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NK-active cytokines IL-2 IL-12 and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells.

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THE ANATOMICAL RECORD 266:87–92 (2002)
DOI 10.1002/ar.10039
NK-Active Cytokines IL-2, IL-12, and
IL-15 Selectively Modulate Specific
Protein Kinase C (PKC) Isoforms in
Primary Human NK Cells
Institute of Human Anatomy, University of Parma, Parma, Italy
Institute of Cytomorphology NP CNR, c/o Research Institute “Codivilla-Putti”,
Bologna, Italy
Institute of Histology, University of Bologna, Bologna, Italy
Department of Morphology and Embryology, University of Ferrara, Ferrara, Italy
Institute of Human Anatomy, University of Bologna, Bologna, Italy
Institute of Morphological Sciences, University of Urbino, Urbino, Italy
Natural killer (NK) cell function is largely modulated by growth factors
and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major
effects on the proliferative and cytotoxic activities of NK cells against tumor
and virus-infected cells. It is thought that the members of the protein kinase
C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have
investigated the downstream effects generated in purified human NK cells
by IL-2, IL-12, and IL-15 on PKC␣ and PKC⑀—a canonical and a novel
isoform of PKC, respectively. By means of Western blotting, PKC activity
assays, and immunofluorescence performed on highly purified preparations
of primary human NK cells, we demonstrate that: 1) the three cytokines
have similar effects on PKC␣ and PKC⑀ activities; 2) whereas PKC⑀ activity
is induced by cytokine stimulation, PKC␣ activity is inhibited; and 3) both
the induction of PKC⑀ and the inhibition of PKC␣ functional activity are
relatively early events in NK cells, while longer cytokine stimulations do
not generate significant variations in enzyme activity, suggesting that the
activation of both the canonical and novel isoforms of PKC are events
required in the early phases of cytokine-induced NK cell stimulation. Anat
Rec 266:87–92, 2002. © 2002 Wiley-Liss, Inc.
Key words: natural killer cells; PKC; IL-2; IL-12; IL-15
Interleukin (IL)-2, IL-12, and IL-15 are cytokines that
are active on natural killer (NK) cells. Although they are
structurally different, IL-2 and IL-15 share the ␤ and ␥
chain of their trimeric receptors (Rebollo et al., 1996),
while the IL-12 receptor is a dimer structurally distinct
from the other two (Gately et al., 1998). In general, the
three cytokines activate NK cells. However, while IL-2
and IL-15 induce proliferation and enhance the cytotoxicity of NK cells, IL-12 alone enhances cytotoxicity but has
a modest proliferative effect on NK cells (Gately et al.,
1998). The binding of each of these cytokines to their
respective receptors activates a number of intracellular
signaling pathways (Gately et al., 1998; Karnitz and Abra©
ham, 1996; Waldmann and Tagaya, 1999). NK cells constitutively express the ␤ and ␥ chains of the IL-2 receptor
Grant sponsor: AIRC; Grant sponsor: MURST Cofin ’99; Grant
sponsor: University of Parma.
*Correspondence to: Marco Vitale, M.D., Institute of Human
Anatomy, University of Parma, Ospedale Maggiore, via Gramsci
14, 43100 Parma, Italy. Fax: ⫹39-0521-988340.
Received 8 March 2001; Accepted 1 August 2001
(IL-2R). The ␣ chain of the receptor is upregulated upon
binding of IL-2 to the IL-2R␤␥ to form the ␣␤␥ heterotrimer that represents the high affinity receptor for IL-2
(Gately et al., 1998). In vivo IL-2 is exclusively produced
by activated T cells.
Similar to IL-2, IL-15 is able to activate NK cells (see for
review Fehniger and Caligiuri, 2001). The receptors for
IL-2 and IL-15 share their ␤ and ␥ chains and activate
similar downstream signal transduction pathways. It has
been demonstrated in T cells that the PKC ␤ and ␨ isoforms are involved in the IL-2/IL-15 ␤␥ intermediate affinity receptor signaling, while PKC ⑀ and ␨ are involved in
the signaling downstream of the high affinity receptor for
IL-2 (Gomez et al., 1995). IL-15, in contrast to IL-2, is
produced by a variety of cells in different anatomical locations, such as bone marrow stromal cells, gut epithelial
cells, macrophages, and dendritic cells— but not activated
T cells. Moreover, the IL-15R␣ chain has a much broader
tissue distribution than the IL-2R␣ chain, being expressed
also by nonhematopoietic tissues such as heart, skeletal
muscle, liver, and endothelial cells. Indeed, it has recently
been reported that IL-15 acts as a survival factor for NK
cells, and is able to drive their differentiation in vitro in
the absence of IL-2, suggesting that IL-2 is not essential
for NK cell differentiation (Fehniger and Caligiuri, 2001).
IL-12, originally identified as an NK cell stimulating
factor (Kobayashi et al., 1989), is a dimeric cytokine secreted by monocytes and dendritic cells. Together with
other accessory signals, such as CD40/CD40L interaction,
it directs the development of IFN-␥-producing Th1 cells.
IL-12-driven Th1 differentiation requires the IL-12-responsive transcription factor signal transducer and activator of transcription 4 (STAT4) (Thierfelder et al., 1996).
The high affinity receptor for IL-12 (IL-12R) is formed by
two subunits, the ␤1 and ␤2 chains (Gately et al., 1998).
NK cells constitutively express the ␤1 chain of the IL-12R.
While T cells do not express the IL-12R␤2 chain, it is not
yet clear whether NK cells express the ␤2 chain or not.
An important role in mediating at least some of the
pleiotropic effects of cytokines on their target cells is
thought to be played by members of the protein kinase C
(PKC) family of serine/threonine kinases (Hug and Sarre,
1993). At least 12 different isoforms of PKC have been
characterized so far (Kiley and Jacken, 1994) and grouped
into three categories based on the Ca2⫹ requirement for
activation and phorbol ester binding activity. Conventional PKCs (␣, ␤⌱, ␤⌱⌱, and ␥) are Ca2⫹-dependent phorbol
ester receptor kinases; novel PKCs (␦, ⑀, ␪, and ␩) are
Ca2⫹-independent phorbol ester receptor kinases; and
atypical PKCs (␨, ␫, ␭, and ␮) are kinases that are independent of both Ca2⫹ and phorbol esters. PKC has been
involved in the regulation of NK cell cytotoxicity (Ting et
al., 1992; Bonnema et al., 1994) and in the regulation of
their activated phenotype. Altogether, data have shown
that PKC is involved in the regulation of natural killing,
but not antibody-dependent cellular cytotoxicity (ADCC),
and that PI3 kinase, which lies upstream of PKC ␨ activation, plays a role in CD16-initiated granule exocytosis
and killing, but not in natural cytotoxicity. More recently,
the role of some PKC isoforms has been studied in greater
detail, demonstrating that in NK cells the PKC isoforms ␣,
␥, ⑀, and ␩ are required for perforin-induced, but not for
Fas-dependent cytotoxicity (Ohmi et al., 1997). In different model systems, the ␣ isoform of PKC has been demonstrated to rescue from apoptosis the IL-3-dependent
myeloid progenitor cell line 32D after IL-3 withdrawal (Li
et al., 1999), while the ⑀ isoform of PKC has been demonstrated to lie downstream of the VEGF receptor in
HUVEC endothelial cells (Wu et al., 2000) and to be downmodulated during early erythroid differentiation (Bassini
et al., 1999).
Given that IL-2, IL-12, and IL-15 all generate activation
of NK cells by inducing, to a different extent, the PI3
kinase and the phospholipase C (PLC) signaling pathways, we studied in detail the effects of these three cytokines on the presence and activity of a canonical (PKC ␣)
and novel (PKC ⑀) isoform in primary human NK cells.
NK Cell Purification and Cytokine Treatment
NK cells were isolated from peripheral blood lymphocytes (PBL) of normal voluntary donors by immunomagnetic negative selection using the Vario-MACS and the
NK cell isolation Kit (Milteny Biotech, Gladbach, Germany), according to the manufacturer’s protocol. Briefly, PBL
were separated from buffy coats by Ficoll-Hypaque gradient centrifugation. After plastic adherence, PBL were
magnetically labeled with a cocktail of hapten-conjugated
mAbs to CD3, CD4, CD19, and CD33 of mouse IgG1 isotype, followed by anti-hapten antibody-coupled MACS microbeads. The magnetically labeled cells were then depleted on a depletion column in the magnetic field of a
Vario-MACS. The purity of the NK cells was immediately
checked by anti-CD16-PE labeling and FACS analysis of a
small aliquot of the obtained sample. Only samples with a
purity exceeding 95% were used.
Purified NK cells (1 ⫻ 106 cells/ml) were stimulated
with either 100 U/ml of IL-2, or 1 ng/ml IL-12 or 10 ng/ml
IL-15 for 120 min or overnight, as indicated. Control samples were incubated for the same time periods in the
absence of the respective cytokine.
Western Blot Analysis
To obtain whole cell homogenates, cells were washed
twice with cold (4°C) PBS, resuspended in 0.25 M sucrose,
10 mM Tris HCl pH 7.4, 5 mM MgCl2, 10 mM NaCl, and
0.5 mM PMSF, plus proteases and phosphatases inhibitors, and sonicated twice for 30 sec in a Dynatech sonic
dismembrator (PBI, Milan, Italy).
Rabbit peptide-specific antibodies (IgG fraction) that
recognize PKC ␣ and PKC ⑀ were obtained from Santa
Cruz Biotechnologies (Santa Cruz, CA). Equal amounts of
protein, determined by Bradford assay (Bio-Rad Labs,
Munchen, Germany) were diluted 1:3 with 4⫻ loading
buffer (250 mmol/L Tris HCl, pH 6.8, 20% ␤-mercaptoethanol, 40% glycerol, 8% Na deoxycholate, and 0.003%
bromophenol blue) and subjected to sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) on
7.5% gel. Gels were transferred to nitrocellulose membrane by Trans-blot (Bio-Rad). SDS-PAGE and transfer
were performed according to the manufacturer’s protocol.
Equal loading of each fraction was verified by staining
duplicate gels with Coomassie Brillant Blue R-250 (Sigma, St. Louis, MO). The membranes were incubated in
PBS, supplemented with 3% (wt/vol) BSA (Sigma) for 30
min at room temperature, and then incubated overnight
at 4°C with anti-PKC␣ (dilution 1:500) and anti-PKC⑀
(dilution 1:250) specific antibodies. After four washes with
PBS/0.1% Tween 20/0.1% BSA, peroxidase-conjugated
goat anti-rabbit IgG antibody (1:1,500; Cappel, Durham,
NC) was applied to the membrane for 60 min at room
temperature. The bound antibody was visualized by the
ECL Western blotting detection reagents and HyperfilmECL luminescence detection film (Amersham Corp, Arlington Heights, IL). Semiquantitative densitometric
analysis was performed with an imaging densitometer
(model GS 670; Bio-Rad) by the Molecular Analist software (Bio-Rad). The following criteria were used to confirm that the immunostaining was specific for the monitored enzymes. No immunostaining was obtained by
antibodies adsorbed with 1 mg of the corresponding immunizing peptide. When the blots were stripped, reblocked, and reprobed with antibodies which had been
previously incubated 1:1 for 10 min with the corresponding immunizing peptide (1 mg/ml), the specific peptide
blocked immunoreactivity.
PKC Catalytic Activity Assay
The in vitro PKC kinase assay was performed according
to a previously described procedure (Marchisio et al.,
1999). Primary NK cells (between 1 and 1.5 ⫻ 106 cells per
experimental point) were treated with IL-2, IL-12, or
IL-15 for the indicated times at 37°C; washed twice with
ice-cold buffer A (137 mM NaCl, 20 mM Tris, 1 mM
MgCl2, 1 mM Vanadate, pH 7.5); and lysed in lysis buffer
(buffer A plus 10% glycerol, 1% NP-40, and 1 mM PMSF).
The content of protein in the lysate was determined by the
Bio-Rad protein assay system. The lysates were incubated
for 2 hr at 4°C with a 1:200 dilution of rabbit polyclonal
anti-PKC-␣ or anti-PKC-⑀ antiserum (Santa Cruz Biotechnologies, Santa Cruz, CA). Protein A-sepharose was then
added to the lysates for 1 hr at 4°C. After precipitation,
the lysates were washed twice in lysis buffer, twice in
buffer containing 0.5M LiCl and 0.2% NP-40, and, finally,
in 10 mM phenyl phosphate.
The five immunoprecipitates were then aliquoted: four
were subjected to a catalytic assay, and one was subjected
to Western blot analysis. For the catalytic assay, immunoprecipitates were incubated in a 50 ␮l reaction mixture,
containing 20 mM Tris-HCl pH 7.4, 5 mM MgCl2, 3 mM
DTT, 100 ␮M vanadate, 13 ␮M ATP, 1 ␮Ci of (32P)-␥-ATP
(3000 Ci/10-3M), and 10 ␮g of a serine-substituted peptide
(RFARKGSLRQKNVHEVKN, corresponding to aminoacids 19 –36 of PKC; U.B.I., Lake Placid, NY).
The reactions were stopped with appropriate volumes of
4⫻ loading buffer (0.25 M Tris-HCl pH 6.8, 8% SDS, 40%
glycerol, 20% ␤-mercaptoethanol, and 0.005% bromophenol blue) and boiled for 3 min. After 18% SDS-PAGE, the
gels were stained with Coomassie R-250, destained, dried,
and autoradiographed with Kodak X-OMATS S films. The
radioactive spots were excised, and radioactivity was
counted in a liquid scintillation counter.
PKC Isoform-Specific Indirect
Rabbit peptide-specific antibodies (IgG fraction) that
recognize PKC ␣ and PKC ⑀ were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). Purified NK cells
were seeded on slides, fixed in 4% paraformaldehyde/PBS
for 10 min at room temperature, washed twice in PBS,
incubated with PBS for an additional 10 min to quench the
remaining paraformaldehyde, and saturated/permeabilized using an NET gel solution (150 mM NaCl, 5 mM
Fig. 1. Activation of purified primary human NK cells induced by
overnight incubation in the presence of IL-2, IL-12, or IL-15. a: 3Hthymidine incorporation in NK cells stimulated without/with 100 U/ml
IL-2, 1 ng/ml IL-12, or 10 ng/ml IL-15. A representative of three independent experiments performed in triplicate (S.D. ⬍ 5%). b: Cytotoxicity
(51Cr-release assay) against K562 target cells (T). Purified NK cells
stimulated as above were used as effectors (E). A representative of three
independent experiments performed in triplicate is shown (S.D. ⬍ 5%).
EDTA, 50 mM Tris-HCl pH 7.4; 0.05% NP-40; 0.25% carrageenan lambda gelatin; and 0.02% Na azide) for 30 min
at room temperature. After two washes with the NET gel
solution cells were treated with anti-PKC IgG (diluted
1:50) for 60 min at room temperature. After two washes
with the NET gel solution, goat anti-rabbit IgG (GAR-Fitc,
diluted 1:150) was added to the cells and incubated for 45
min at room temperature. After three additional washes
(two with NET gel solution, one with PBS) slides were
mounted in DABCO (Sigma)-glycerol-PBS. The following
criteria were used to confirm the specificity of the immunostaining. No staining was obtained using as primary
antibodies: 1) anti-PKC ␣ and ⑀ (diluted 1:50) antibodies
preadsorbed for 30 min at room temperature with 1 ␮g of
Fig. 2. Western blot analysis of PKC␣ and PKC⑀ isoforms in NK cells.
Untreated: purified NK cells without cytokines (control). IL-2: purified NK
cells stimulated with 100 U/ml of rIL-2 for 2 hr. IL-12: purified NK cells
stimulated with 1 ng/ml of rIL-12 for 2 hr. IL-15: purified NK cells
stimulated with 10 ng/ml of rIL-15 for 2 hr. PKC␨ does not change with
2-hr cytokine stimulation and is used as internal control. A representative
of three independent experiments is shown.
the corresponding immunizing peptide (Santa Cruz, CA);
or 2) normal rabbit serum (diluted 1:50).
Statistical Analysis
Results are expressed as means ⫾ S.D. of three or more
experiments performed in duplicate. Statistical analysis
was performed using the two-tailed Student’s t-test.
Cytokine Effect on NK Cell Activity
Overnight incubation in the presence of IL-2 or IL-15
induced the proliferation of purified NK cells. The proliferative effect of IL-12 was lower than that of IL-2 and
IL-15, while it stimulated the NK cytotoxicity at the same
level of IL-2 and IL-15 (Fig. 1a and b). After an incubation
period as short as 2 hr, none of the three cytokines produced detectable levels of proliferation or cytotoxicity enhancement in NK cells.
Modulation of PKC␣ and ⑀ Expression and
Activity in NK Cells by Cytokine Stimulation
The expression of PKC␣ and ⑀ was investigated in primary human NK cells stimulated with IL-2, IL-12, or
IL-15 for 2 hr or overnight, by Western blotting. After 2 hr
of stimulation, both PKC isoforms were relatively increased by the three cytokines (Fig. 2). On the contrary,
overnight incubation with the cytokines did not produce
any relevant variation of PKC␣ or ⑀ expression (not
shown). This kinetic suggests that PKC activation is required early in the response of NK cells to cytokines, while
Fig. 3. Assay for (A) PKC␣ and (B) PKC⑀ catalytic activity in homogenates obtained from purified NK cells stimulated for 30 min (solid bars),
2 hr (striped bars), or 6 hr (dotted bars) with IL-2, IL-12, or IL-15. Data are
reported as percentage of control from three separate experiments ⫾
it is lowered back to baseline when the proliferative and
cytotoxic effects start to be visible at the cellular level
(overnight). However, since the variation of the presence
of an enzyme does not necessarily imply its activation, we
further analyzed the PKC activity on specific PKC␣ and
PKC⑀ immunoprecipitates obtained from the cytokinestimulated purified NK cells and examined it by adding a
specific peptide substrate, Ca⫹⫹, and lipid cofactors to the
cell homogenates. Interestingly, while PKC⑀ activity was
significantly increased (P ⬍ 0.05) after 2 hr of stimulation
with the cytokines, the PKC␣ activity was significantly
inhibited (P ⬍ 0.05) at this time interval. Shorter (30 min)
or longer (6 hr) incubations in the presence of the cytokines did not reveal significant variations of enzyme activity (Fig. 3A and B).
It has been demonstrated in different hematopoietic
models that translocation of PKC isoforms to specific cellular domains is required for their biological activation. In
Fig. 4. Immunofluorescent photomicrographs of freshly purified human peripheral blood NK cells treated
with IL-2, IL-12, or IL-15, examined for PKC␣ and PKC⑀ expression. After 2 hr of incubation with or without
(control) each cytokine, cells were collected and cytospin slides were made by cytocentrifugation.
particular, PKC␣ has been demonstrated to migrate to the
cell nucleus upon activation by different growth factors
(Neri et al., 1998), while PKC⑀ associates with the membrane fraction upon activation (Johnson et al., 1996).
Therefore, in another group of experiments we studied the
localization of PKC␣ and PKC⑀ in purified NK cells stimulated with IL-2, IL-12, or IL-15. Our results show that
PKC␣ and PKC⑀ are resident in the cytoplasmic compartment of resting NK cells, and that upon stimulation with
cytokines, their overall fluorescence increases, in agreement with the data obtained by Western blot. However,
PKC␣ does not appear to localize to the nucleus of stimulated NK cells, while the fluorescence of PKC⑀ dramatically increases in the cytoplasm (Fig. 4), apparently associating with the membrane compartment which is
compatible with the active form of the enzyme.
PKC enzymes are conserved among species, participate
in signal transduction in many cells, and mediate a wide
number of intracellular functions. Most PKC isoforms exist in an inactive form in the cytosol of resting cells, with
an amino-terminal pseudosubstrates sequence occupying
the active site. The generation of diacylglycerol (DAG) as
a result of membrane phospholipid breakdown induced by
cytokine receptor triggering causes redistribution of conventional and novel PKC isoforms to the membrane by
binding the regulatory domain of the kinase. PKC activity,
in fact, is controlled by its subcellular compartmentalization, bringing the enzyme close to substrates and regulators, and by phosphorylation of the enzyme. It has recently been demonstrated that the phosphorylation of
canonical and novel PKC isoforms requires either PDK-1,
which is upstream of PI3 kinase, or the induction of the
PLC signaling pathway, which generates the DAG required for PKC activation (Toker and Newton, 2000; Capitani et al., 2000).
The involvement of PKC enzymes in the functional activity of NK cells has been established in the past few
years. General PKC activity or the involvement of some
specific PKC isoforms have been shown to be necessary for
the execution of natural cytotoxicity, while ADCC and
Fas-mediated toxicity appear to be PKC-independent processes (Ting et al., 1992; Ohmi et al., 1997). On the contrary, little is known about the differential involvement of
PKC isoforms in the response to the main NK-active cytokines, IL-2, IL-12, and IL-15. This relative lack of information may be due to the paucity of NK-like cell lines
compared to the considerable number of cells needed to
perform the immunoprecipitation experiments. The
highly efficient negative immunomagnetic sorting system
allowed us to purify NK cells without touching any surface
activatory molecule, such as CD16, and to design the
enzyme catalytic assays on two PKCs (␣ and ⑀)—a classical Ca⫹⫹- and PMA-dependent, and a novel Ca⫹⫹-independent/PMA-dependent isoform, respectively. The intracellular signaling generated by the three cytokines had
effects on both PKC␣ and ⑀, with a peak at 2 hr. Shorter
(30 min) or longer (6 hr or overnight) stimulation, in fact,
did not generate significant variations of PKC activity.
After 2 hr of incubation with cytokines, PKC␣ activity was
decreased, while the PKC⑀ activity was increased. Following cell activation by growth factors and cytokines in different model systems, different PKC isoforms reach their
substrates localizing in specific cellular domains. Our
data, obtained by Western blot and catalytic assay performed on PKC⑀ immunoprecipitates, were paralleled by
immunofluorescence that showed a strong increase and a
specific cytoplasmic and membrane localization of the fluorescence upon stimulation of NK cells with the cytokines,
which is compatible with the activation of the enzyme. On
the contrary, the apparent increase in the presence of
PKC␣ observed by Western blot, which was not paralleled
by an increase in the enzyme activity, generated only a
relative increase in its immunofluorescence, with an evident lack of specific nuclear localization.
Altogether, our data show that IL-2, IL-12, and IL-15
activate the ⑀ isoform of PKC with a relatively rapid
kinetic, while inhibiting the activity of PKC␣. Further
experiments of pharmacological inhibition of PKC⑀ and
PKC␣ overexpression in NK cell lines, and possibly in
primary NK cells, will clarify whether both a PKC␣ functional inhibition and a PKC⑀ activation or just the latter
are essential for NK cell activation after cytokine stimulation.
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