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Department of Microbiology and Immunology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIIA-induced granule exocytosis and both spontaneous and
Ab-dependent cytotoxicity were reduced in a dose-dependent manner in
cells pretreated with either of two specific inhibitors of this kinase.
Target cell-induced IFN- and FcRIIIA-induced TNF-
mRNA
accumulation was similarly affected under the same conditions. Lack of
inhibition of NK cell cytotoxicity in cells overexpressing an inactive
form of JNK1 indicates that this kinase, activated only upon FcRIIIA
ligation, does not play a significant role in cytotoxicity. These data
underscore the involvement of p38, but not JNK1, in the molecular
mechanisms regulating NK cell cytotoxicity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIIA) is responsible for triggering
Ab-dependent cell-mediated cytotoxicity
(ADCC)3 (reviewed in
Ref. 3). Early biochemical events induced upon
cross-linking the receptors involved in binding IgG Ab-coated or
NK-sensitive target cells include protein tyrosine kinase (PTK)
activation and increased intracellular Ca2+
concentration as a consequence of phospholipase C-1 and -2
activation (4, 5, 6, 7). Intermediate molecules in the elicited
signaling cascades have been reported to modulate NK cell cytotoxicity.
The PTK Syk (8), the adapter protein LAT (linker for
activation of T cells) (9), and the Vav-Rac1 pathway
(10, 11) play roles in both types of cytotoxicity, which
are abolished upon PTK inhibition or activation of protein tyrosine
phosphatases (4, 5, 12, 13). Biochemical pathways
induced preferentially during ADCC or spontaneous cytotoxicity have
also been identified. For example, granule exocytosis-mediated
spontaneous cytotoxicity against the prototypic K562 target cells
depends on activation of protein kinase C, but not phosphatidylinositol
3-kinase, whereas the reverse is true for ADCC (14). Mitogen-activated protein kinases (MAPK) transduce signals that regulate cell growth and differentiation (15). They are serine-threonine kinases, the enzymatic activity of which is elicited upon phosphorylation of threonine and tyrosine residues in a Thr-X-Tyr motif in their regulatory domain (16). This is mediated by dual specificity MAPK kinases, which also become activated, following phosphorylation, under the same conditions. The MAPK family includes the extracellular signal-regulated kinases (ERK), the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase, and the p38 MAPK (p38). For most part, ERK are activated by mitogenic factors, while JNK and p38 are activated by stress-inducing agents or proinflammatory cytokines. Although in most cell types each MAPK is activated by specific nonoverlapping kinases, named MEK, activation of both p38 and JNK by a single MEK4 kinase has also been reported (17). In different cell types the different MAPKs may act antagonistically or cooperate with each other to regulate different cell functions (15). Examples for this are the opposite effects of ERK (facilitating) and JNK (protecting) to control B cell receptor-induced apoptosis (18) and the requirement for both kinases in TCR- and CD28-dependent T cell activation and IL-2 production (19).
We and others have shown that ERK-2 activation occurs in NK cells upon
target cell binding or FcRIIIA stimulation, and that cytokine mRNA
accumulation, spontaneous cytotoxicity, ADCC, and FcR-induced
degranulation depend at least in part on ERK-2 function
(20, 21, 22, 23). The possible role of the other MAPK family
members is unknown. The observation that p38 plays a role in actin
reorganization leading to formation of filamentous actin in endothelial
cells upon platelet-derived growth factor (PDGF) (24) and
vascular endothelial growth factor (25) stimulation
suggests the possibility that the same kinase may be activated to play
a similar role in NK cells upon target cell binding and thus may be
involved in regulating granule exocytosis-mediated cytotoxicity.
Increased JunB mRNA and emergence of JunB:Fos heterodimers with
increased AP-1-activity during spontaneous cytotoxicity have been
reported in the human NKL cell line (26). Because ERK, but
not JNK, is involved in the regulation of JunB transcription (27, 28), a possible differential role for distinct MAPK in
cytotoxicity may be envisaged.
To define the role of non-ERK-2 MAPK in the regulation of NK cell
cytotoxicity, we investigated the involvement of p38 and JNK kinases.
Our data indicate that both FcRIIIA triggering and NK cell
recognition of nonsensitized target cells generate signals leading to
activation of p38, whereas only FcRIIIA stimulation activates JNK1.
Inhibition experiments indicate that only p38 plays a role in ADCC and
spontaneous cytotoxicity and exclude a role for JNK1 in ADCC,
demonstrating a differential role for p38 and JNK1 in regulating NK
cell cytotoxic functions. Similar to ERK, p38 is also involved in the
regulation of target cell-induced cytokine expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human monocytic THP-1, erythroleukemic K562, T lymphoid Jurkat (clone J32), B lymphoblastoid RPMI-8866 and 721.221 cell lines were maintained in culture in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated FBS (Sigma, St. Louis, MO) and 100 µg/ml L-glutamine (Life Technologies, Gaithersburg, MD).
Homogeneous NK cell preparations were obtained from 10-day cocultures of PBL from healthy individuals with 30-Gy irradiated RPMI-8866 cells following negative selection using a mixture of anti-CD14, -CD3, and -CD5 mAb and indirect anti-Ig rosetting as previously described (29). The cell preparations contained >98% CD16+/CD56+/CD3- and <3% CD3+ cells, as determined by indirect immunofluorescence (flow cytometry) using a panel of mAb.
Monoclonal and polyclonal Abs
mAb 3G8 (anti-CD16), B159.5 (anti-CD56), B36.1 (anti-CD5), OKT3 (anti-CD3), and B52.1 (anti-CD14) have been previously described (29); the anti-FLAG M2 mAb was obtained from Sigma. The polyclonal rabbit sera anti-MAPK p38 and JNK1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); the polyclonal rabbit serum detecting enzymatically active, Thr183- and Tyr185-phosphorylated JNK1 and JNK2 was obtained from Promega (Madison, WI); that anti-Thr180- and Tyr182-phosphorylated active p38 was purchased from New England Biolabs (Beverly, MA); the anti-phospho-c-Jun mAb was obtained from Santa Cruz Biotechnology; the anti-HSP90 Ab was purchased from Transduction Laboratories (Lexington, KY).
Cell stimulation
Cells were incubated (5 x 106/ml; 37°C) for the indicated times with the different stimuli. These were: K562 and 721.221 cells (5:1, NK to target cell ratio), PMA (50 ng/ml) and ionomycin (1 µM; both from Sigma), immune complexes (rabbit IgG-sensitized bovine erythrocytes (EA)), or E (negative control; 0.5% suspension) prepared as previously described (30), and biotin-labeled mAb 3G8 or B159.5 (both 20 µg/ml) with added streptavidin (50 µg/ml; Sigma). In the samples used for Western blotting, K562 and 721.221 cells were fixed (3 x 106 cells/ml 1% paraformaldehyde, 30 min on ice) and washed extensively before use. This treatment prevents possible activation of endogenous kinases and has no effect on target cell binding to NK cells or subsequent stimulation of early biochemical events and ERK activation in NK cells (23, 21) (data not shown). When target cells were used, effector/target cell contact was facilitated by centrifugation (600 rpm, 2 min) before incubation. When indicated, the p38 inhibitors SB203580 and SB202190 (Calbiochem, San Diego, CA) were added to the effector cells at the indicated concentrations for 1 h at 37°C before stimulation.
Western blotting and kinase assays
After stimulation the cells were lysed
(108 cells/ml lysis buffer: 1% Nonidet P-40, 10
mM HEPES (pH 7.5), 0.15 M NaCl, 10% glycerol, 10 µg/ml each
aprotinin and leupeptin, 1 mM PMSF, 1 mM
Na3VO4, 50 mM NaF, and 1 mM
EDTA). Western blotting was performed according to our published
protocols (20), and Ab-reactive proteins were detected
with HRP-labeled sheep anti-rabbit Ig sera and enhanced
chemiluminescence (Amersham, Arlington Heights, IL). Two methods were
used to assess kinase activity: 1) expression (detected by Western
blotting) of the enzymatically active phosphorylated forms of p38 and
JNK, and 2) JNK1 and p38 kinase assays, performed according to the
protocols of Hibi and Rose (31, 32), respectively. For
these each kinase was immunoprecipitated from 5 x
106 NK cell lysate equivalent using the specific
Ab (1 µg) and protein A-Sepharose (Pharmacia, Uppsala, Sweden). After
four washes with lysis buffer and two with 10 mM HEPES (pH 7.5), 25 mM
MgCl2, 50 mM NaCl supplemented with 1 mM
Na3VO4, 50 mM sodium
fluoride, and 1 mM PMSF, the protein A-Sepharose beads were incubated
(30 min, 30°C) with 30 µl of reaction buffer and occasional
tapping. The kinase buffer for the JNK1 assays was 20 mM HEPES (pH
7.5), 2 mM DTT, 20 mM ß-glycerol phosphate, 20 mM
MgCl2, 0.1 mM
Na3VO4, 20 mM ATP, 10 µCi
[-32P]ATP (sp. act., 4000 Ci/mmol; ICN,
Costa Mesa, CA). GST-ATF2 (aa 1–96; Santa Cruz Biotechnology) and
GST-c-Jun (aa 1–223) (31), each 1 µg, were used as
interchangeable substrates for JNK because both polypeptides have
sequences specifically recognized by this kinase (33). The
kinase buffer for the p38 kinase assays was 20 mM HEPES (pH 7.5), 25 mM
ß-glycerol phosphate, 25 mM MgCl2, 2 mM DTT,
0.1 mM Na3VO4, 20 mM ATP,
10 µCi [-32P]ATP. The substrate was
GST-ATF2, as described above. After the reaction the kinases were
eluted from the beads by heating and were analyzed in 10% SDS-PAGE
(reducing conditions). Western blots were performed to verify that
equal amounts of precipitated MAPK were loaded per sample, and in vitro
phosphorylation of the kinase substrates was detected after exposure of
the filters to X-AR films (Eastman Kodak, Rochester, NY). MAPKAP
kinase-2 assays were performed using a commercial kit following the
manufacturer’s recommendations (MAPKAP kinase-2 IP-Kinase Assay Kit,
Upstate Biotechnology, Lake Placid, NY; with a sheep serum reacting
with both rabbit and human MAPKAP kinase-2 for
immunoprecipitation, and the KKLNRTLSVA peptide as a substrate).
Vaccinia virus (Vac) recombinant preparations and NK cell infection
To generate JNK1 recombinant Vac, cDNA fragments encoding the FLAG-tagged wild-type (wt) and dominant negative (APF) Ala183 and Phe185 JNK1 (33) were generated after BamHI and HindIII, or XbaI and HindIII digestion, respectively. The blunt-ended cDNA were inserted into the NheI cloning site of the psC11 vector and introduced into Vac, WR strain, by homologous recombination, as previously described (34). For infection, NK or Jurkat T cells were incubated with the indicated Vac recombinant (10–20 multiplicity of infection, 37°C, 1.5 h, 107 cell/ml, and an additional 4 h in 2 x 106/ml RPMI 1640 medium supplemented with 10% FBS). The cells were used immediately after washing. Expression of the wt or dominant negative JNK1 fusion recombinant proteins was confirmed by Western blot with anti-JNK1 or anti-FLAG Ab, and the effect of the expression of APF JNK1 on c-Jun phosphorylation was determined by Western immunoblotting with anti-phospho c-Jun Ab. Cell lysates for the latter were in 0.5 M NaCl.
Sodium benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT)-esterase release assay
This was performed as described by Visonneau et al. (35) using as stimuli plastic-immobilized mAb 3G8, B159.5 as a negative control, and PMA (50 ng/ml) and ionomycin (1 mM; both from Sigma) as the positive control. Cell-free supernatants were collected after 4-h incubation at 37°C. The percentage of released BLT esterase activity was calculated for each sample according to the formula (S/S + C) x 100, where S is OD in the supernatant, and C is that in the corresponding cell lysate.
Cytotoxicity assays
K562, THP-1, and 721.221 cells, as indicated, were used as the target in 3-h 51Cr release assays (30). For redirected ADCC, mAb 3G8 or B159.5 as the control (both supernatants, 1/4 predetermined optimal concentration) was present throughout the assay with THP-1 cells. These were not lysed in the presence of the control B159.5 mAb (not shown). A constant number of target cells (5 or 10 x 103/well, as indicated) and serial dilutions of effector cells were used in triplicate. Spontaneous release from any target cell used was <10%; lytic units (36) were calculated at 40% cytotoxicity.
F-actin detection
After stimulation, NK cells were labeled with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (Molecular Probes, Eugene, OR) and analyzed by flow cytometry as described by Salmon et al. (37). Briefly, 3.5 x 105 cells/sample were fixed (10 min, 20°C) in 3.7% formaldehyde in PBS (Sigma), permeabilized, and labeled (20 min, 4°C) in PBS containing 0.5% saponin (Sigma), 0.2% FBS, 0.005% Tween-20, 0.01% NaN3, and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin-phallacidin (1 U/sample). Relative F-actin content is expressed as the ratio between the mean fluorescence intensity of NDB staining in stimulated and nonstimulated control NK cells.
Northern blot analysis
After 1-h incubation at 37°C with or without the p38 inhibitor
SB202190 (50 µM), NK cells (5 x 106/ml)
were cultured with the indicated stimuli for 1.5 h, and Northern
blot analysis was performed as previously described (38),
with slight modifications. Briefly, total RNA was extracted using
TRIzol reagent (Life Technologies, Gaithersburg, MD), size fractionated
in 1% agarose-formaldehyde gels, transferred to Hybond-nylon membranes
(Amersham), and hybridized to cDNA probes specific for human IFN-,
TNF-, and TCR ß-chain (detecting a nonfunctional, truncated,
1.0-kb mRNA species in NK cells) for normalization. cDNA probes were
labeled with [-32P]dCTP (spec. act., 3000
Ci/mmol; ICN) by nick translation (Roche, Indianapolis, IN) (20, 21). Hybridization was detected and quantitated using a
PhosphorImager (PhosphorImager SI, Molecular Dynamics, Sunnyvale, CA)
with proprietary software (ImageQuant).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIIA- and target cell-induced p38 and JNK1 kinase activation
in NK cells
To determine whether FcRIIIA ligation induces p38 and JNK1 MAPK
activation, Western blot analysis was performed with anti-active
p38 or anti-active JNK Ab on lysates from NK cells nonstimulated or
stimulated with immune complexes (EA), E, or PMA and ionomycin as a
control (Fig. 1
A, left
panels). Minimal levels of active p38 and JNK1 were detected in NK
cells before stimulation or after 10-min incubation with control E. As
with ERK1 and ERK2 (20, 22) (not shown), the levels of
active p38 and JNK1 significantly increased to plateau by 5–10 min,
started declining within 20 min (not shown), and returned to control
levels by 1-h stimulation with EA (first and
third panels). All samples expressed similar levels of total
p38 and JNK1 (second and fourth panels). Active
p38 and JNK1 were also detected upon stimulation with PMA/ionomycin. As
expected, p38 and JNK1 immunoprecipitated from EA-stimulated NK cells
were able to phosphorylate the exogenous substrates ATF2 and GST-c-Jun,
respectively, in in vitro kinase assays (Fig. 1B). The same
substrates were only minimally phosphorylated by the kinases
immunoprecipitated from E-stimulated cells.
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A, right, first and third
panels). Comparable levels of p38 and JNK1 were detected in all
samples (right, second and fourth
panels). No active JNK1 was detected upon longer (1-h) stimulation
of NK with the target cells (data not shown), and the
immunoprecipitated JNK1 did not phosphorylate recombinant ATF2 used as
the substrate in in vitro kinase assay (Fig. 1C).
Role of p38 in FcRIIIA-induced granule exocytosis and
cytotoxicity
The pyridinyl imidazoles SB203580 and SB202190 specifically
inhibit p38, but not other MAPK activity (39), by
occupying p38 ATP-binding sites (40). Low doses of
SB203580 (1 µM) were sufficient to inhibit by 50% the p38 activity
in lysates of NK cells stimulated with PMA/ionomycin (not shown). The
ability of SB203580 to inhibit the p38-dependent pathway in NK cells
was assessed using as a readout the induced activation of MAPKAP
kinase-2 (K-2), a physiological substrate of p38 (40, 41)
activated in NK cells upon stimulation with immune complexes or target
cells (data not shown). Addition of 13 µM SB203580 to intact cells
inhibited by 50% the PMA/ionomycin-induced MAPKAP K-2 activity (Fig. 2, inset). Further inhibition
was obtained at higher doses. Concentrations of this inhibitor up to 50
µM did not affect activation of ERK and JNK MAPK (data not shown).
Both FcRIIIA- and PMA/ionomycin-induced BLT esterase secretion were
inhibited in a dose-dependent manner in NK cells preincubated with
SB203580 (Fig. 2) or SB202190 (data not shown).
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). The levels of redirected ADCC against
THP-1/3G8 (left panels) and of spontaneous
cytotoxicity against K562 and 721.221 target cells (middle
and right panels) mediated by NK cells pretreated with
SB203580 or SB202190 (top and bottom,
respectively) were significantly lower than those mediated by control
nontreated cells. Based on calculation of lytic units at 40%
cytotoxicity in the two experiments reported, 50 µM SB203580 or
SB202190 inhibited ADCC by 77 and 84%, respectively, spontaneous
cytotoxicity against K562 by 90 and 91%, and spontaneous
cytotoxicity against 721.221 target cells by 64 and 71%. In the same
conditions, cell viability, expression of FcRIIIA, LFA-1, CD18, and
CD11b, and formation of conjugates with any of the target cells used
were not affected (not shown).
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To determine whether FcRIIIA ligation mediates induction of
actin polymerization and, if so, whether p38, alone or with ERK
kinases, regulates it, F-actin content was analyzed in NK cells treated
with inhibitors of p38 (SB203580, or SB202190; Table I) and/or of MEK (PD098059; not shown)
and stimulated for 5 min with anti-FcRIIIA mAb (Table I).
Similar to the changes reported for granulocytes (37) in
similar experimental conditions, the F-actin levels detectable in NK
cells were significantly increased upon PMA/ionomycin or CD16, but not
CD56, stimulation. The levels of CD16-induced actin polymerization did
not change in cells pretreated with any of the inhibitors, alone or in
combination (not shown).
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As detected in Western blotting with an Ab recognizing
specifically Ser63-phosphorylated c-Jun (Fig. 4), expression of the FLAG-tagged,
Vac-encoded, recombinant inactive APF mutant (33), but not
of the wt JNK, correlated with lack of c-Jun phosphorylation induced by
PMA-ionomycin stimulation in J32 cells and by EA in NK cells. This
confirms that JNK1 activity is significantly reduced in the
APF-inactive mutant-expressing cells, indicating that overexpression of
APF inhibits the enzymatic activity of endogenous JNK on its natural
substrate (c-Jun) in intact cells. ADCC and spontaneous cytotoxicity
were tested in NK cells expressing wt JNK1 kinase or its inactive
mutant (APF; Fig. 5). Expression of the
exogenous proteins was confirmed by Western blot using an anti-JNK1
or an anti-FLAG Ab (Fig. 5A, left panels). An
46-kDa band corresponding to endogenous JNK1 was detected in all
lysates analyzed, while a slower migrating band, corresponding to
FLAG-tagged APF or wt JNK1, was detected only in the lysates from cells
infected with the wt or the mutant JNK1 Vac recombinant. Similar levels
of wt and APF JNK1 were expressed in the Vac-infected cells. To confirm
that overexpression of the kinase-inactive form of JNK1 results in
inhibition of endogenous JNK1 activation, the expression of active JNK1
was analyzed by Western blot in NK cells noninfected or infected
with recombinant Vac encoding APF or wt JNK1 and stimulated
with EA. In cell expressing the kinase inactive or the wt form of JNK1
the levels of active JNK1 were, respectively, significantly lower or
higher than those in noninfected cells (Fig. 5A, right
panel). Additionally, CD16-redirected and spontaneous cytotoxicity
were not inhibited in APF-JNK1-expressing NK cells or in NK cells
infected with wt (Fig. 5B) or empty Vac (not shown). Similar
results were obtained using the human NK cell line NKL infected with
the JNK1 recombinant viruses (not shown).
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RIIIA- and target cell-induced cytokine mRNA
accumulation
To determine whether p38 plays a role in regulating cytokine
expression induced by FcRIIIA ligation or target cell binding, we
analyzed the effects of the p38 inhibitor SB202190 on IFN-
(Fig. 6) mRNA accumulation. In
two separate experiments, no significant variations in basal cytokine
mRNA levels were observed in SB202190-pretreated cells, whereas EA- and
K562- induced IFN- mRNA accumulation were inhibited by 86 and 83%
and by 71 and 74%, respectively, compared with those in nontreated
cells. In the same experiments EA-induced TNF- mRNA accumulation was
inhibited by 64 and 79%, whereas the levels of target cell-induced
TNF- mRNA were too low to allow meaningful comparison (not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIIA ligation and target
cell binding, and that, like ERK-2 (21, 23), it plays a
role in cytotoxicity and cytokine expression, whereas JNK, activated
exclusively upon FcRIIIA ligation, does not play a role in either
ADCC or spontaneous cytotoxicity. These observations serve to establish
that all MAPK family members can be activated upon ligand binding to
FcRIIIA and, among the MAPK family, identify p38 and ERK as key
molecules in the biochemical pathway(s) that regulates activation of
two of the NK cell functions, namely cytotoxicity and cytokine
production.
All MAPK family members have been previously shown to be activated in
murine macrophages (32) upon FcR cross-linking.
However, the identity of the FcR type responsible for this effect
was not definitively established. Using NK cells, we extend those data
to report that FcRIIIA is, in itself, capable of this effect. Upon
ligand binding, other immune receptors structurally similar to
FcRIIIA, e.g., the B cell Ag receptor, also transduce signals
resulting in activation of the same three kinases
(42).
We previously reported that FcRIIIA-dependent ERK-2 activation plays
a role in cytokine production, ADCC, and spontaneous cytotoxicity in NK
cells (20, 21). The kinetics of FcRIIIA-induced
activation and inactivation (dephosphorylation) of p38 and JNK in NK
cells are very similar, if not identical, to those of ERK, and it is
likely that a specific phosphatase(s) induced via FcRIIIA
stimulation controls activation of all MAPK. The most likely candidate
for this is the dual specificity protein MKP-1, which dephosphorylates
ERK-2, JNK, and p38 MAPK in PMA-stimulated U937 cells
(43).
Unlike immune complexes, tumor target cells binding to NK cells induce
activation of p38, but not JNK1 kinase. One or more activating
receptors may be sensitive to fixation, as used here. However, neither
ERK (21, 23) nor p38 activation is prevented under these
conditions. This indicates that at least one of the target surface
molecules triggering spontaneous cytotoxicity is still capable of
responding to fixed cells to induce early biochemical events and
activation of at least two MAPK family members, making it unlikely that
lack of JNK activation depends on lack of NK cell activation by the
fixed target cell. Thus, we favor the hypothesis that, unlike
FcRIIIA, the receptor(s) triggering spontaneous cytotoxicity
transduces signals leading specifically to the activation of ERK and
p38, but not JNK, similar to the PDGF receptor (24) or the
TCR (19). Whatever the reason for the lack of JNK
activation upon target cell binding, our data support a nonredundant
role of individual MAPK members in NK cells.
We have used pharmacologic inhibitors to determine the necessary role of p38 in ADCC and spontaneous cytotoxicity and in cytokine expression, similar to ERK2. Several lines of evidence support that the observed inhibition does not depend on toxicity of the inhibitors or on inhibition of ERK or other biochemical pathways upstream of MAPK. Specifically, 1) cell viability, conjugate formation, and expression of several adhesion molecules involved in spontaneous cytotoxicity or ADCC are not modified in the inhibitor-treated cells; 2) the same treatment does not affect PMA/ionomycin-induced ERK activation (not shown); and 3) both inhibitors used, which specifically bind the ATP-binding site of the p38 kinase, do not affect the activity of the closely related ERK and JNK or other serine-threonine kinases, such as c-Raf, p90 S6kinase, and P70 S6 kinase (41). The possibility that in our previous report using a MEK inhibitor to prevent ERK activation (21), p38 inhibition was responsible for the almost complete inhibition of both ADCC and spontaneous cytotoxicity can be discounted based on the observation that p38, but not ERK, phosphorylation (and thus activation) is maintained in cells treated with a MEK inhibitor (not shown).
Similar to what we (21) and others (23)
previously reported for the ERK pathway, our findings indicate that the
inhibition of ADCC following p38 inactivation depends on a regulatory
effect of this kinase on NK cell degranulation. Our data suggest that
both ERK- and p38-mediated signals, although necessary, are not
sufficient alone to activate FcRIIIA-mediated degranulation and the
lytic process. As discussed above, sequential and/or interdependent
activation of the two kinases is unlikely, and their roles do not
appear to be redundant. Whether the two kinases phosphorylate distinct
substrates, or both kinases are needed to phosphorylate a single
substrate remains to be determined. Among specific p38 substrates,
MAPKAP K-2, activated both upon FcRIIIA stimulation and target cell
binding (data not shown), may represent a biochemical mediator common
to the two types of cytotoxicity.
Direct detection of perforin- and granzyme B-containing intracellular
granules has indicated the ERK2 dependence of FcRIIIA-induced
granule migration along cytoskeletal structures in NK cells
(23). Here we show that, as previously reported for
FcRIIIB and FcRIIA in neutrophils (37), FcRIIIA
stimulation induces actin polymerization in NK cells. Activation of p38
is required for PDGF-induced cell motility responses such as cell
migration and actin reorganization (24) and mediates the
vascular endothelial growth factor-induced ERK-independent actin
reorganization in endothelial cells (25) and the
TGF-ß1-induced actin polymerization in neutrophils (44).
However, we obtained no evidence of a role for p38 (this manuscript),
ERK (21), or the two kinases combined (not shown) in
target cell-, FcRIIIA-, or PMA/ionomycin-induced actin
polymerization in NK cells. In cytotoxic T cells degranulation is
regulated by the motor protein kinesin, and several kinesin-associated
proteins have been identified, the state of phosphorylation of which
affects the extent of kinesin motor activity and subsequent granule
release (45, 46). Kinesin and/or kinesin-associated
proteins might be among the direct or indirect targets of the Ser/Thr
kinase phosphorylation cascade induced by p38 or ERK activation during
NK cell cytotoxicity.
Our data indicate that both p38 and ERK MAPK activation regulate at
least in part the immune complex-induced IFN- and TNF- mRNA
accumulation and the target cell- induced IFN- mRNA accumulation in
human NK cells. These data add to a recent report indicating a role for
p38 MAPK in integrin-triggered IL-8 production by human NK cells
(47). FcRIIIA stimulation induces AP-1-dependent
transcription of the cytokines tested (48), and both ERK
and p38 phosphorylate and regulate the activity of this and other
transcription factors (49, 50). Thus, in this case,
cytokine regulation may occur at least in part at the transcriptional
level. However, post-transcriptional regulation of the expression
of several cytokines has been reported for p38 (39, 51), and the level(s) at which this kinase regulates FcRIIIA-
and/or target cell induced IFN- and TNF- mRNA remains to be
determined. The possibility that JNK1 may also be involved (at least in
cytokine expression induced upon FcRIIIA stimulation) is not
excluded by our data and unfortunately cannot be tested at present.
No specific inhibitors of this kinase are available, and the use of
cells infected with Vac-encoding inactive kinase is inappropriate to
study nonimmediate events that require host cell RNA transcription and
protein translation, both known to be subverted by the vaccinia
virus.
Collectively, our data serve to establish that only p38 and ERK, among MAPK, play a role in NK cell cytotoxicity and cytokine production induced upon target cell recognition. They also open the way to future studies to define the mechanism(s) through which p38 regulates NK cell lytic functions, the common or specific MAPK substrates involved, and the possible role of JNK1 specifically in functions other than cytotoxicity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bice Perussia, Jefferson Medical College, Kimmel Cancer Center, BLSB 750, 233 South 10th Street, Philadelphia, PA 19107.
3 Abbreviations used in this paper: ADCC, Ab-dependent cell-mediated cytotoxicity; BLT, sodium benzyloxycarbonyl-L-lysine thiobenzyl ester; EA, IgG-sensitized E; ERK, extracellular signal-regulated kinase; FcR, receptor for the Fc fragment of IgG; F-actin; filamentous actin; JNK, c-Jun N-terminal kinase; K-2, kinase 2; MAPK, mitogen-activated protein kinase; MAPKAP kinase-2, MAPK-activated-protein kinase-2; PDGF, platelet-derived growth factor; PTK, protein tyrosine kinase; Vac, vaccinia; wt, wild type.
Received for publication December 20, 1999. Accepted for publication May 26, 2000.
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  M. Lagadari, K. Truta-Feles, K. Lehmann, L. Berod, M. Ziemer, M. Idzko, D. Barz, T. Kamradt, A. A. Maghazachi, and J. Norgauer Lysophosphatidic acid inhibits the cytotoxic activity of NK cells: involvement of Gs protein-mediated signaling Int. Immunol., June 1, 2009; 21(6): 667 - 677. [Abstract] [Full Text] [PDF]
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 D. Kumar, J. Hosse, C. von Toerne, E. Noessner, and P. J. Nelson JNK MAPK Pathway Regulates Constitutive Transcription of CCL5 by Human NK Cells through SP1 J. Immunol., January 15, 2009; 182(2): 1011 - 1020. [Abstract] [Full Text] [PDF]
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 H. Guo, A. Samarakoon, B. Vanhaesebroeck, and S. Malarkannan The p110{delta} of PI3K plays a critical role in NK cell terminal maturation and cytokine/chemokine generation J. Exp. Med., September 29, 2008; 205(10): 2419 - 2435. [Abstract] [Full Text] [PDF]
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R. Trotta, J. D. Col, J. Yu, D. Ciarlariello, B. Thomas, X. Zhang, J. Allard II, M. Wei, H. Mao, J. C. Byrd, et al. TGF-{beta} Utilizes SMAD3 to Inhibit CD16-Mediated IFN-{gamma} Production and Antibody-Dependent Cellular Cytotoxicity in Human NK Cells J. Immunol., September 15, 2008; 181(6): 3784 - 3792. [Abstract] [Full Text] [PDF]
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S. M. Shahjahan Miah, T. L. Hughes, and K. S. Campbell KIR2DL4 Differentially Signals Downstream Functions in Human NK Cells through Distinct Structural Modules J. Immunol., March 1, 2008; 180(5): 2922 - 2932. [Abstract] [Full Text] [PDF]
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 C. Li, B. Ge, M. Nicotra, J. N. H. Stern, H. D. Kopcow, X. Chen, and J. L. Strominger JNK MAP kinase activation is required for MTOC and granule polarization in NKG2D-mediated NK cell cytotoxicity PNAS, February 26, 2008; 105(8): 3017 - 3022. [Abstract] [Full Text] [PDF]
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D. G. T. Hesslein, R. Takaki, M. L. Hermiston, A. Weiss, and L. L. Lanier Dysregulation of signaling pathways in CD45-deficient NK cells leads to differentially regulated cytotoxicity and cytokine production PNAS, May 2, 2006; 103(18): 7012 - 7017. [Abstract] [Full Text] [PDF]
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 R. Parihar, R. Trotta, J. M. Roda, A. K. Ferketich, S. Tridandapani, M. A. Caligiuri, and W. E. Carson III Src Homology 2-Containing Inositol 5'-Phosphatase 1 Negatively Regulates IFN-{gamma} Production by Natural Killer Cells Stimulated with Antibody-Coated Tumor Cells and Interleukin-12 Cancer Res., October 1, 2005; 65(19): 9099 - 9107. [Abstract] [Full Text] [PDF]
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N. D. Huntington, Y. Xu, S. L. Nutt, and D. M. Tarlinton A requirement for CD45 distinguishes Ly49D-mediated cytokine and chemokine production from killing in primary natural killer cells J. Exp. Med., May 2, 2005; 201(9): 1421 - 1433. [Abstract] [Full Text] [PDF]
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 R. Trotta, R. Parihar, J. Yu, B. Becknell, J. Allard II, J. Wen, W. Ding, H. Mao, S. Tridandapani, W. E. Carson, et al. Differential expression of SHIP1 in CD56bright and CD56dim NK cells provides a molecular basis for distinct functional responses to monokine costimulation Blood, April 15, 2005; 105(8): 3011 - 3018. [Abstract] [Full Text] [PDF]
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S. Pisegna, G. Pirozzi, M. Piccoli, L. Frati, A. Santoni, and G. Palmieri p38 MAPK activation controls the TLR3-mediated up-regulation of cytotoxicity and cytokine production in human NK cells Blood, December 15, 2004; 104(13): 4157 - 4164. [Abstract] [Full Text] [PDF]
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C. Ohlen, M. Kalos, L. E. Cheng, A. C. Shur, D. J. Hong, B. D. Carson, N. C.T. Kokot, C. G. Lerner, B. D. Sather, E. S. Huseby, et al. CD8+ T Cell Tolerance to a Tumor-associated Antigen Is Maintained at the Level of Expansion Rather than Effector Function J. Exp. Med., June 3, 2002; 195(11): 1407 - 1418. [Abstract] [Full Text] [PDF]
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K. Jiang, B. Zhong, D. L. Gilvary, B. C. Corliss, E. Vivier, E. Hong-Geller, S. Wei, and J. Y. Djeu Syk Regulation of Phosphoinositide 3-Kinase-Dependent NK Cell Function J. Immunol., April 1, 2002; 168(7): 3155 - 3164. [Abstract] [Full Text] [PDF]
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 J. Y. Djeu, K. Jiang, and S. Wei A View to a Kill: Signals Triggering Cytotoxicity Clin. Cancer Res., March 1, 2002; 8(3): 636 - 640. [Abstract] [Full Text] [PDF]
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S. S. Chuang, P. R. Kumaresan, and P. A. Mathew 2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN-{gamma} Release in Human NK Cells Involves Distinct Pathways J. Immunol., December 1, 2001; 167(11): 6210 - 6216. [Abstract] [Full Text] [PDF]
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Y. M. Vyas, K. M. Mehta, M. Morgan, H. Maniar, L. Butros, S. Jung, J. K. Burkhardt, and B. Dupont Spatial Organization of Signal Transduction Molecules in the NK Cell Immune Synapses During MHC Class I-Regulated Noncytolytic and Cytolytic Interactions J. Immunol., October 15, 2001; 167(8): 4358 - 4367. [Abstract] [Full Text] [PDF]
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F. Colucci, E. Rosmaraki, S. Bregenholt, S. I. Samson, V. Di Bartolo, M. Turner, L. Vanes, V. Tybulewicz, and J. P. Di Santo Functional Dichotomy in Natural Killer Cell Signaling: Vav1-Dependent and -Independent Mechanisms J. Exp. Med., June 18, 2001; 193(12): 1413 - 1424. [Abstract] [Full Text] [PDF]
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) or with (
and •) the indicated
concentrations of SB203580 (top) or SB202190
(bottom). Fc
, none) or
infected with Vac recombinants encoding FLAG-tagged wt or dominant
negative JNK1 (APF) and stimulated with E or EA. B, NK
cells were left uninfected (