From the Department of Medicine, ¶ Department of
Pediatrics,
Program in Cell Biology, and ** Program in Molecular
Signal Transduction, National Jewish Medical and Research Center and
the § Departments of Medicine and
Pharmacology, University of Colorado School
of Medicine, Denver, Colorado 80206
Received for publication, August 17, 2000
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ABSTRACT |
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The intensity and duration of an
inflammatory response depends on the balance of factors that favor
perpetuation versus resolution. At sites of inflammation,
neutrophils adherent to other cells or matrix components are exposed to
tumor necrosis factor- The rapid influx of polymorphonuclear leukocytes (neutrophils)
into sites of injury is an important component of the acute inflammatory response in humans (1). The resulting adherence and
chemotaxis of leukocytes in the appropriate cytokine milieu are not
only necessary for efficient clearance of microorganisms (2) but may
also participate in appropriate resolution of the inflammatory response
through the subsequent induction of cell death by apoptosis.
Tumor necrosis factor- Whereas TNF Activation of tyrosine kinases plays a central role in human neutrophil
integrin signaling (14). Studies have linked adherence-associated neutrophil responses in TNF Recent studies have identified the integrin-associated
cytoplasmic tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2 (24)), also known as CAK Three distinct MAPk families have been described to date that may act
downstream of tyrosine kinases. The prototypical p42/p44 ERK MAPk are
activated in response to growth factors and chemoattractants, whereas
the p38 MAPk and c-Jun N-terminal kinase (JNK) families modulate
stress-activated responses (32, 33). Each MAPk cascade requires
sequential activation of a MAPk kinase kinase (or equivalent) which can
then activate a MAPk kinase, resulting in turn in the phosphorylation
and activation of a MAPk (34, 35). In the neutrophil, only the p38 MAPk
cascade has been clearly shown to be utilized in response to TNF Apoptosis is an important mechanism for regulating the extent of an
inflammatory response, making it self-limiting and thus preventing
massive tissue damage. TNF The present study was undertaken to determine the conditions under
which TNF Materials--
Endotoxin-free reagents and plasticware were used
throughout the experimental process. Neutrophils, prepared by
previously described methods (40), were resuspended in Krebs-Ringer
phosphate buffer, pH 7.2, with 0.2% dextrose (5% dextrose in 0.2%
NaCl, injectable, Abbott) KRPD. KRPD salts were purchased from
Mallinckrodt Chemical Works, and all components were diluted in
endotoxin-free saline (0.9% saline for irrigation, Abbott). Aprotinin,
leupeptin, PMSF, bovine serum albumin fraction V, sodium orthovanadate
(Na3VO4), p-nitrophenyl phosphate
(pNPP), Brij 97, protein A-Sepharose, wortmannin, and
propidium iodide were purchased from Sigma. Recombinant human TNF Antibodies and Protein Fragments--
Anti-JNK1 (C-17),
phosphospecific anti-JNK (G-7), anti-MEKK (C-22), anti-Syk (C-20),
anti-MKK7 (T-19), and c-Jun-(1-79) were all from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA). Anti-Lyn was from Transduction
Laboratories (San Diego, CA). Anti-MKK4 and anti-caspase-3 were
purchased from PharMingen, and the phosphospecific anti-SEK1 was from
New England Biolabs (Beverly, MA). Anti-CR3 receptor (anti-CD11b) was
purchased from Dako (Denmark). Mouse IgG was from Upstate Biotechnology
Inc. (Lake Placid, CA). Anti-CD11b F(ab')2 fragments were
generated with an Immunopure IgG1 F(ab) and F(ab')2
Preparation kit, using immobilized ficin (Pierce).
Histidine-tagged Recombinant Proteins--
Wild type SEK1
(SEK1wt), wild type JNK1 (JNK1wt), and
kinase-inactive JNK1 (JNK1km) were expressed in
Escherichia coli and purified as described previously (40).
Preincubation of Neutrophils to Promote
Adherence--
Neutrophils were resuspended at 20 × 106/ml in complete KRPD containing 0.25% human serum
albumin, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin. Following this, 20 × 106 cells were either
preincubated in a tube under static conditions for 55 min at 37 °C
(cell-cell/homotypic adherence; hereafter referred to as adherent
neutrophils) or preincubated in a tube under static conditions for 30 min at 37 °C and then aliquoted into 1 well of a 12-well plate
(cell-substratum adherence) for an additional 25 min prior to
stimulation with 10 ng/ml TNF JNK1 Immunoprecipitation--
Cell pellets were lysed with 500 µl of cold JNK lysis buffer, JLB (50 mM Tris, pH 7.5, 10% glycerol, 1% Nonidet P-40, 137 mM NaCl, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, 2 mM NaF, 1 mM Na3VO4)
and centrifuged at 15,000 rpm for 10 min at 4 °C. Triton-soluble
cell lysates were precleared with protein A-Sepharose for 15 min,
4 °C, prior to immunoprecipitation with anti-JNK1 for 2 h at
4 °C.
JNK1 in Vitro Kinase Assay--
Beads were washed once in JNK
lysis buffer and twice in JNK reaction mix (20 mM
JNK activity was expressed as a ratio percentage of unstimulated
control (% BL). Baseline-BL.
MKK4/MKK7 in Vitro Kinase Assays--
Adherent neutrophils were
exposed to TNF MEKK1-coupled in Vitro Kinase Assay--
Adherent neutrophils
were stimulated with TNF Syk/Lyn in Vitro Kinase Autophosphorylation
Studies--
Adherent neutrophils were stimulated with TNF Inhibitor Studies--
Neutrophils were preincubated with the
PI3-kinase inhibitor wortmannin and tyrosine kinase inhibitor genistein
as described (40, 41). 10 and 30 µM piceatannol were used
for Syk and Lyn in vitro kinase autophosphorylation studies.
Neutrophils were preincubated with tyrphostin A9 (5 µM)
or tyrphostin A63 (50 µM) a negative control, for 1 h at 37 °C prior to stimulation with TNF Apoptosis Assessment--
For annexin V binding, externalized
phosphatidylserine was determined by annexin V binding. Adherent
neutrophils were exposed to TNF TNF The Role of Adherence in JNK Activation--
JNK activation was
studied in neutrophils preincubated under different conditions that
promote adherence and then exposed to TNF
To validate the specificity of blocking by anti-CD11b, two types of
control experiments were performed. The first, using mouse IgG as an
isotype-matched antibody, ruled out the possibility that the inhibition
observed was due to anti-CD11b competition for protein A-Sepharose
(preventing anti-JNK1 binding), whereas it also tested the blocking
capabilities of anti-CD11b per se (Fig. 3, A and
B). The second, carried out to determine whether anti-CD11b
inhibited the TNFR and thus its downstream signaling, examined a
TNF MKK7 and MKK4 Activate JNK1--
Whereas JNK activation has been
reported as a consequence of MKK4/SEK1 activation in many cell systems
(46), the recent description of MKK7 (homologous to
hemipterous, the Drosophila JNK kinase) (47) as a
downstream effector of TNF TNF
By having determined the TNF Involvement of Tyrosine Kinases in the
In initial studies, the tyrosine kinase activities of Syk and Lyn were
determined in vitro by autophosphorylation. Anti-Syk immunoprecipitates from TNF
Since integrin signaling may proceed through Syk activation (19), we
next questioned whether Syk activation in TNF
To test this hypothesis, we studied the effects of a Syk-specific
inhibitor, piceatannol, on the TNF
Recent studies have linked activation of another tyrosine kinase, Pyk2,
a FAK family member, to TNF TNF
Caspase-3 cleavage has been widely described in cells undergoing
apoptosis, including neutrophils. We observed a
time-dependent cleavage of pro-caspase-3 (32 kDa) to a
caspase-3 cleavage product (17 kDa) in lysates from adherent
neutrophils that had been exposed to TNF Regulation of cellular responses to specific stimuli occurs in
part through selective activation of MAPk-signaling cascades. In human
neutrophils, chemoattractant-induced activation of c-Raf and MEKK1
leads to the activation of p42/p44 ERK MAPk pathways (40, 41, 61, 62),
whereas chemoattractants and lipopolysaccharide activate p38 MAPk as
well (63, 64). In contrast, studies to date in human neutrophils have
not as yet provided evidence for activation of JNK and its associated
signaling partners. This study, performed in primary human neutrophils,
describes key elements leading to activation of the JNK pathway
following stimulation with TNF (TNF
). Although TNF
has been implicated
in induction of pro-inflammatory responses, it may also inhibit the
intensity of neutrophilic inflammation by promoting apoptosis. Since
TNF
is not only an important activator of the stress-induced
pathways leading to p38 MAPk and c-Jun N-terminal kinase (JNK)
but also a potent effector of apoptosis, we investigated the effects of
TNF
on the JNK pathway in adherent human neutrophils and the
potential involvement of this pathway in neutrophil apoptosis. Stimulation with TNF
was found to result in
2
integrin-mediated activation of the cytoplasmic tyrosine kinases Pyk2
and Syk, and activation of a three-part MAPk module composed of MEKK1,
MKK7, and/or MKK4 and JNK1. JNK activation was attenuated by blocking antibodies to
2 integrins, the tyrosine kinase
inhibitors, genistein, and tyrphostin A9, a Pyk2-specific inhibitor,
and piceatannol, a Syk-specific inhibitor. Exposure of adherent
neutrophils to TNF
led to the rapid onset of apoptosis that was
demonstrated by augmented annexin V binding and caspase-3
cleavage. TNF
induced increases in annexin V binding to neutrophils
were attenuated by blocking antibodies to
2 integrins,
and the caspase-3 cleavage was attenuated by tyrphostin A9. Hence,
exposure of adherent neutrophils to TNF
leads to utilization of the
JNK-signaling pathways that may contribute to diverse functional
responses including induction of apoptosis and subsequent resolution of
the inflammatory response.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF
),1 a pluripotent
cytokine, is produced by a variety of leukocytes in response to
stimulation by lipopolysaccharide (3). In turn, exposure to TNF
induces wide ranging biological effects including cell differentiation, proliferation, apoptosis, and multiple pro-inflammatory effects. TNF
exerts potent effects on neutrophils that have been suggested to
contribute to the inflammatory response in sepsis (4, 5) and the adult
respiratory distress syndrome (6). However, TNF
also exerts
anti-inflammatory effects as demonstrated by an enhanced inflammatory
response in TNF
/
mice after infection (7), an effect that has
been ascribed to the ability of TNF
to induce apoptosis in
neutrophils (8).
can prime neutrophils in suspension (9, 10), many
TNF
-induced cellular responses, including oxidant release, have been
reported in association with
2-mediated,
integrin-mediated adherence (11-13). Neutrophils utilize a variety of
adhesion molecules (14), but studies employing either genetically
manipulated mice (15), antibody blocking studies (16), and studies with
adhesion molecule-deficient patients (17) have shown that integrins, in
particular
2 and
3, are fundamentally
important in adhesive events (16, 18). Integrin ligation in neutrophils
results in reorganization of the cytoskeleton, cell spreading across an adhesive surface, and release of O2
and granule constituents.
stimulated neutrophils to
2 integrin-dependent phosphorylation of
tyrosine kinase substrates and augmentation of
p58c-Fgr and p53/56Lyn kinase activity
(19-22). In addition, neutrophils induced to spread over fibrinogen in
the presence of TNF
show integrin-dependent activation
of Syk (a non-Src-like cytoplasmic) protein tyrosine kinase (PTK) which
associates with p58c-Fgr and p53/56Lyn in
co-localized complexes. The TNF
-induced phosphorylation of another
tyrosine kinase, Pyk2, has also observed (23).
-(cell adhesion kinase
(25)), RAFTK-(related adhesion focal tyrosine kinase (26)),
CADTK-calcium-dependent tyrosine kinase CADTK (27)), as a
member of the FAK family. Pyk2 has been identified in brain cells, cell
lines and hematopoietic cells (24, 25, 28, 29). Studies in adherent
neutrophils exposed to TNF
demonstrate that tyrosine phosphorylation
of Pyk2 is
2 integrin-dependent and that
Pyk2 appears to lie downstream of Lyn, Syk, protein kinase C, and
reorganization of the cytoskeleton (30). Furthermore, overexpression of
Pyk2 in PC12 cells, which have been stimulated with TNF
, induces JNK
activation in a calcium-independent manner (31). Thus the altered
response of the adherent neutrophil to TNF
may reflect in part
differences in tyrosine kinase signaling through
2
integrin-mediated mechanisms.
stimulation (36). Involvement of the p42/p44 ERK MAPk pathway in
response to TNF
is controversial. Activation of a JNK pathway in the
neutrophil has not as yet been shown in any context. Furthermore,
regulation of apoptosis may represent a functional consequence of
JNK, and this has yet to be delineated in the context of the neutrophil
inflammatory response.
is a potent inducer of apoptosis in
neutrophils, but its mechanisms of action are unknown. Recent reports
have suggested that TNF
in conjunction with
2 integrins induces apoptosis in a tyrosine kinase-dependent
manner (37), whereas other studies have shown activation of the
cysteine proteases, caspase-3 and -8 (8). The role of MAPK members, in
particular JNK pathway activation, in the TNF
induction of apoptosis
remains controversial (38, 39).
might activate the JNK pathway in human neutrophils, to
define components of a putative MAPK-module, and to delineate key
upstream components, as well as downstream functional consequences (in
particular apoptosis). We report that in adherent neutrophils, activation of JNK1 in response to TNF
results from the mobilization of an integrin-mediated cascade involving activation of tyrosine kinases, in particular Syk and Pyk2, and activation of a MAPk module in
which MEKK1 activates either or both of the MAPk kinases, MKK7 and
MKK4. Among possible functional consequences of this pathway, we show
that TNF
-elicited JNK activation is accompanied by a dramatic
acceleration of neutrophil apoptosis which is modulated both by
integrins and tyrosine kinases. Together, these results provide the
first description to date of the activation of the JNK MAPk cascade in
the human neutrophil, while providing a potential mechanism for
TNF
-induced apoptosis in the human neutrophil.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was from PharMingen (San Diego, CA). Genistein was purchased from Life
Technologies, Inc., and piceatannol, tyrphostin A9, and tyrphostin A63
were from Calbiochem. Human serum albumin was from Intergen (Purchase,
NY). NBD-phallacidin was from Molecular Probes (Eugene, OR). ECL
reagents and [
-32P]ATP were from Amersham Pharmacia Biotech.
for the intervals indicated hereafter.
Cells were either harvested from wells into a microcentrifuge
tube and pelleted at 15,000 rpm for 20 s or those stimulated in
microcentrifuge tubes were directly pelleted.
-glycerophosphate, pH 7.2, 50 mM Hepes, pH 7.6, 50 µM Na3VO4, 1 mM
dithiothreitol, 10 mM MgCl2, 12 mM
pNPP). 40 µl of in vitro kinase assay mix
containing 10 µCi of [32P]ATP and 500 ng of
c-Jun-(1-79) in JNK reaction mix were added to samples that were
incubated for 30 min at 30 °C. Reactions were terminated with
addition of 5× Laemmli sample buffer, subjected to SDS-PAGE, and
proteins transferred to nitrocellulose. c-Jun-(1-79) phosphorylation
was quantified by PhosphorImager (Molecular Dynamics) analysis and
visualized by autoradiography.
, lysed in JNK lysis buffer, and immunoprecipitated
with either anti-MKK4 or anti-MKK7. MKK4-bound beads or MKK7-bound
beads were then washed once in JNK lysis buffer and twice in JNK
reaction mix. For direct in vitro kinase assays, samples
were incubated with 40 µl of JNK reaction mix containing 10 µCi of
[
-32P]ATP and 500 ng of JNK1km for 30 min
at 30 °C. For coupled in vitro kinase assays, samples,
immunoprecipitated and washed as above, were incubated with 40 µl of
JNK reaction mix containing 10 µCi of [
-32P]ATP, 250 µM ATP, 50 ng JNK1wt, and 500 ng of
c-Jun-(1-79) for 30 min at 30 °C. All reactions were terminated
with the addition of 5× Laemmli sample buffer, subjected to SDS-PAGE,
and proteins transferred to nitrocellulose. Phosphorylation of
substrates (JNK1km, c-Jun-(1-79), and JNK1wt)
was quantified by PhosphorImager (Molecular Dynamics) analysis and
visualized by autoradiography.
, pelleted, lysed in RIPA buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 0.1% SDS, 0.5%
sodium deoxycholate, 1% Triton X-100, 10 mM sodium
pyrophosphate, 25 mM
-glycerophosphate, 1 mM
Na3VO4, and 2.1 µg/ml aprotinin), and
immunoprecipitated with anti-MEKK1. MEKK1-bound protein A-Sepharose beads were washed once in RIPA, once in PAN (10 mM Pipes pH
7.0, 100 mM NaCl, 21 µg/ml aprotinin), and then incubated
with 48 µl of the MEKK in vitro kinase mix (25 mM
-glycerophosphate, pH 7.2, 20 mM Hepes,
pH 7.6, 100 µM Na3VO4, 2 mM dithiothreitol, 20 mM MgCl2, and
100 µM ATP) containing 10 ng of SEK1wt and
100 ng of JNK1wt for 10 min at 30 °C. 2 µl of a mix
containing 15 µCi of [
32P]ATP and 500 ng of
c-Jun-(1-79) were added to the samples which were further incubated
for 30 min at 30 °C. Reactions were terminated with the addition of
5× Laemmli sample buffer, and proteins were separated by SDS-PAGE.
Phosphorylation of JNK1wt and c-Jun-(1-79) was quantified
by PhosphorImager analysis and visualized by autoradiography.
and
lysed in Syk lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Brij 97, 1 mM
Na3VO4, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin). Brij-soluble lysates were protein A-Sepharose
immunoprecipitated with either anti-Syk or anti-Lyn. Syk-bound beads or
Lyn-bound beads were washed twice in wash buffer A (25 mM
Hepes, pH 7.5, 150 mM NaCl, 0.1% Brij 97, 1 mM
Na3VO4, 10 µg/ml aprotinin, 10 µg/ml
leupeptin) and twice in wash buffer B (25 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM NaVO4, 10 µg/ml
aprotinin, and 10 µg/ml leupeptin). Beads and kinase mix (25 mM Hepes, pH 7.5, 2 mM MnCl2, 20 mM pNPP, and 25 µCi of
[
-32P[ATP]) were preincubated separately for 5 min at
30 °C and then 40 µl of kinase mix added to beads for 1 min at
30 °C. The reaction was terminated with 5× Laemmli sample buffer.
Samples were boiled for 5 min at 100 °C, subjected to SDS-PAGE, and
transferred to nitrocellulose by immunoblotting. Autophosphorylation
was detected and quantified by PhosphorImager Analysis.
.
for 1, 1.5, and 2 h. Aliquots
containing 2.5 × 105 cells were resuspended in 100 µl of NBD buffer, pH 7.5 (137 mM NaCl, 2.7 mM
KCl, 2 mM MgCl2, 5 mM glucose, 10 mM Hepes, and 2.5 mM CaCl2),
incubated with 400 ng of annexin V fluorescein (Caltag Laboratories,
Burligame, CA) and 500 ng of propidium iodide for 15 min at room
temperature and placed on ice with the addition of 400 µl of cold NBD
buffer. FACs analysis was performed by flow cytometry on a FACScan
(Becton Dickinson), and annexin V binding was determined from the
non-necrotic cell population, which stains negatively for propidium
iodide. 5000 cells were analyzed in each sample. For caspase-3
cleavage, 20 × 106 adherent neutrophils were exposed
to TNF
, 10 ng/ml for 0, 1, 1.5, and 2 h. Cells were lysed in
RIPA, and whole cell lysates were prepared from the Triton-soluble
fraction. Samples were subjected to SDS-PAGE using a 14% gel, and
proteins were transferred to nitrocellulose membrane that was then
probed with antibodies to caspase-3 by Western blot analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Activates JNK in Adherent Neutrophils--
Adherent human
neutrophils were stimulated with TNF
, and JNK1 was
immunoprecipitated from Triton-soluble cell lysates, and JNK activation
was measured by the phosphorylation of an N-terminal fragment of c-Jun
(c-Jun-(1-79)) in the presence of [32P]ATP.
Phosphorylation of c-Jun-(1-79) by JNK1 immunoprecipitates was
augmented following TNF
stimulation of neutrophils (Fig. 1A). Activation was detected
at 5 min and was maximal between 10 and 15 min. Immunoblots, probed
with anti-JNK1 antibody, confirmed that equivalent amounts of JNK were
immunoprecipitated and that in neutrophils the predominant
immunoreactive JNK1 species is a 55-kDa protein (Fig. 1C). A
change in the electrophoretic mobility of JNK1, consistent with
phosphorylation, was detected as early as 1 min after TNF
exposure.
JNK1 activation was further supported by immunodetection with a
phosphospecific anti-JNK1 antibody, indicating increased
phosphorylation of JNK in the Triton-soluble fraction of JNK1
immunoprecipitates 5 min after TNF
stimulation of neutrophils (Fig.
1B). The time course of JNK1 phosphorylation mirrored the
phosphorylation of c-Jun-(1-79).
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Fig. 1.
TNF -stimulated
activation of JNK. A, autoradiograph depicting the time
course of activation of JNK1. JNK was immunoprecipitated with anti-JNK1
from the lysates of neutrophils, exposed to TNF
(10 ng/ml for 1, 5, 10, 15, and 30 min) or buffer (30 min) and subjected to in
vitro kinase assay using c-Jun-(1-79) as a substrate. Samples
were separated on SDS-PAGE and transferred to nitrocellulose. The
phosphorylated c-Jun-(1-79) is visualized in the autoradiograph.
B, anti-phosphospecific JNK immunoblot of Triton-soluble
whole cell lysates (WCL) from neutrophils, stimulated with
TNF
(10 ng/ml) for 1, 5, 10, 15, and 30 min. C, anti-JNK1
immunoblot of TNF
-stimulated adherent neutrophils (10 ng/ml for 1, 5, 10, 15, and 30 min). Anti-JNK1 immunoprecipitates were subjected to
SDS-PAGE and transferred to nitrocellulose. Membranes were probed with
anti-JNK-1 (B, buffer). Figure depicts a representative
result from three consecutive experiments.
. Cells were maintained
either stationary in a tube under conditions that favor cell-cell
interactions or stimulated on a surface, favoring cell-substratum
interactions (Fig. 2A).
Neutrophil exposure to TNF
induced comparable amounts of JNK1
activation under both sets of conditions (Fig. 2B). Since
integrins, in particular
2 and
3, are
important components of neutrophil-adhesive interactions, we questioned
whether
2 integrins were up-regulated by TNF
on the
neutrophil surface. Neutrophils, exposed to TNF
for various time
intervals, were stained with anti-CD11b, an antibody directed against
2 integrins, and their fluorescence was analyzed by flow cytometry. We observed a time-dependent up-regulation of
2 integrins on the neutrophil surface in those cells
that had been exposed to TNF
(Fig. 2C).
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Fig. 2.
Role of adherence in JNK activation.
A, autoradiograph depicting JNK activity under two
conditions that promote adherence. Phosphorylation of the substrate
c-Jun-(1-79) was determined by JNK in vitro kinase assay on
JNK1 immunoprecipitates from neutrophils preincubated in one of two
ways: (i) under stationary conditions in a test tube for 55 min
(cell-cell adherence); or (ii) in a tube for 30 min and then plated in
a well for 25 min (cell substratum). Following preincubation cells were
stimulated with TNF (10 ng/ml for 15 min), lysed, and
immunoprecipitated with anti-JNK. B, buffer and
T, TNF
. B, graph represents mean c-Jun-(1-79)
phosphorylation ± S.E. from PhosphorImager analyses of
phosphorylated c-Jun-(1-79) band under the conditions described above.
By using the Student t test, we determined a lack of
statistically significant differences in JNK activation. C,
graph depicts TNF
-induced up-regulation of CD11b. Neutrophils,
exposed to TNF
(10 ng/ml) for 15, 30, and 60 min or buffer and
stained with anti-CD11b, were analyzed by fluorescence analysis on the
FACScan (Becton Dickinson) for surface expression of CD11b. CD11b
up-regulation was expressed as a relative fluorescence index
(RFI), (
, buffer;
, TNF
).
2 Integrin Dependence of JNK
Activation--
Previous studies have indicated a role for integrins
in mediating a variety of neutrophil responses, including the activity of the Na/H+ antiport and oxygen radical release (12, 42).
To determine whether the TNF
-induced JNK activation we had observed
was
2 integrin-mediated, neutrophils were pretreated
with a blocking antibody directed against CD11b, prior to TNF
stimulation, and JNK activity was determined on JNK immunoprecipitates.
The resultant JNK activation was significantly diminished compared with
controls (Fig. 3, A and
B).
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Fig. 3.
2 integrin
involvement in JNK activation. A, autoradiograph
showing JNK activation following preincubation with anti-CD11b.
Neutrophils were pretreated with either buffer (
), isotype control
antibody, mouse IgG 20 µg/ml (IgG), or the
2 integrin
blocking antibody, anti-CD11b10 µg/ml, 20 µg/m,(CD11b) for 55 min
at 37 °C, stimulated with buffer (B) or TNF
(T, 1 ng/ml for 15 min). JNK activity was determined on JNK
immunoprecipitates by in vitro kinase assay, in which
c-Jun-(1-79) was used as substrate. Samples were separated by
SDS-PAGE, transferred to nitrocellulose, and the phosphorylated
c-Jun-(1-79) visualized by autoradiography. B, graph
depicting JNK activity under the same conditions demonstrated in
A. JNK activation was determined from c-Jun-(1-79)
phosphorylation by the protocol described for samples from (Fig.
2A) and was quantified by PhosphorImager analysis. The graph
depicts mean JNK activity ± S.E. of three experiments.
Statistical analysis employing a one-way ANOVA determined that in the
presence of 20 µg/ml anti-CD11b, JNK activation was significantly
reduced compared with control, * (p < 0.01).
C, graph depicts TNF
-induced increase in actin assembly
in the absence and presence of anti-CD11b. Neutrophils were pretreated
in the absence (
) and presence (
) of anti-CD11b (10 µg/ml),
stimulated with TNF
(10 ng/ml) for 0, 2, 5, and 15 min, and then
stained with NBD-phallacidin. The fluorescence was analyzed by flow
cytometry on an Epics (Coulter) for determination of actin assembly as
described previously (45).
-induced function, actin assembly. We have previously shown in
neutrophils and HL-60 promyelocytic cells, stimulated with either
chemoattractants or lipopolysaccharide, that actin assembly is
differentiable from integrin-mediated adherence (43-45). Neutrophils,
pretreated in the absence and presence of anti-CD11b and stimulated
with TNF
, demonstrated identical actin assembly in both cases (Fig.
3C), indicating that anti-CD11b does not modulate the TNFR
or its immediate downstream signaling.
suggests its potential role (48).
Activation of MKK4 and MKK7 following TNF
exposure was determined in
MKK4 and MKK7 immunoprecipitates by direct phosphorylation of a
kinase-inactive recombinant human JNK1 (JNK1km). A
time-dependent increase in JNK1km
phosphorylation by both MKK4 and MKK7 immunoprecipitates from
TNF
stimulated neutrophils was detected (Fig.
4A). To determine whether the
JNK1km phosphorylation induced by MKK4 and MKK7 resulted in
enhanced activation of JNK1, we determined whether these MKK homologues could initiate a coupled reaction leading to c-Jun-(1-79) via kinase-active JNK1. Both MKK4 and MKK7 immunoprecipitates from TNF
exposed neutrophils induced phosphorylation of the
JNK1wt and c-Jun-(1-79) substrates with a time course
identical to that shown for JNK1km (Fig. 4B).
However, MKK7 induced much stronger phosphorylation than did MKK4.
Immunodetection of immunoprecipitated MKK4 and MKK7 confirmed that
within each experiment, equivalent amounts of each kinase were
immunoprecipitated (data not shown). Since MKK4 appeared to be only a
weak activator of JNK1, we further verified that MKK4 itself was being
activated by probing with an antibody specific for the phosphorylated
(active) form of MKK4. MKK4 was activated in a
time-dependent manner in TNF
stimulated neutrophils
(Fig. 4C). Immunoblotting the same samples with an antibody
to MKK4 confirmed that equivalent amounts of MKK4 had been
immunoprecipitated.
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Fig. 4.
TNF activates MKK4
and MKK7. A, MKK4/MKK7 direct in vitro
kinase assay. Autoradiograph of JNKkm phosphorylated
directly by either MKK4 or MKK7 immunoprecipitates from neutrophils
stimulated with TNF
(10 ng/ml) for 0, 5, 10, and 15 min is shown.
B, autoradiograph of coupled MKK4/MKK7 activation assays.
MKK4 and MKK7 immunoprecipitates from neutrophils, stimulated with
TNF
(10 ng/ml) for 0, 5, 10 and 15 min, and then subjected to
in vitro kinase assay (30 min, at 30 °C) using
JNKwt and c-Jun-(1-79) as substrates are shown.
Phosphorylated proteins were separated by SDS-PAGE, transferred to
nitrocellulose, and visualized by PhosphorImager analysis.
C, anti-MKK4 immunoblots from neutrophils exposed to TNF
(10 ng/ml) for 5 and 15 min. Triton-soluble lysates were
immunoprecipitated with anti-MKK4, subjected to SDS-PAGE, and
transferred to nitrocellulose. Membranes were probed with either
anti-phosphospecific MKK4 or anti-MKK4.
Induces MEKK1 Activation--
MAP kinase kinase kinases
reported to activate MKK4 include MLK, MEKK1, and MEKK4 (49-51). Since
it has been shown that MEKK1 activates JNK1 in TNF
-stimulated
macrophages (52), we employed a coupled assay to determine whether
MEKK1 activity was induced in neutrophils by TNF
exposure. MEKK1
immunoprecipitates were incubated together with the kinase-active (wild
type) recombinant human kinase substrates MKK4 and JNK1 and a c-Jun
fragment, c-Jun-(1-79). The consequence of this coupled co-incubation
is visualized in a representative autoradiograph (Fig.
5A) wherein increased
phosphorylation of both the c-Jun-(1-79) and JNK1wt
species was detected, peaking 5 min after TNF
stimulation of
adherent neutrophils. The time course of MEKK1 activity, determined
from several experiments, was quantified from c-Jun-(1-79)
phosphorylation (Fig. 5B). MEKK1 activation peaked 5 min after TNF
stimulation of neutrophils and declined to a plateau
between 10 and 15 min. Phosphorylation of wild type recombinant JNK1
was also detected, confirming that JNK1 is itself a substrate for
recombinant MKK4 following MEKK1 activation in neutrophils.
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Fig. 5.
TNF activates MEKK1
in neutrophils. A, autoradiograph showing time course
of MEKK1 activation. Neutrophils were stimulated with TNF
(10 ng/ml)
for 0, 5, 10, and 15 min. MEKK1 immunoprecipitates from Triton-soluble
lysates were subjected to coupled in vitro kinase assay
using MKK4wt, JNK1wt, and c-Jun-(1-79) as
substrates. Samples were separated by SDS-PAGE, transferred to
nitrocellulose, and phosphorylated JNK1wt and c-Jun-(1-79)
visualized. B, MEKK1 time course. Above samples were
subjected to PhosphorImager analysis, and phosphorylation of band
representing c-Jun-(1-79) was quantified. Graph depicts mean
c-Jun-(1-79) phosphorylation ± S.E. for 3 consecutive
experiments. C, autoradiograph displaying
2
integrin-associated MEKK1 activity. Neutrophils, pretreated in the
absence (
) or presence (+) of anti-CD11b (10 µg/ml), were exposed
to TNF
(10 ng/ml) for 5 min, and lysates were immunoprecipitated
with anti-MEKK1. Immunoprecipitates were subjected to coupled in
vitro kinase assay as described under "Experimental
Procedures." Samples were subjected to SDS-PAGE and transferred to
nitrocellulose. Phosphorylated c-Jun-(1-79) and JNKwt were visualized
by autoradiography (B, buffer and T,
TNF
).
-stimulated JNK1 activation in
neutrophils to be
2 integrin-dependent, we
next questioned whether integrin input into the signaling pathway
leading to JNK1 activation might occur upstream from MEKK1 or between
MEKK1 and JNK1. MEKK activity was determined in response to TNF
in
the presence and absence of anti-CD11b by a coupled in vitro
kinase assay. MEKK1 immunoprecipitates were prepared from neutrophils,
pretreated in the absence and presence of anti-CD11b, and then exposed
to TNF
. The phosphorylation of c-Jun-(1-79) and JNK1wt
induced by these immunoprecipitates is shown (Fig. 5C). We
observed greatly decreased phosphorylation of both the c-Jun-(1-79)
and JNK1wt species in TNF
stimulated neutrophils that
had been pretreated with anti-CD11b, suggesting that the
integrin-dependent activation of JNK occurs at, or upstream
of, MEKK1.
2
Integrin-mediated JNK Activation--
Since studies in adherent
neutrophils exposed to TNF
have shown that phosphorylation and
activation of tyrosine kinases are associated with integrin activation,
we sought to determine whether tyrosine kinases were involved in the
TNF
induced JNK activation in adherent neutrophils. Activation of
JNK1 from TNF
stimulated neutrophils was assessed following
pretreatment with genistein, an inhibitor of tyrosine kinases, and
wortmannin, a PI3-kinase inhibitor. TNF
induced JNK activation was
significantly inhibited in cells that had been pretreated with
genistein but unaffected by wortmannin (Fig.
6). Since the Src family kinase member,
Lyn, and the non-Src cytoplasmic tyrosine kinase, Syk, have been
implicated in the stress-activated JNK pathway in B-cells (53-56) and
in integrin signaling in platelets (57) and monocytes (58), we
questioned whether either kinase was activated in adherent neutrophils
exposed to TNF
and, if so, whether this activation contributed to
JNK pathway activation in the neutrophil.
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Fig. 6.
Effects of genistein (tyrosine kinase
inhibitor) and wortmannin (PI3-kinase inhibitor on JNK1
activation. Neutrophils, pretreated with either genistein (100 µM), wortmannin (50 nM), or 0.1%
Me2SO control for 55 min were stimulated with TNF
(10 ng/ml) for 15 min. JNK activity was determined on JNK1
immunoprecipitates by in vitro kinase assay using
c-Jun-(1-79) as substrate. Samples were separated by SDS-PAGE,
transferred to nitrocellulose, and analyzed by PhosphorImager analysis.
Graph depicts mean phosphorylated c-Jun-(1-79) ± S.E.
Statistical analysis employing a one-way ANOVA determined that in
presence of genistein for JNK activation was significantly reduced
compared with control, * (p < 0.001), whereas
there no significant difference with wortmannin.
-stimulated adherent neutrophils
exhibited tyrosine kinase activity with greatly enhanced
autophosphorylation of a 40-kDa species, consistent with the almost
universal proteolytic cleavage product of Syk (59) (Fig.
7A). Incubation with
piceatannol, a Syk-specific inhibitor (60), inhibited this
autophosphorylation in a dose-dependent fashion. In
contrast to the augmented Syk activation, we observed minimal
activation of the Src family kinase, Lyn, in TNF
-stimulated adherent
neutrophils (Fig. 7B). Additionally, the autophosphorylation
activity of immunoprecipitated Lyn was diminished only slightly at the
highest concentration of piceatannol.
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Fig. 7.
TNF -induced Syk
activation and
2 integrin
association. A, autoradiograph of TNF
-induced Syk
autophosphorylation and its inhibition in the presence of piceatannol.
Neutrophils were stimulated with either buffer (
) or TNF
(+), 10 ng/ml for 5 min, and Syk immunoprecipitates were subjected to in
vitro kinase assay in the presence of 0.1% Me2SO
(solvent control), piceatannol (10 or 30 µM). Samples
were subjected to SDS-PAGE, transferred to nitrocellulose, and the
autophosphorylated p40Syk visualized by PhosphorImager
analysis. B, autoradiograph depicting Lyn activity in
TNF
-stimulated (10 ng/ml, 5 min) adherent neutrophils.
P53/56Lyn autophosphorylation was determined on anti-Lyn
immunoprecipitates in presence of 0.1% Me2SO, piceatannol
(10 or 30 µM). C,
2
integrin-associated inhibition of Syk autophosphorylation. Neutrophils
were pretreated with anti-CD11b (10 µg/ml for 55 min) exposed to
TNF
(10 ng/ml) for 5 min, and an in vitro kinase assay
was performed on the anti-Syk immunoprecipitates. Samples were
subjected to SDS-PAGE, transferred to nitrocellulose, and the Syk
autophosphorylation visualized (B, buffer and T,
TNF
).
-stimulated neutrophils
was also integrin-dependent. The Syk autophosphorylation activity, determined on Syk immunoprecipitates, was diminished by
preincubation of neutrophils with anti-CD11b antibody prior to TNF
exposure (Fig. 7C). This observation provided a link between
2 integrins and Syk activation in our system, raising
the possibility that Syk may be a component of the integrin-signaling
pathway leading to JNK1 activation.
induced activation of MEKK1 and
JNK1 in adherent neutrophils. Piceatannol pretreatment of
TNF
-stimulated neutrophils resulted in reduced MEKK1 activation (Fig. 8A) as determined by the
diminished phosphorylation of both the c-Jun-(1-79) and
JNK1wt species. Similarly, piceatannol pretreatment induced
a concentrationdependent inhibition of JNK1 activation in
TNF
-stimulated neutrophils (Fig. 8B). Pretreatment of
neutrophils with 10 µM piceatannol, a concentration that
significantly inhibited Syk autophosphorylation but had no effect on
Lyn autophosphorylation, decreased JNK1 activation by 66%.
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Fig. 8.
Syk involvement in JNK pathway
activation. A, MEKK activation following piceatannol
pretreatment. Neutrophils, preincubated in the absence ( ) and
presence (+) of piceatannol (30 µM), were stimulated with
TNF
(10 ng/ml for 5 min), and anti-MEKK1 immunoprecipitates were
exposed to coupled in vitro kinase assay (as described under
"Experimental Procedures"). Samples were subjected to SDS-PAGE and
transferred to nitrocellulose. Phosphorylated substrates were
visualized by autoradiography (B, buffer and T,
TNF
). B, JNK activity following pretreatment with
piceatannol. Neutrophils, pretreated with 0.1% Me2SO
(control) or piceatannol (3, 10, 30, and 100 µM) for 55 min, were stimulated in the absence (
) or presence of TNF
(
),
10 ng/ml, for 15 min. JNK1 immunoprecipitates were subjected to
in vitro kinase assay using c-Jun-(1-79) as substrate, and
samples separated by SDS-PAGE were transferred to nitrocellulose.
Phosphorylated c-Jun-(1-79) was quantified by PhosphorImager analysis.
Graph represents mean c-Jun phosphorylation ± S.E. of three
consecutive experiments.
-induced JNK signaling in PC12 cells
(31), whereas
2 integrin-mediated Pyk2 phosphorylation in adherent neutrophils, exposed to TNF
(23) has been also shown.
Furthermore, in the latter system, Pyk2 has been shown to lie
downstream of Syk and Lyn (30). Hence, we questioned whether Pyk2 was
phosphorylated under our experimental conditions and, if so, was this
linked to the JNK activation we had observed earlier. Enhanced tyrosine
phosphorylation of Pyk2 was detected in Pyk2 immunoprecipitates from
TNF
-stimulated neutrophils compared with the unstimulated controls
(Fig. 9A). Tyrphostin A9, a
tyrosine kinase inhibitor, has been shown to inhibit specifically Pyk2 phosphorylation in TNF
-stimulated neutrophils (23). Adherent neutrophils, pretreated with tyrphostin A9 and stimulated with TNF
,
exhibited reduction in JNK activation compared with nonpretreated controls (Fig. 9B).
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Fig. 9.
TNF induced Pyk2 activation
and association with the JNK pathway. A, Western blot
analysis of tyrosine-phosphorylated Pyk2. Neutrophils, stimulated
with either buffer (B) or TNF
(T), 10 ng/ml,
for for 5 and 15 min were immunoprecipitated (IP) with
anti-Pyk2. Samples, separated by SDS-PAGE, were transferred to
nitrocellulose and membranes probed with anti-phosphotyrosine.
B, autoradiography showing dose response of tyrphostin A9
inhibition on JNK activation. Neutrophils, pretreated with piceatannol
(10 µM), tyrphostin A9 (1 µM, 5 µM), or solvent control (0.1% Me2SO) were
either stimulated with either buffer (B) or TNF
(T), 10 ng/ml, for 15 min. A JNK in vitro kinase
assay was performed on JNK1 immunoprecipitates, and the phosphorylation
of a c-Jun-(1-79) fragment substrate was visualized by PhosphorImager
analysis.
induced Apoptosis in Adherent Neutrophils--
Exposure to
TNF
induces apoptosis in many cells by mechanisms that, while
controversial, may involve the JNK pathway. Annexin V binding and
caspase-3 cleavage were used to examine the onset of apoptosis in human
neutrophils. Annexin V binding to neutrophils, studied at 1, 1.5, and
2 h post-TNF
stimulation was significantly increased at 1.5 and
2 h post-TNF
(Fig.
10A). Pretreatment of neutrophils with anti-CD11b F(ab')2 fragments prior to
TNF
stimulation attenuated this increase that was significant at
2 h post-TNF
. The onset of nuclear condensation, as determined
from cytospin preparations, and the susceptibility of this process to
anti-CD11b fragments were similar to annexin V binding (data not
shown).
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Fig. 10.
TNF induced apoptosis.
A, effect of anti-CD11b on TNF
-induced annexin V binding.
Unstimulated neutrophils (
) or neutrophils exposed to TNF
(10 ng/ml, for 1, 1.5, and 2 h) in the absence (
) and presence
(
) of anti-CD11b F(ab')2 fragments (2 µg/ml) were
incubated with a mixture of annexin V fluorescein and propidium iodide,
and their fluorescence was analyzed by flow cytometry. (10,000 cells
were analyzed, and the percent of cells binding annexin V was
determined from the propidium iodide-negative staining population.)
Graph represents mean annexin V binding ± S.E. of three
experiments. Statistical analysis was carried out by two-way ANOVA. *
designates comparison of (i) TNF
/buffer with
p < 0.0136 at 1.5 h and p < 0.0001 for 2 h and (ii) TNF
-CD11b/buffer with p < 0.002. ** represents (p < 0.01) for samples
stimulated with TNF
for 2 h in the presence and absence of
anti-CD11b F(ab')2 fragments. B, TNF
induces
caspase-3 cleavage in neutrophils. Anti-caspase-3 immunoblot shows the
appearance of the 17-kDa cleavage product with time. Triton-soluble
lysates were prepared from neutrophils stimulated with either buffer
(B) or TNF
(T) 10 ng/ml for 0, 60, 90, and 120 min. Samples were separated by SDS-PAGE, transferred to nitrocellulose,
and probed with anti-caspase-3. The 32-kDa proenzyme is cleaved into a
heterodimer of 17-21 and 12 kDa. In neutrophils we have only observed
the 17-kDa species. C, immunoblot showing the effect of
tyrphostin A9 on caspase-3 cleavage. Neutrophils were preincubated in
the absence (
) or presence of 5 µM tyrphostin A9 (+)
prior to stimulation with buffer (B) or TNF
(T), 10 ng/ml, for 1, 1.5, and 2 h. Triton-soluble
lysates were prepared, and samples separated by SDS-PAGE were
transferred to nitrocellulose and probed with anti-caspase-3 by Western
blotting.
(Fig. 10B). When
neutrophils were preincubated with tyrphostin A9 (the Pyk2-associated
tyrosine kinase inhibitor shown in Fig. 9B to inhibit JNK
activation) and then stimulated with TNF
, the cleavage of caspase-3
was greatly reduced (Fig. 10C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
under adherent conditions. Our data
support the conclusion that integration of two signals, one from
integrin-mediated adherence and the other through the TNF receptor(s),
regulates a pathway in which activation of tyrosine kinases, Syk and
Pyk2, is linked to activation of a MAPK module consisting of MEKK1,
MKK4/7, JNK1, and associated with cleavage of caspases-3 and
externalization of phosphatidylserine (Fig.
11). We propose that induction of
apoptosis, a critical mechanism for maintaining a self-limiting
inflammatory response, may be associated with activation of these
elements. This pathway not only constitutes the first report of
activation and utilization of a JNK pathway in human neutrophils but
joins a very limited group of studies on apoptosis induced through
stimulation of the JNK pathway.
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Fig. 11.
Proposed scheme for activation of JNK
pathway in TNF -stimulated adherent
neutrophils. TNF
-stimulated activation of JNK1 proceeds through
TNFR and
2 integrin-mediated activation of the Syk and
Pyk2 tyrosine kinases, MEKK1 and MKK4/MKK7. Functional outcomes of JNK1
activation may be associated with induction of apoptosis.
JNK activation in response to TNF exposure has been reported for a
variety of cell types (65). Ten different human JNK isoforms, found as
either 46- or 55-kDa proteins, have been described, arising from the
alternative splicing of the three genes jnk1, jnk2, and
jnk3. We detected by Western blot analysis both the 46- and
55-kDa species, although the predominant species was a 55-kDa protein.
This p55 splice variant we detected is probably JNK1
2 or
JNK1
2 (65, 66). Evidence of TNF
-induced neutrophil JNK activation comes both from observations that immunoprecipitated, endogenous JNK is itself specifically phosphorylated following TNF
stimulation and that it phosphorylates a c-Jun fragment substrate.
The MAP kinases kinases reported to be directly upstream of JNK include
MKK4 and the recently cloned MKK7 (67). Overexpression of MKK4 in a
variety of cell types leads to activation of JNK(s) (46, 68), and its
targeted deletion blocks the activation of JNK in embryonic fibroblasts
(69). Recent studies, however, suggest that MKK7 may be the preferred
MKK leading to JNK activation following TNF exposure (48). In our
studies, both MKK4 and MKK7 were activated in a
stimulus-dependent manner when neutrophils were exposed to
TNF
, indicating the likelihood that both MKK4 and MKK7 are involved
in JNK1 activation in the neutrophil. Our data, while suggesting that
MKK7 may be a more potent activator of JNK1 than MKK4 in this system,
do not preclude the possibility that immunoprecipitation may not allow
detection of the full extent of activation. In keeping with the
upstream location of MKK4 and MKK7, their peak activation (5 min)
precedes that of JNK1 (10-15 min). Similar time courses of activation
for JNK and MKK4 have been reported in macrophages following TNF
exposure (52).
MAP kinase kinase kinases shown to act upstream of JNK include MEKK
family members (49, 50, 70, 71), MLK3 (72), ASK-1 (51), and NIK (73).
We have focused on MEKK1, which phosphorylates and activates MKK4
in vitro (49). Overexpression of MEKK1 in a variety of cell
types results in activation of both MKK4 and JNK(s) (49, 74) and also
leads to apoptosis in fibroblasts (75). In this study, we demonstrate,
with the use of recombinant constituents of the JNK pathway, MEKK1
activation in adherent neutrophils following exposure to TNF. In
light of our previous findings that
formyl-methionyl-leucyl-phenylalanine induces MEKK1-associated p42/44
ERK MAPk activation (conditions in which MKK4 is not activated (40)),
we speculate that the neutrophil may respond to specific stimuli by
using the same upstream kinase but allowing different downstream
effectors to be activated. The yeast accomplishes such specificity
through the use of scaffolding proteins (76). Additionally, or
alternatively, other MEKK family members may also activate MKK4 and
MKK7 in response to TNF
.
Signaling events upstream of MEKK remain to be elucidated, although the
pleiotropic actions of TNF (7) are known to be mediated by two TNF
receptors, RI and RII, that belong to a superfamily of cell surface
receptors including Fas and CD40 (77). Although most of the biological
effects of TNF
are ascribed to TNFRI (78, 79), neutrophils express
both types.2 Binding of
TNF
induces receptor aggregation and recruitment of TNFR-associated
proteins (80-82), including TRAF2 and RIP, thought to be points of
divergence that lead to many downstream events, including activation of
the JNK pathway and of the transcription factor NF-
B (83).
In spite of the many activities linked to activation of TNFRI and -II,
we demonstrate that an additional component, neutrophil adherence, is
necessary for JNK1 activation in TNF-stimulated neutrophils.
Although neutrophils utilize a variety of adhesion molecules,
2 integrins are fundamentally important in both
homotypic and cell-substratum adhesive events, and neutrophil
interactions with the extracellular matrix have been shown to require
integrins (16). In our studies, both the JNK and MEKK1 activation in
TNF
-stimulated adherent neutrophils could be blocked by pretreatment
of cells with an antibody to CD11b, the
subunit of the
2 integrin, Mac-1 (CD11b/CD18, aM
2).
Although JNK activation following integrin activation alone has been
reported (84), we found that in neutrophils adherence in the absence of
TNF
stimulation was insufficient to initiate activation of JNK1. By
using an isotype control antibody, we demonstrated that the anti-Cd11b
blocking was indeed specific. Additionally, we present evidence that
anti-CD11b does not block the TNFR itself by demonstrating that
TNF
-induced actin assembly in neutrophils is an integrin-independent
function (Fig. 2C). Initial actin assembly in the neutrophil
in response to variety of agonists occurs in suspension and is
independent of
2 integrin blockade. Hence, this provides
a system in which integrin-independent signaling events downstream of
the TNFR and its expression can be assessed.
Neutrophil interactions with the extracellular matrix have been shown
to induce phosphorylation of specific cytoplasmic tyrosine kinases,
including the Src family member p58Fgr (19-22).
Fuortes and colleagues (20) have demonstrated that TNF activation of
adherent neutrophils resulted in tyrosine phosphorylation of several
protein species that were not seen in suspended cells. Our studies show
that the tyrosine kinase inhibitor, genistein, attenuated
TNF
-induced JNK activation, providing initial evidence for upstream
participation of tyrosine kinases. A recent study in T-cells reports
that activation through the T-cell receptor, if associated with
ligation of a co-receptor, results in activation of Syk and downstream
activation of the JNK pathway (85), whereas Syk has also been reported
to be associated with integrins (57, 58). We have not only demonstrated
Syk activation, which could be inhibited by anti-CD11b antibodies, but
also inhibition of both Syk and JNK activation by piceatannol, the
Syk-specific inhibitor, thus linking Syk and JNK activation in
neutrophil. Although we were unable to ascertain from our present
studies clear involvement of the Src family member, Lyn, we feel that
at present we cannot discount its contribution.
Recent studies, however, have shown that another tyrosine kinase, the
FAK family Pyk2, localizes to podosomes and focal adhesion-type structures in TNF-stimulated adherent neutrophils (23).
TNF
-induced Pyk2 phosphorylation in neutrophils is decreased in the
presence of
2 integrin blocking antibodies (30) and Pyk2
integrates signals from integrins and TNFR (23). In B-cells,
aggregation of
1 integrins (86) and in T-cells,
3 integrins (87) results in the phosphorylation of Pyk2.
Our studies in adherent neutrophils show both phosphorylation of
immunoprecipitated Pyk2 following TNF
stimulation and inhibition of
JNK activation by the tyrphostin A9, shown to inhibit specifically Pyk2
(23). These observations provide preliminary evidence that Pyk2 may be
associated with JNK activation in the neutrophil and are in agreement
with studies in which Pyk2 overexpression in 293 cells leads to
activation of co-expressed JNK, whereas dominant-negative Pyk2 inhibits
JNK in PC12 (31). Pyk2 has been linked to JNK via p130Cas
and p130Crk (87, 88). Whereas in THP1 cells the PI3-kinase
pathway is associated with Pyk2 (89), it is not necessary for Pyk2
activation in neutrophils (30). This observation agrees with our
findings that wortmannin does not affect TNF
-induced JNK activation
in neutrophils. Our studies on Syk and Pyk2, together with our CD11b blocking studies in TNF
-induced JNK activation in the neutrophil, further strengthen our hypothesis that the JNK pathway may require two
interacting signals, one of which involves
2 integrins
whose actions are mediated in part by activation of tyrosine kinases. However, the TNFR- and integrin-mediated signals may not necessarily be
independent, since they may reflect the action by TNF
to modify integrin function (19) as demonstrated by both the capability of TNF
to induce up-regulation of CD18/CD11b (Fig. 3C) and
stimulate
2 integrin-mediated adherence in neutrophils
(90). Since both antibodies to
2 integrins and
piceatannol inhibited MEKK1 activation (Fig. 5 and Fig. 8), these data
indicate that protein tyrosine kinases and
2 integrins
effects impinge on the JNK pathway upstream of MEKK1.
In the neutrophil, as in many other cells, TNF enhances apoptosis.
We have shown herein that in adherent neutrophils TNF
induces
apoptosis with a rapid time course comparable to that induced by UV
exposure (91). Whereas recent studies invoke a role for JNK as a
co-participant of apoptosis in response to ionizing radiation, UV,
TNF
(38), and trophic factor deprivation (92), the role of JNK in
apoptosis may be stimulus- and cell-specific. Certain studies suggest
the possibility that JNK lies upstream of caspase-3 (93). Our studies
on caspase-3 cleavage and annexin V binding demonstrated the induction
of apoptosis in TNF
-stimulated adherent neutrophils.
Walzog and colleagues (37) have previously suggested that integrin
involvement is important in TNF-induced apoptosis and, furthermore,
that tyrosine kinase inhibitors modulate the induction of apoptosis.
Similarly, it has been demonstrated that wound macrophages induce
neutrophil apoptosis through a mechanism requiring both TNF
and
integrin-ligand interactions (94). Of considerable interest, Coxon and
colleagues (95) have reported that CD11b
/
mice exhibit an increase
in elicited neutrophils, ascribed, in part, to a failure of apoptosis.
Similarly, we found that preincubation of neutrophils with
F(ab)'2 fragments of anti-CD11b partially inhibited annexin
V binding. Furthermore, a recent report that overexpression of Pyk2
leads to apoptosis in rat and mouse fibroblasts (27) supports our
studies in which caspase-3 cleavage is both attenuated and delayed
following preincubation of neutrophils with tyrphostin A9. These
effects on caspase-3 cleavage were induced by the same concentrations
of tyrphostin A9 that inhibited JNK activation.
While TNF alone is a potent inducer of apoptosis in transformed
cells, an additional insult is required to induce apoptosis in normal
cells (e.g. cycloheximide). We propose that in human neutrophils, integrin involvement is required to induce apoptosis in
the presence of TNF
. Hence, unlike many cells, in which
integrin-mediated detachment from substratum leads to apoptosis
(anoikis), we have found, in contrast, that the neutrophil requires
integrin-mediated attachment for apoptosis to occur. As we have
previously reported, UV-induced apoptosis of neutrophils involves the
MAP kinase family member p38 (91). Thus, neutrophil apoptosis may
involve different MAP kinase family members for apoptotic responses
under diverse conditions.
The acute inflammatory response is characterized both by large numbers
of adherent neutrophils and the presence of TNF. It is precisely
these conditions, as we have demonstrated here, that allow the
neutrophil to utilize the JNK pathway. The consequences of such
activation including apoptosis may act to down-regulate the
intensity of the inflammatory response. Although further studies, including investigations using genetically manipulated mice, are required to determine the relationship between the JNK pathway and
apoptosis, this complex activation sequence provides a mechanism for
enabling the neutrophil to both acquire specific functional capabilities and to ensure they are only used transiently.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. D. W. Riches, E. D. Chan, S. C. Frasch, P. Gerwins, and K. C. Malcolm for helpful discussions and Dr. D. L. Bratton and D. A. Richter for advice on the annexin V binding assay. We also thank Leigh Landskroner and Barry Silverstein for assistance with illustrations and Brenda Sebern for assistance with the manuscript.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§§ Supported by National Institutes of Health Grants HL40784, HL61407, and HL13403. To whom correspondence should be addressed. Tel.: 303-398-1171; Fax: 303-398-1851; E-mail:worthens@njc.org.
Published, JBC Papers in Press, October 25, 2000, DOI 10.1074/jbc.M007527200
2 G. S. Worthen, N. J. Avdi, M. M. K. Gillespie, and P. E. Parsons, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor
necrosis factor-
;
MAP, mitogen-activated protein;
MAPk, MAP kinase;
ERK, extracellular signal-regulated kinase;
pNPP, p-nitrophenyl phosphate;
MKK, MAPk/ERK kinase (MEK);
MEKK, MEK kinase (MAPk kinase kinase);
JNK, c-Jun N-terminal kinase;
JNKwt, recombinant wild type JNK1;
JNKkm, recombinant kinase-inactive JNK1;
SEK, SAPK/ERK kinase;
PI3-kinase, phosphatidylinositol 3-kinase;
PAF, platelet-activating factor;
Pyk2, Proline-rich tyrosine kinase 2;
TNFR, TNF receptor;
PMSF, phenylmethylsulfonyl fluoride;
PAGE, polyacrylamide gel
electrophoresis;
Pipes, 1,4-piperazinediethanesulfonic acid;
ANOVA, analysis of variance;
NBD, 12-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)).
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REFERENCES |
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