ + +
-
Oh 4h
IL2R
FIG.10. Staurosporine (Stauro)
inhibitssecretion
of soluble IL2
receptors but not expression of
membrane bound IL2 receptors or
of the IL2 receptor gene. A, expression of IL2 receptor (Y mRNA in unstimulated 532 cells, or in cells stimulated for
G h by PHA (5 pg/ml) + TPA (20 nM)
with staurosporine (10 nM) added as indicated, was determined by Northern
blotting witha
human IL2 receptor
cDNA.Theexposure of the autoradiogram was 72 h, and the results arerepresentative of two experiments done. R,
expression of membrane-bound IL2
receptor (Y (p55/Tac) by PHA-stimulated PRL, in theabsence (circles) or
presence (squares) of staurosporine (10
nM), was determined by indirect immunofluorescent analysis. The
results
shown were compiled from three independent
experiments.
Unstimulated
I'BL routinely exhibited 4 % CD2'/
IL2Rn+ cells. C , theconcentration of
soluble IL2 receptors in culture supernatants from PBL stimulated by PHA
in the absence (circles) or presence of
staurosporine which was added to the
cells either at the onset of culture (triangles) or 4 h later (squares) were measured by a sandwich enzyme-linked immunosorbent assay. No soluble IL2
receptors were detected in the supernatants from unstimulated PBL. The results shown were compiled from two experiments; similar results were obtained
in two additional experiments using532
cells.
100 - IB
90
YI
..
80
0-0
PHA
0-0
PHA+Stauro
70
cv
a 30
0
be 20
10
0
0
bound IL2 Receptors-To begin to determine whether these
multiple effects of PKC in the control
of IL2 productionwere
specific, we analyzed whether staurosporine also affected the
expression or the secretionof IL2 receptorsby human T cells
stimulated by mitogen. The expression of IL2receptor
mRNAs (3.5- and 1.5-kilobase transcripts) wasinducedby
stimulation of 532 cells with PHA+TPA (Fig. lOA, lune Z),
24
48
72
Time (hours)
and the steady state level of IL2 receptor mRNAs was only
inhibited by staurosporine (25% in these cells (Fig. lOA, lune
3).
Similarly, stimulationof PBL by PHA induced the expression of IL2 receptor a (p55/Tac) on thecell surface, and this
was maximal by 50-72 h as determined by indirect immunofluorescence (Fig. 10B). In thesecells, staurosporine affected
PKC Regulates IL2 Secretion
10559
of IL2
the expression of membrane-bound IL2 receptor a only min- itors of PKC also appeared to interfere with secretion
imally (<15%) at 50 h or not at all (Fig. 10B). Staurosporine and of soluble IL2 receptors.Specifically, staurosporine or H7
also did not appear to affect the expression
of surface IL2 could essentially abrogate IL2 activity in culture supernatants
a
receptors as measuredby binding of radiolabeled IL2. In one of PBL or 532 cellsstimulated by mitogens with only partial
experiment, the specific binding of IL2 to PHA-stimulated reduction in the accumulation of IL2 mRNA. Particularly,
PBL showed 2.6 X lo3molecules/cell versus 2.1 X lo3 mole- stimulated 532 cells (as well as PBL or H33 cells), to which
cules/cell a t a concentration of 5 PM IL2 and 2.7 x lo6 staurosporine or H7were added afterIL2 gene expression had
molecules/cell versus 3.3 X lo6 molecules/cell a t a concentra- already occurred still showed a 50% decrease in the amount
tion of 20 nM IL2 without and with staurosporine, respec- of IL2 protein and activity in culture supernatants, concurrent
with a 30-40% increase in the amount of intracellular IL2,
tively.
PHA also induced secretion of soluble IL2 receptor a by but notof other cytosolic proteins. Also, whereas the absolute
from stimulated 532 cells
PBL as measuredby a sandwich enzyme-linked immunosor- level of IL2 in the supernatants
bent assay(Fig. 1OC).In contrast to the
effect of staurosporine increased linearly between 6 and 15 h, the amountof IL2 in
the supernatants fromcells which received staurosporine 4 h
on the expression of membrane-bound IL2 receptors, this
PKC inhibitor reduced by 50-80% the amountof soluble IL2 after stimulation, reached a plateau 2 h later. This suggests
that essentially all secretion of IL2 ceased within two hours
receptor a in culture supernatants from PHA-stimulated PBL
when added either at the onset of culture or 4 h after stimu- after exposure to staurosporine.
Soluble IL2receptorsrepresent
amodifiedform
of the
lation (Fig. 1OC). The secretion of soluble IL2 receptor LY by
mitogen-stimulated 532 cells wasinhibited toa similar extent membrane-bound IL2 receptor a chain, and they appear to
be associated with activated or rapidlydividing lymphocytes
(not shown).
(Rubin et al., 1985; Nelson, et al. 1986; Robb and Kutny,
DISCUSSION
1987). Staurosporine alsoreduced, byup to80%, the secretion
To study the role of PKC in the activation of human T of soluble IL2 receptors by PBL or 532 cells stimulated by
and
cells, we assessed the effect of two chemically distinct inhib- mitogen. As noted above, IL2 receptor gene expression
itors of PKC on proliferation of normal human PBL and on transport of the membrane-bound form of this protein were
not significantly affected by staurosporine.
IL2 production and IL2 receptor expression in PBL and in
Taken together, these data suggest that PKC may importwo human T cell lines.
of IL2 by mitogen-stimulated human
Our results show that proliferation of PBL stimulated by tantly regulate secretion
PHA was inhibited by staurosporinein a dose-dependent T cells both indirectly anddirectly, acting at the level of gene
fashion. This was associated with impaired function of PKC expression and on the release of the protein from the cell.
from these cells, as measured in vitro and in intact lympho- Additionally, PKC may also control the secretion of soluble
cytes. Staurosporine did not inhibit tyrosine kinase function IL2 receptor a molecules by these cells. Furthermore, this
in these cells; thus, itseffect on proliferation of PBL may be effect may be limited tomolecules that utilize secretory pathmediated a t least in part by inhibiting PKC and not through ways, since the buddingof virus from MLA-144 cells, and the
a nonspecific inhibition of protein kinases. This is consistent expression of PKC itself and of cell surface proteins including
with previous observations which suggested PKC was impor- CD2, CD3, and membrane-bound IL2 receptors in human T
tant in the
proliferative response of PBL tomitogens (Manger cells, were either not affectedor decreased only minimally by
staurosporine.
et al., 1987; Ne1 et al., 1987).
These conclusions are consistent with resultsfrom experiOur data also indicate that thisdecrease in the responseof
PBL to PHA could reflect primarily an effect on IL2, since mental systems using platelets (Kaibuchi et al., 1983; Kajistaurosporine inhibited profoundly the elaborationof IL2 by kawa et al., 1983; Higashihara et al., 1985), neutrophils (KaPHA-stimulated PBL and by both cell lines stimulated by jikawa et al., 1983; Pontremoli et al., 1986), or cells of the
PHA plus TPA. The observed inhibition of IL2 production nervous system (Zurgil and Zisapel, 1985; reviewed in Kikby staurosporine was partially explained by a reduction of kawa et al., 1986), in which PKC agonists induce the release
-50% in IL2 gene expression in PBL or 532 cells. These data of serotonin, serine proteinase, or neurotransmitters, respecbe
by inhibarecompatiblewiththeobservations
of Yamamoto et al. tively, and these secretory responses can blocked
(1986), where the disruption of pathways related to the acti- itors of PKC. Thus, PKC may have a widespread role in the
regulation of secretory pathways in a variety of cell types
vation of PKC, but not to PKA, PKG, or prostaglandins,
(reviewed inNishizuka, 1986).At this point, however, we
inhibited IL2 gene expression. Furthermore, in our experiments, the observed decrease in IL2 production appeared to cannot rule out that chronic exposure to staurosporine may
be directly related to the inhibition of PKC; IL2 production also affect synthesis of IL2 or the post-transcriptional modiwas reduced both by staurosporine and by H7, a chemically fication(s) of the soluble IL2 receptor, directly or indirectly.
It should be noted that some studies
have suggestedanother
distinct inhibitor of PKC, but not by Ha1004, a preferential
inhibitor of cyclic nucleotide-dependent protein kinases.
role for PKC in human lymphocytes thatmay not be related
An effect on IL2 receptor expression or functioncould also to the earliest steps signaling;
in
PBL stimulatedby anti-CD3
have accountedfor the inhibitionof the proliferative response antibodies, exhibited in addition to the immediate, transient
of PBL to PHA. But in contrast to the
decrease inIL2
translocation of PKC, a second prolonged phase of up to 4 h
production, the expression of IL2 receptor a mRNA and of in which the PKC enzyme(s)
were found to associate primarily
both this protein and high affinity IL2 receptors on the cell with the particulate fraction from these cells (Szamel et al.,
surface were either not affected or decreased only slightly by 1989). The time course through which staurosporine diminstaurosporine. The signaling function of these receptors did ished IL2 secretion in our experiments
could be consistent
not appear to
be significantly affectedby staurosporine either, with an inhibitoryeffect on this second phase of PKC funcsincerecombinantIL2
largelyreversed thestaurosporinetion. Additionally, recombinant IL2 has been shown to be a
inducedinhibition of proliferation of PBLstimulated by natural substrate for PKC in bacterial and in mammalian
PHA.
cells (Kung et al., 1989). The physiological importance of this
Perhaps most interestingwas the fact thatchemical inhib- phosphorylation of IL2 at serine 6 is unclear; although it did
10560
PKC Regulate s IL2 Secretion
not affect the biological activity of the protein, its ability to
be exported to theextracellular space was not investigated.
It is interesting to speculate whether the two independent
regulatory steps (gene expression and secretion) involved in
IL2 production are mediated by different isoforms of PKC.
Human lymphocytes contain the a and B isoforms of PKC in
approximately equimolar concentrations (Beyers et al., 1988;
Berry et al., 1989).3Studies analyzing the intracellular distribution of different PKC isoforms in thecentral nervous
system (Brandt et al., 1987; Huang et al., 1987; Mochyl-Rosen
et al., 1987) suggest there maybe a degreeof functional
heterogeneity associated with them, such that one (possibly
p) maybe important in signal transductionandanother
(possibly a?) may be important for neurotransmitter release.
Results from our laboratory and others (Berry et al., 1989)
have not yet demonstrated any major differences in the relative quantityor subcellular localization of either isoform with
respect to the other in stimulated PBL. However, the functional importance of each isoform at different stages of T cell
activation remains to be determined.
In general, the present data confirm and extend previous
studies in human lymphocytes which indicated that PKC is
important in the regulation of IL2 production. These reports
showed a high positive correlation among the subcellular
localization of the enzyme (“translocation”; Isakov and Altman, 1987; Ne1 et al., 1987), the relative rates of PKC substrate phosphorylation in situ (Cantrell et al., 1985; Shackelford and Trowbridge, 1986; Ne1 et al., 1987; Friedrich and
Gullberg, 1988))and the level of IL2 produced by stimulated
human T cells (Yamamoto et al., 1986; Friedrich and Gullberg,
1988; Subramaniam et al., 1988; Szamel et al., 1989). Limited
studies in murine cell lines have been less consistent. In one
study, pharmacologic depletion of PKC from a cell line abrogated the elaboration of IL2 (andthe expression of IL2
mRNA) in response to stimulation by lectin (concanavalin A)
or by the combination of calcium ionophore plus phorbol ester
and also decreased significantly the expression of the IL2
receptor gene (Valge et al., 1988). In a study of a different
murine cell line, however, inhibiting PKC by staurosporine
or depleting it by chronic treatment with TPA did not affect
IL2 production stimulated by lectin (PHA) or anti-CD3 antibodies in combination with IL1 (Mills et al., 1989). It remains to be determined whether these variable findings reflect
inherent differences among the murine cell lines used for
these experiments or whether different modes of stimulation
may preferentially utilize different signalling pathways.
Acknowledgments-We wish to thank G. Gaulton, J. Monroe, M.
Greene, and C. Deutsch for helpful discussions; T. Waldmann, M.
Nakada, and M. Kamoun for providing reagents and cells; E. Stolzenberg and C. Cunningham for technical assistance, and L. Delpino
for editorial assistance.
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