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INTRODUCTION |
Detection of microbial products such as lipopolysaccharide
(LPS)1 and
N-formyl peptides (fMLF) by neutrophils is an essential
event that triggers the microbicidal functions of these cells.
Activation of neutrophils by such stimuli results in chemotaxis to
inflammatory sites, cytokine production, release of degradative granule
enzymes, and generation of toxic oxygen intermediates.
The MAP kinases are members of discrete signaling cascades that form
focal points for diverse extracellular stimuli and function to regulate
fundamental cellular processes. Three distinct classes within the MAP
kinase family have been described, including ERK, c-jun-NH2 kinase, and p38, each having different
physiological roles (1-4). The p38 MAP kinase is associated with
immune cell activation, because this kinase is activated by a variety
of inflammatory mediators (5, 6). It is well established that p38 has
an important role in gene expression in monocytes and macrophages (7-9). A specific function for p38 in neutrophils remains unclear, but
it has been suggested to play an important role in the respiratory burst, interleukin 8 production, and apoptosis (10-13). Activation of
p38 occurs after phosphorylation of a distinctive TGY motif by the
upstream kinases MEK3 or MEK6 (6, 14-16). Stimulation of human
neutrophils with classical chemoattractants, including fMLF, results in
a rapid and transient phosphorylation and activation of p38 (12, 13,
17-20). It has also been demonstrated that challenge of neutrophils
with LPS leads to activation of p38 but with slower kinetics than
G-protein-coupled chemoattractant receptors (17, 19).
The molecular mechanism(s) leading to activation of ERK have been
largely unraveled, but the regulation of components upstream of p38
remain to be clarified. Although recent reports have examined signaling
downstream of fMLF, little attention has focused on the mechanism for
LPS-mediated activation of p38 in neutrophils (18, 20). In Jurkat
T-cells, long-term exposure to nitric oxide (NO) leads to activation of
several MAP kinases, with ERK being the most responsive (21). LPS
induces NO production in neutrophils, and this process is thought to
mediate many of its inflammatory activities (22-27). Stimulation of
neutrophils with fMLF can also induce NO production, which is thought
to play an important role in chemotaxis and degranulation elicited by
this agent (28-32). The mechanism of action for NO to regulate cell physiology is not clear; however, a key mediator is cGMP, which is
generated by direct activation of soluble guanylyl cyclase.
In this study we examined the role of NO in the activation of p38 MAP
kinase by LPS and by fMLF in human neutrophils. It was found that NO
formation is sufficient to cause phosphorylation of p38 and that its
formation is a prerequisite to p38 activation by LPS but not by fMLF.
Furthermore, it was found that the mechanism of action involves an
increase in intracellular cGMP and possibly activation of protein
kinase G.
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EXPERIMENTAL PROCEDURES |
Reagents--
Inhibitors of the nitric oxide system, including
N-nitro-L-arginine methyl ester,
2-phenyl-tetramethylimidazoline-1-oxyl-3-oxide, and the NO releasing
agents S-nitroso-N-acetylpenicillamine, sodium nitroprusside, and N-acetyl-L-cysteine, the
phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and
4-(methylenedioxy)-benzylamino-6-methoxyquinazoline, RO-20-1724, and
the protein kinase G inhibitor KT5823 were purchased from Calbiochem.
The guanylyl cyclase stimulator YC-1 was from Alexis Biochemicals (San
Diego, CA). Dibutyryl cGMP and fMLF were from Sigma; LPS from
Salmonella Minnesota was purchased from Calbiochem. Rabbit
anti-p38 serum was a generous gift from Dr. J. Han (Scripps Research
Institute). All other antibodies, including those directed against the
phosphorylated forms of MEK3,6, MEK1,2, and p38, were purchased from
New England Biolabs (Beverly, MA) unless otherwise indicated.
Preparation of Neutrophils from Whole Blood--
Neutrophils
were prepared from the blood of healthy donors using Percoll gradient
(Amersham Pharmacia Biotech) as published elsewhere (33). In brief,
coagulation of the blood was prevented using citrate-dextrose during
collection, and the erythrocytes were sedimented by adding 0.5 volume
of 6% heta-starch (Abbott) at room temperature for ~45 min. The
erythrocyte-depleted supernatants were then layered on top of 55%
isotonic Percoll containing a 74% cushion and centrifuged at 12 °C
for 40 min. After collection of the granulocytes from the cushion
interface, they were diluted 4-fold in ice-cold phosphate-buffered
saline and washed twice by centrifugation at 500 × g.
Neutrophils were then resuspended in serum-free RPMI 1640 medium at a
density of 5 × 106 cells/ml and maintained at
37 °C.
Assay for Phosphorylation of p38 and MEK--
In experiments
requiring pretreatment with inhibitors, cells were incubated in
microtubes for 30 min (unless otherwise indicated) rotating at
37 °C. In all cases cells were pelleted and resuspended in 0.25 ml
of the same medium, (in some cases the stimulating agents were added at
this time), and the cells were incubated for various periods in a
37 °C water bath. Incubations were terminated by placing tubes on
ice and diluting rapidly with 1 ml of ice-cold phosphate-buffered
saline. Cells were then pelleted for 5 s in a microcentrifuge at
4 °C, and the supernatant was replaced with SDS-polyacrylamide gel
electrophoresis sample buffer.
Gel Electrophoresis and Western Blotting--
After resuspension
in sample buffer, cell extracts were prepared for electrophoresis by
boiling for 6 min followed by clarification by centrifugation. Equal
amounts of protein (~20 µg in 10 µl) of each extract were then
loaded onto 10% polyacrylamide minigels and separated using 30 mA/gel.
Protein profiles were transferred to supported nitrocellulose
membranes, which were then blocked by incubating in phosphate-buffered
saline containing 0.025% Tween 20 and 4% bovine serum albumin (PTS
buffer) for 20 min at room temperature. In all cases the blots were
probed by incubating without agitation with 1/1000 primary antibody in
PTS at 4 °C overnight. MAP kinases on the blots were then visualized
by rotating for 1 h at room temperature in PTS containing 1/2000
peroxidase-conjugated goat anti-rabbit IgG (PharMingen, La Jolla, CA)
followed by chemiluminesence using enhanced reagents according to the
manufacturer's instructions (Pierce). Between incubation steps blots
were washed in excess PTS buffer three times for 5 min each. To
standardize for protein loading in each lane, blots were stripped by
incubation in a buffer containing 62.5 mM Tris-HCl, pH 6.8, 100 mM 2-mercaptoethanol, and 2% SDS for 30 min at
50 °C. Blots were then washed extensively in PTS, followed by
reprobing with antibody directed against the unphosphorylated form of p38.
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RESULTS |
Role of Nitric Oxide in Formyl Peptide- and
Lipopolysaccharide-mediated Phosphorylation of p38 in Human
Neutrophils--
It has been shown previously that LPS and the
N-formyl peptide fMLF stimulate the phosphorylation and
activation of p38 in human neutrophils (17, 18, 20, 34). MEK3 and MEK6
are upstream kinases that, on activation by phosphorylation, can
recognize p38 as a substrate both in vitro and in
vivo. The presence or activation of either of these kinases has
not previously been demonstrated in neutrophils. As shown here,
challenge with 100 nM fMLF caused a rapid and transient
phosphorylation of MEK3,6 in neutrophils, with a maximum within 1 min
of stimulation and a return to basal levels within 15 min (Fig.
1A). This phosphorylation leads to activation of MEK3,6, as indicated by the equally rapid and
transient appearance of phosphorylated p38. Stimulation of neutrophils
with LPS (10 µg/ml) also resulted in phosphorylation of MEK3,6, with
a concomitant appearance of phospho-p38 (Fig. 1B). In
agreement with previous findings (17), the LPS-mediated phosphorylation
of p38 and MEK3,6 was slower than that of fMLF, increasing after 5 min,
and was maximal between 15 and 30 min after stimulation. Because the
kinetics of MEK3,6 phosphorylation paralleled the appearance of
phospho-p38 for both of these stimuli, it is likely that MEK3,6 is
upstream of p38 in neutrophils as in other systems. In this study we
did not address whether MEK3 or MEK6 (or both) was important in
neutrophils, because our antibodies did not distinguish these closely
related species. The signal detected on Western blots probed with
phospho-MEK3,6 antibodies was very weak when compared with similar
blots probed with phospho-MEK1,2 antibodies (see below), which suggests
that these proteins are less abundant. This point supports previous
findings, which have determined that MEK1 and MEK2 are the major
isoforms found in neutrophils (35-37).

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Fig. 1.
Activation of MEK3,6 by fMLF and LPS in
neutrophils. Freshly isolated neutrophils were prepared as
detailed under "Experimental Procedures" and incubated in the
presence of 100 nM fMLF (A) or 10 µg/ml LPS
(B). At the indicated times cells were harvested, and the
levels of phosphorylated MEK3,6 and p38 were measured using Western
blotting with antibodies directed against the phosphorylated forms of
these proteins. The blots showing MEK3,6 and p38 represent parallel
gels run using the same samples. The lower blot was
generated by reprobing the blot above with antiserum specific for the
unphosphorylated form of p38 and reflects the protein loading of each
lane. Gels shown reflect similar patterns obtained from at least three
independent experiments.
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Both LPS and fMLF are able to induce NO formation in neutrophils, and
this function contributes to the biological activity of these agents.
Furthermore, in Jurkat cells NO can activate several MAP kinase
pathways (21). To examine the relevance of NO formation in the
phosphorylation of p38, neutrophils were treated with either the NO
scavenger 2-phenyl-tetramethylimidazoline-1-oxyl-3-oxide or the
NO-synthase inhibitor N-nitro-L-arginine methyl
ester before stimulation with fMLF and LPS. The increase in phospho-p38
levels measured 20 min after stimulation with LPS was
dose-dependently inhibited by both
2-phenyl-tetramethylimidazoline-1-oxyl-3-oxide and
N-nitro-L-arginine methyl ester (Fig.
2, A and B). In
contrast, neither of these drugs was capable of reducing fMLF-induced
phosphorylation of p38 even at doses 100-fold over the published
EC50 (Fig. 2, C and D). These data
support a physiological role for NO formation in the activation of p38
in neutrophils by LPS but not by fMLF.

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Fig. 2.
Nitric oxide formation is necessary for
activation of p38 by LPS. Neutrophils were preincubated with
different doses of the nitric oxide scavenger
2-phenyl-tetramethylimidazoline-1-oxyl-3-oxide (A and
C) or the inhibitor of nitric-oxide synthase
N-nitro-L-arginine methyl ester (B
and D) for 30 min. Cells were then stimulated with either 10 µg/ml LPS (A and B) for 20 min or 100 nM fMLF for 1 min (C and D), and the
cells were harvested. The levels of phosphorylated p38 were then
determined using Western blotting with phospho-specific antisera as
detailed under "Experimental Procedures." The lower
panel in each case shows the same blot reprobed for total p38 as
an indicator of protein loading. Data shown were reproduced using three
different donors with similar results.
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Because NO production appeared to have a role in the activation of p38
MAP kinase, it was of interest to determine whether NO was sufficient
to stimulate this pathway. Although several NO-releasing compounds are
commercially available, some of these are problematic in neutrophils
(38). We chose to use agents that have been shown previously to be
effective in neutrophils, including
S-nitroso-N-acetylpenicillamine, and a
combination of sodium nitroprusside (an NO releasing agent) and
N-acetyl-L-cysteine (increases availability of
NO) (26, 27). Under the conditions used here, incubation of neutrophils
in medium without NO-releasing agents for periods of up to 30 min did
not result in significant levels of phospho-p38 (Fig.
3A). However, in the presence
of the NO-releasing agents
S-nitroso-N-acetylpenicillamine and sodium nitroprusside/N-acetyl-L-cysteine, there was a
notable increase in the phosphorylation of p38 (Fig. 3, B
and C). In both cases the increase in phospho-p38 was
detectable within 20 min and increased in amount throughout the period
investigated.

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Fig. 3.
Nitric oxide formation is sufficient for p38
phosphorylation in neutrophils. Freshly isolated neutrophils were
either left untreated (A) or incubated in the presence of
the nitric oxide releasing compounds sodium nitroprusside
(SNP1) and N-acetyl-L-cysteine
(SNP1+NAC; B) or
S-nitroso-N-acetylpenicillamine (SNAP;
C). At the indicated times cells were harvested, and the levels of
phosphorylated p38 were determined by Western blotting as detailed
under "Experimental Procedures." The same blots were stripped and
reprobed with antibodies against unphosphorylated p38 as a measure of
protein loading. Results shown are representative of experiments from
three different donors.
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Exogenous Elevation of Intracellular cGMP Levels Results in
Activation of the Pathway Leading to Phosphorylation of p38--
The
biological effects of NO formation are generally assumed to be
attributable to the activation of soluble guanylyl cyclase, leading to
cGMP accumulation (39-41). If this mechanism underlies the NO-mediated
activation of p38, as measured here, then it follows that cGMP should
also play an important role. To examine this question we used several
different methods to increase the intracellular concentration of this
messenger. Initial studies used phosphodiesterase (PDE) inhibitors,
which presumably lead to an increase in basal cGMP levels by preventing
cyclic nucleotide degradation. Treatment of neutrophils with the
broad-range PDE inhibitor IBMX resulted in an increase in
phospho-MEK3,6 and phospho-p38 after 1 min of incubation in the drug
with maximal levels at 15 min (Fig.
4A). Although IBMX has been
reported to have agonist activity to adenosine receptors, the
phosphorylation of p38 by this drug was not affected by other adenosine
agonists (data not shown). There are several PDEs known to be expressed
in different mammalian tissues. In neutrophils the expression of both
the cAMP-specific (PDE-IV) and the cGMP-specific (PDE-V) isoforms have
been demonstrated (42-44). Treatment of neutrophils with RO-20-1724, a
specific inhibitor of the cAMP-specific PDE-IV, was unable to increase
the level of phosphorylated MEK3,6 in similar experiments (Fig.
4B). In contrast, it was found that the specific inhibitor
of PDE-V (4-(methylenedioxy)-benzylamino-6-methoxyquinazoline) was
equal to IBMX in its ability to increase levels of the phosphorylated forms of both MEK3,6 and p38 in neutrophils, which indicated an important role for cGMP in this phenomenon (Fig. 4C).

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Fig. 4.
Inhibition of cGMP-phosphodiesterase leads to
activation of MEK3,6 in neutrophils. Freshly isolated neutrophils
were incubated with the nonspecific phosphodiesterase inhibitor IBMX
(A), with the cAMP-specific inhibitor RO-20-1724 (RO2;
B), or with the cGMP-specific phosphodiesterase inhibitor
4-(methylenedioxy)-benzylamino-6-methoxyquinazoline (MBQ; C)
for up to 15 min at 37 °C. At the indicated times cells were
harvested, and the phosphorylated forms of MEK3,6 and p38 were measured
by Western blotting as detailed under "Experimental Procedures."
The lower panel reflects the protein-loading pattern of the
above gels as determined by staining for total p38. Results shown are
representative of at least three donors.
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To lend support to the idea that cGMP but not cAMP is important to the
activation of p38 in neutrophils, neutrophils were treated with
activators of soluble guanylyl cyclase or of adenylyl cyclase.
Activation of guanylyl cyclase by adding YC-1 to neutrophils resulted
in a rapid phosphorylation of both p38 and MEK3,6, and this increased
level remained stable within the 15-min period investigated (Fig.
5A). In contrast, increasing
the intracellular cAMP concentration by treatment with forskolin did
not change the levels of the phosphorylated forms of either p38 or
MEK3,6 (Fig. 5B). These studies demonstrate that an
elevation in the intracellular concentration specifically of cGMP is
sufficient to cause phosphorylation of p38 in neutrophils.

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Fig. 5.
Increased cGMP levels lead to phosphorylation
of p38 in neutrophils. The intracellular cyclic nucleotide levels
were increased by incubating freshly isolated neutrophils in the
activator of soluble guanylyl cyclase YC-1 (A) or the
activator of adenylyl cyclase forskolin. At the indicated times cells
were harvested for analysis of phospho-p38 content using Western
blotting as detailed under "Experimental Procedures." The
lower panel shows the same gel reprobed with an
antibody directed at the unphosphorylated form of p38. Gels shown were
reproduced with similar results in three independent experiments.
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Increasing the intracellular concentration of cGMP by treating cells
with increasing doses of the membrane-permeable analog dibutyryl cGMP
resulted in phosphorylation of both MEK3,6 and p38 (Fig.
6A). In these studies cGMP was
effective at concentrations as low as 250 nM, and at 250 mM the resulting phosphorylation of p38 and MEK3,6 was
equal to or greater (respectively) than levels produced by fMLF
stimulation of the cells. The similarity in the phosphorylation of
MEK3,6 and p38, both in terms of the effect of PDE inhibition and the
dose response for dibutyryl cGMP, further supports the role of MEK3,6
in the regulation of p38 activity in neutrophils. In addition, these
data point to cGMP-sensitive regulation of components upstream of
MEK3,6 in neutrophils. Treatment of freshly isolated neutrophils with
250 mM dibutyryl cGMP caused a rapid and transient
phosphorylation of MEK3 and p38, with maximal levels detected within 1 min after addition of this reagent to the cells. Using antibodies
specific to the phosphorylated domain of other MEK isoforms, we found
that the effect of cGMP was specific to MEK3,6, because neither
MEK1,2 (Fig. 6) nor MEK4 (not shown) was affected by dibutyryl
cGMP.

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Fig. 6.
The cGMP effect is specific to
phosphorylation of MEK3,6. The intracellular levels of cGMP were
increased by treating neutrophils with different concentrations of
dibutyryl cGMP (as indicated). After 1 min the cells were analyzed for
phospho-MEK3,6 and phospho-p38 levels by Western blotting
(A). The ability of dibutyryl cGMP to induce phosphorylation
specifically of MEK3,6 was assessed by adding a 250 mM
concentration of the nucleotide to freshly isolated neutrophils
(B). At the indicated times, cells were harvested, and equal
amounts of protein were loaded onto acrylamide gels for analysis by
Western blotting with phospho-specific MEK3,6 (upper panel)
or phospho-specific MEK1,2 (lower panel). Neutrophils
treated with 100 nM fMLF for 1 min are shown as a positive
control and are marked F. Data shown are representative of
at least two independent experiments.
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Importance of Protein Kinase G in the Pathway Leading to p38
Phosphorylation in Neutrophils--
The translation of elevated
intracellular cGMP levels into a physiological response is often
mediated by activation of protein kinase G (41). To determine whether
activation of PKG is important to the phosphorylation of p38,
neutrophils were treated with a specific inhibitor of this kinase
before stimulation with LPS and fMLF (Fig.
7). It was found that this inhibitor
(KT5823) could dose-dependently block the accumulation of
phospho-p38 when stimulated with LPS for 20 min. Furthermore, maximal
doses reduced the phospho-p38 in response to LPS close to basal levels.
The increases in phospho-p38 levels in neutrophils stimulated with fMLF
for 1 min were only slightly reduced by blocking PKG activation even at
maximal doses of this inhibitor (Fig. 7B). This finding
supports the observation that NO formation and elevation of cGMP levels
do not contribute significantly to the activation of p38 by fMLF.
Although the previous data favor a role for cGMP rather than cAMP in
the activation of p38 downstream of LPS, higher concentrations of this
nucleotide might stimulate the cAMP-dependent protein
kinase in addition to PKG. To test this hypothesis, the inhibitor of
cAMP-dependent protein kinase (H89) was compared with the
PKG inhibitor KT5823 with respect to its ability to block
LPS-stimulated phosphorylation of p38 (Fig. 7C). In these
experiments inhibition of PKG using KT5823 at 100 times the reported
EC50 completely blocked the increase in p38 phosphorylation
by LPS. In contrast, blockade of cAMP-dependent protein
kinase function with similar concentrations of H89 were ineffective.

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Fig. 7.
Involvement of protein kinase G in LPS- but
not fMLF-induced phosphorylation of p38 in neutrophils. Freshly
isolated neutrophils were incubated for 30 min in the presence of
different concentrations of the protein kinase G inhibitor KT5823 (as
indicated). Cells were then stimulated for 20 min with 10 µg/ml LPS
(A) or for 1 min with 100 nM fMLF
(B). Cells were then harvested and analyzed for the
phosphorylation of p38 as described under "Experimental
Procedures." C, neutrophils were preincubated with 2,5 µM KT5823 or the protein kinase A inhibitor H89 (5 µM) for 30 min before stimulation with 10 µg/ml LPS for
20 min. The effectiveness of each inhibitor was then determined by
Western blotting as above. The lower panel shows the same
blot reprobed for total p38 content as a measure of equal loading. The
gel shown is representative of two independent experiments.
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DISCUSSION |
Stimulation of neutrophils with LPS or fMLF leads to activation of
several MAP kinase pathways, although only MEK1,2 has been identified
in these cells. In this study it was demonstrated that both LPS and
fMLF were able to transiently activate MEK3,6 kinase activity in
neutrophils. Both fMLF and LPS cause endogenous nitric oxide formation
in neutrophils. Recent work by Lander and colleagues (21) has
demonstrated activation of ERK,
c-jun-NH2-kinase, and p38 kinase activities
after stimulation of Jurkat T-cells with NO. As shown here in human
neutrophils, inhibition of the NO pathway was able to inhibit
phosphorylation of p38 downstream of LPS but not fMLF. This result
indicates that NO functions specifically downstream of LPS in p38 MAP
kinase activation, and that fMLF must use different signaling pathways
to achieve the same end. The different kinetics of p38 phosphorylation
with fMLF reaching a maximum at 1-2 min, whereas LPS activated p38
only after 10-20 min, might reflect these underlying differences in
signaling mechanism. Other work by Lander et al. (45-47)
has shown that p21ras is a target for NO-related free radicals,
which might explain the activation of ERK in this system. It is not
clear from their studies how NO might stimulate p38 MAP kinase activity
in their system, or which stimuli might use this pathway to activate
MAP kinases. Because activation of soluble guanylyl cyclase by NO is
well documented, in this report we examined the role of cGMP in the
signaling pathway leading to activation of p38 in neutrophils. It was
found that phosphorylation of MEK3,6 was induced by increasing intracellular cGMP levels in several different ways: by inhibition of
PDEs, by ligand-independent activation of soluble guanylyl cyclase, or
by addition of dibutyryl cGMP. The increase in phospho-MEK3,6 levels
initiated by each of these treatments corresponded with activation of
MEK3,6 kinase activity, as measured by the in vivo phosphorylation of its physiological substrate p38. Because both LPS
and fMLF stimulation of neutrophils leads to the phosphorylation of
p38, it would follow that signals generated by each ligand must
converge at some point upstream of p38. Results shown here suggest that
this point of convergence is likely to be at, or upstream of, MEK3,6
activation. In contrast to the previous studies using Jurkat cells,
results shown here demonstrate that the MEK3,6/p38 pathway is
specifically phosphorylated downstream of the NO system and that
activation of the MEK1,2/ERK pathway or the
MEK4/c-jun-NH2-kinase pathway was not affected.
This result suggests that NO-related species might signal to the MAP
kinases using more than one mechanism and indicates cell type-specific
differences in the responsive signaling pathways. To extend these
findings, we show that the cGMP-dependent phosphorylation
of p38 in neutrophils requires activation of PKG. The lack of effect of
cAMP-elevating agents and of the inhibitor of
cAMP-dependent protein kinase suggests that this pathway
does not play an important role in the activation of p38 by LPS in this
system. It is not known from our studies where in the p38 signaling
pathway PKG is likely to function, because many proteins have been
reported to have a role upstream of MEK3,6. Because the number of
substrates for PKG that have been characterized in any system is few,
and those that might contribute to activation of MAP kinase pathways
have not been explored, this remains a question for future endeavors.
LPS and fMLF use fundamentally different signaling mechanisms; the
former is incompletely understood, whereas that latter uses activation
of G-proteins. The differential effect of NO inhibitors on fMLF- and
LPS-stimulated p38 phosphorylation reveals that there are different
biochemical pathways capable of activating p38 phosphorylation in
neutrophils. It remains to be determined whether other stimuli such as
tumor necrosis factor
or interleukin 1
, which activate p38 with
similar kinetics to LPS as measured here, also use NO generation as a
mediator of MAP kinase activation. The mechanism used by fMLF to
activate p38 in neutrophils requires both phosphatidylinositol 3-kinase
and calcium-dependent activation of protein kinase C (18,
20). Other work has shown that fMLF causes PKG to co-localize with
intermediate filaments and to phosphorylate vimentin in a calcium-dependent manner, and speculation has suggested
that in this context, PKG is involved in cytoskeletal rearrangement
associated with the degranulation process (28, 40, 48, 49). However, results shown here would suggest that PKG does not contribute to the
phosphorylation of p38 in response to stimulation with fMLF.
Previous work has shown that NO can contribute to cytokine gene
expression in neutrophils (26, 50). Nitric oxide has also been
associated with the regulation of gene expression in endothelial cells
(51-54). Activation of p38 has also been associated with transcription
factor activation (1, 8). Recent work has demonstrated that in
LPS-stimulated neutrophils and monocytes, p38 is involved in cytokine
production (9, 55). Thus it is likely that the activation of p38 MAP
kinase by NO contributes to the inflammatory response in these cells
through regulation of cytokine gene expression. It has been shown that
p38 activation is important to the up-regulation of iNOS in glial cells
and astrocytes (52, 56). Although this has not been demonstrated for
myeloid cells, it is an intriguing possibility that longer-term
activation of p38 might create an autonomous regulatory loop where the
increased NO would help maintain or augment cytokine synthesis in these cells.
In summary, we have investigated the role of NO production on the
activation of the p38 MAP kinase pathway in human neutrophils. It was
found that exogenous NO caused phosphorylation of p38 in these cells.
In addition, using inhibitors of NO synthesis it was shown that LPS but
not fMLF used this pathway to activate p38. Preliminary investigations
of the mechanism of action for NO indicated that cGMP accumulation was
sufficient to cause phosphorylation of p38 and its upstream regulatory
kinase MEK3,6 but not MEK1,2 and MEK4, which are associated with ERK
and c-jun-NH2-kinase MAP kinase pathways,
respectively. Inhibition of protein kinase G attenuated LPS- but not
fMLF-induced phosphorylation of p38, suggesting that it might function
downstream of NO formation but upstream of MEK3,6 in this pathway.