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From the
In human polymorphonuclear leukocytes (PMNs), mitogen-activated
protein kinases (MAPKs), also known as extracellular signal-regulated
kinases (Erks), are activated within minutes upon stimulation with
either chemoattractant formyl-Met-Leu-Phe (fMLP) or phorbol
12-myristate 13-acetate (PMA). This activation of MAPKs coincides with
the formation of superoxide anion, which occurs through the activation
of a multiple-component NADPH oxidase pathway. MAPKs have thus been
suggested to be involved in signal transduction leading to the
oxidative burst. To investigate whether MAPK activation plays a central
role in the oxidative burst, we evaluated the effect of cAMP on MAPK
activation induced by fMLP and PMA. cAMP inhibits many PMN functional
responses, including the oxidative burst, and has recently been shown
to reduce growth factor- and PMA-induced MAPK activities in a variety
of cells. We found that in differentiated, neutrophil-like HL-60 cells,
while cAMP reduced PMA-induced MAPK activation, it had no effect on
fMLP-induced MAPK activation. Despite the presence of unchanged levels
of activated MAPKs, the fMLP-induced oxidative burst was substantially
diminished by cAMP. By contrast, O
When activated by the chemotactic peptide fMLP,
Several recent studies have demonstrated that the MAPK pathway is
activated upon stimulation by fMLP(7, 8, 9) .
The kinetics of MAPK activation coincide with the oxidative burst in
PMNs(7, 9) . MAPKs are cytoplasmic protein
serine/threonine kinases that phosphorylate and regulate other protein
kinases, cytoskeletal proteins, and transcription
factors(10, 11) . MAPK kinase (MEK), a dual specificity
kinase that phosphorylates MAPKs on threonine and
tyrosine(12, 13, 14, 15, 16) ,
has been shown to be directly activated by Raf-1
kinase(17, 18, 19) . Raf-1 kinase, in turn, is
modulated by Ras(20, 21, 22, 23) , which
serves as a common point of convergence for many growth
factors(24) . Interestingly, it has been demonstrated that an
increase in intracellular concentration of cAMP inhibits transmission
of growth-stimulatory signals through the Ras-Raf-MEK-MAPK
pathway(25, 26, 27) . cAMP appears to inhibit
Raf activation by preventing Ras from transmitting a signal to
Raf-1(26, 27) .
More recently, Grinstein et al.(28) demonstrated that MEK is activated by fMLP in PMN.
Because tyrosine kinase inhibitors block the oxidative burst, and MEK
phosphorylates MAPKs on tyrosine and threonine, their finding is
consistent with the possibility that MEKs/MAPKs are involved in
activating the NADPH oxidase. MEK activation, as suggested from their
results, might be mediated by protein kinase C, which appears to be
important for the oxidative burst. In addition, another recent study by
Worthen et al. (29) indicated that fMLP activates Ras and
Raf-1 in PMN. Moreover, cAMP inhibits Raf-1 activation in this
system(29) . It has been suggested from these studies that in
PMN, the MAPK pathway may play a central role in PMN functional
responses. Nevertheless, a direct relationship between the oxidative
burst and MAPK activation has not been demonstrated.
In the present
study, we examined the effect of cAMP on MAPK activation and the
oxidative burst in Me
Human
PMNs were isolated from peripheral blood as described previously (31).
Briefly, fresh whole blood was obtained by venipuncture from healthy
volunteers and immediately added to acid citrate dextrose. The PMNs
were purified by dextran sedimentation, followed by hypotonic lysis to
remove the majority of erythrocytes, and then centrifuged through
Ficoll-Paque (Pharmacia Biotech Inc.) to remove contaminating
mononuclear cells. This purification scheme yields at least 98% PMNs
with few contaminating platelets and a viability of >95% as
determined by trypan blue exclusion. For some experiments, PMNs were
incubated with 5 mM diisopropyl fluorophosphate at 4 °C
for 10 min, washed, and resuspended in activation buffer as outlined
below.
PMNs were
suspended at 10
For PMNs, 4
Immunoblot analyses were carried out essentially as
described previously(23) . Briefly, cell lysates were separated
through 10% SDS-polyacrylamide gels and electrophoretically transferred
onto nitrocellulose paper. MAPKs were detected by polyclonal anti-Erk
antiserum 642 (1:500 dilution) (generously provided by S. Decker,
Parke-Davis Pharmaceutical Research Division), followed by secondary
horseradish peroxidase-conjugated goat anti-rabbit antibody (Amersham
Corp.) (1:5,000 dilution), which was then detected by enhanced
chemiluminescence (ECL) reagents (Amersham Corp.).
To examine whether the MAPK phosphorylation patterns observed in Fig. 2B were consistent with MAPK activity, in vitro kinase assays using MBP as substrate were carried out following
immunoprecipitation of MAPK. Because the anti-MAPK antiserum TR10
reacts predominantly with Erk2, the kinase assays measured primarily
Erk2 activity. MBP was only slightly phosphorylated in unstimulated
HL-60 cells in the presence or absence of Bt
Cellular mechanisms that mediate functional responses of
PMNs, such as the oxidative burst, enzyme secretion, and actin
assembly, are not completely defined. A number of recent studies have
implicated the MAPK signal transduction pathway in PMN activation
responses(7, 8, 9, 28, 29) . In
this pathway, Raf-1 phosphorylates and activates MEK, which in turn
phosphorylates and activates MAPK(24) . The oxidative burst, in
particular, has been suggested to be mediated through this MAPK pathway
because the kinetics of superoxide anion production coincide with the
kinetics of MAPK activation(7, 9) . In the present
study, we compared the effects of increased intracellular cAMP levels
on the oxidative burst and MAPK activation in differentiated HL-60
cells and PMNs. Our results show that while increased intracellular
levels of cAMP concentration can markedly inhibit the fMLP-induced
oxidative burst in both HL-60 cells and PMNs, cAMP had no detectable
effect upon the fMLP-induced MAPK activation in either cell type. By
contrast, cAMP could partially inhibit PMA-induced MAPK activation in
HL-60 cells but had no effect on the oxidative burst. Conversely,
blocking protein kinase C activation with staurosporine inhibited
O
Recently,
Worthen et al.(29) demonstrated that Raf-1 kinase
activation was inhibited by elevated intracellular concentrations of
cAMP in PMNs. This finding is consistent with the observation that cAMP
blocks Raf-1 activation in response to growth-stimulatory signals in
fibroblast cells(26, 27) . In fibroblasts, however, the
Raf-MEK-MAPK pathway is blocked by cAMP, whereas in fMLP-stimulated
PMNs we have shown that MAPK activation is not inhibited by this
treatment. Combined with the finding that cAMP blocks Raf-1 activation
in PMNs, this latter result suggests that Raf-1 kinase is not essential
for MEK and MAPK activation in these cells. Nevertheless, because
inhibition of Raf-1 activation by cAMP does coincide with inhibition of
O
In
summary, results of this study demonstrate that MAPK activation, even
though it coincides with PMN functional responses when stimulated by
fMLP or PMA, can be dissociated from the oxidative burst. This finding
raises the question as to which, if any, of the other biological
responses in PMNs is mediated by MAPK. Furthermore, our study, combined
with other recent studies in PMNs(28, 29) , suggests
that while Raf-1 kinase activity may be essential for the oxidative
burst, it is not required for MEK and MAPK activation. Instead, our
results are more consistent with the existence of a Raf-1 substrate
other than MEK that mediates the oxidative burst in PMNs. The
approaches used here may also be applicable to analysis of the
relationship between the Raf-MEK-MAPK signaling pathway and other
biological responses of PMNs.
We thank P. J. Mansfield for excellent technical
assistance; S. Decker and M. Weber for anti-MAPK antibodies; K.
Pumiglia, M. Stofega, and H.-Y. Chow for stimulating discussions; E.
Crockett-Torabi and P. Freeman for the superoxide assay protocol; and
M. Hetzel for superb assistance in preparing this manuscript.
Volume 270,
Number 26,
Issue of June 30, pp. 15719-15724, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
production induced by PMA remained the same even though MAPK
activation was inhibited. In PMNs, although the levels of
O
induced by either 10 ng/ml or 100 ng/ml
PMA were similar, only 100 ng/ml could stimulate MAPK activation,
suggesting that the oxidative burst could occur in the absence of
detectable activation of MAPKs. As in HL-60 cells, cAMP inhibited the
O
production in fMLP-stimulated PMNs but
had no effect on MAPK activity. These results demonstrate that, while
MAPK activation coincides with PMN activation, it can be dissociated
from the oxidative burst.
()PMNs rapidly generate toxic oxygen derivatives,
including superoxide anion and hydrogen peroxide, catalyzed by NADPH
oxidase(1, 2) . Formation of such reactive oxygen
species by PMNs is essential for host defense against bacterial
infection(1) . The cellular mechanisms underlying PMN responses
to fMLP are not yet fully defined. However, it is known that activation
of PMNs is initiated by binding of fMLP to a specific serpentine
receptor and mediated by heterotrimeric GTP-binding
proteins(3) . This activation is accompanied by an increase in
phosphorylation on multiple polypeptides (4). Because the intracellular
concentration of Ca and diacylglycerol increase upon
stimulation, and since phorbol esters like PMA that act directly on
protein kinase C are potent stimulants of the PMN oxidative
burst(5, 6) , it has been suggested that protein kinase
C plays an important role in the generation of oxidative radicals.
SO-differentiated, neutrophil-like
HL-60 cells and in PMNs. Surprisingly, contrary to what was predicted
from the earlier studies, we find that O production does not correlate with either fMLP- or PMA-induced
MAPK activation in either HL-60 cells or PMNs. These results suggest
that MAPK activation is not required for the oxidative burst in
neutrophils.
Cells
HL-60 cells were obtained from the
American Type Culture Collection (Rockville, MD) and grown in RPMI
medium with glutamine (Life Technologies, Inc.) supplemented with 20%
fetal calf serum (Hyclone Laboratories). HL-60 cells were
differentiated into neutrophil-like cells by growth in medium
containing 1.3% MeSO (30) and harvested for assays
after 6 days. 60-70% of the cells were determined to be
differentiated by the nitroblue tetrazolium reduction assay.
Assay for Superoxide
Superoxide production was
determined using an end point assay to measure the superoxide
dismutase-sensitive reduction of ferricytochrome c(30, 32) . Briefly, HL-60 cells were washed
twice and resuspended in Hanks' buffered salt solution with
Mg and Ca
at 10
cells/ml and then incubated with or without 1 mM BtcAMP for 10 min or with 0.5 mM IBMX or 0.05
mM forskolin for 15 min. After stimulating the cells for 5 min
with cytochalasin B (5 µg/ml final concentration), 50 µl of
cells were then added to each well of a 96-well microtiter plate (flat
bottom wells) containing cytochrome c (55 µM)
with or without 250 µg/ml superoxide dismutase (final concentration
in a total of 200 µl). O formation
was initiated by addition of 1 µM fMLP or 1 µg/ml PMA.
After incubating at 37 °C for 10-20 min, cytochrome c reduction at 550 nm was determined on an enzyme-linked
immunosorbent assay plate reader (SLT Labinstruments). Superoxide
production was calculated using an extinction coefficient of 21
mmol/liter
cm
.
cells/ml in phosphate-buffered saline
containing 1 mM CaCl, 1 mM MgCl, and 5 mM glucose and then added to the
assay mixture at a final cell concentration of 10
cells/ml.
In experiments using cell-permeant cAMP analogues and IBMX, PMNs were
preincubated with these agents for 15 min at 37 °C in
phosphate-buffered saline containing Ca,
Mg
, and glucose. Assay mixtures consisted of
phosphate-buffered saline containing Ca
,
Mg
, glucose, and cytochrome c (75
µM) ± superoxide dismutase (60 µg/ml), in the
presence or absence of Bt
cAMP or IBMX. PMNs were stimulated
with cytochalasin B (5 µg/ml for 3 min at 37 °C) followed by
fMLP (10M for 5 min at 37 °C), or with
10 or 100 ng/ml PMA for 5 min at 37 °C. Following activation, cells
were removed by centrifugation, and the absorbance of the supernatants
was measured at 550 nm. O
production was
calculated as outlined above. The protocol for the continuous assay was
the same except that O
was monitored
continuously during activation.
Cell Activation and Immunoblotting
For HL-60
cells, 2 10
differentiated cells/ml in Hanks'
buffered salt solution with Mg and Ca
were incubated at 37 °C with or without Bt
cAMP (1
mM, 10 min) or with IBMX (0.5 mM, 15 min) or
forskolin (0.05 mM, 15 min). Samples were then treated for 5
min with cytochalasin B (5 µg/ml) and stimulated by adding fMLP
(10M, 5 min) or PMA (1 µg/ml, 10 min)
followed by addition of 4
SDS-sample buffer. The samples were
well mixed and boiled for 10 min prior to loading on SDS-polyacrylamide
gels. Controls consisted of differentiated HL-60 cells incubated at 37
°C in the absence of agonist.
10
/ml cells were suspended in
Ca/Mg
-containing phosphate-buffered
saline, prewarmed to 37 °C for 3 min in the presence or absence of
5 µg/ml cytochalasin B, and stimulated with either 10
M fMLP or 10-100 ng/ml PMA for 5 min at 37 °C.
The reaction was stopped by the addition of volume of 4
SDS-sample buffer. The samples were vortexed, boiled for 10 min, and
stored at -80 °C prior to loading on SDS-polyacrylamide gels.
Controls consisted of PMNs incubated at 37 °C in the absence of
agonist. Treatment of PMNs with Bt
cAMP (1 mM, 15
min) or IBMX (0.5 mM, 15 min) occurred prior to agonist
addition.
Immunocomplex Kinase Assay
HL-60 cells were
activated as described above and lysed at 3 10
cells/250 µl in lysis buffer (50 mM HEPES, pH 7.5,
100 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1 µM pepstatin, 1 µg/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium
orthovanadate, 2 µg/ml aprotinin, and 40 mMp-nitrophenyl phosphate). Cleared lysates were incubated
with 4 µl of polyclonal anti-Erk2 antiserum TR10 (kindly provided
by M. Weber, University of Virginia) (33) for 1 h and then with 20
µl of protein-A Sepharose for 30 min at 4 °C with rotation. The
antibody-antigen complex was washed twice with lysis buffer and then
kinase buffer (50 µM ATP, 10 mM MgAc, 10
mM HEPES, pH 7.5). Immune complexes were then resuspended in
20 µl of 3 kinase buffer, 20 µl of 2 mg/ml myelin basic
protein (MBP), and a total of 5 µCi of
[
P]ATP. Reactions were carried out at 30 °C
for 5 min and stopped by the addition of 20 µl of 4
SDS-sample buffer. Samples were separated through 15%
SDS-polyacrylamide gels and transferred electrophoretically to
nitrocellulose. Phosphorylation of MBP was visualized by
autoradiography. Nitrocellulose membranes from the kinase assays were
also probed with anti-MAPK antibodies to ensure equivalent levels of
Erk2 proteins in the kinase assays. Quantification of kinase assays was
performed using an AMBIS 4000 radioisotope detector.
Increased cAMP Inhibits fMLP-induced but Not
PMA-induced O
It has been established that increased intracellular cAMP
levels in PMNs inhibit O Formation in HL-60
Cells
formation
induced by fMLP(34, 35) . On the other hand, PMA-induced
PMN functional responses are not sensitive to
cAMP(34, 35) . We examined whether cAMP might similarly
affect the fMLP- and PMA-induced oxidative burst in differentiated
HL-60 cells. The HL-60 cell line consists predominantly of
promyelocytes and proliferates continuously in suspension culture. This
cell line, when induced by Me
SO, differentiates into a
neutrophil-like cell and develops a fully activated NADPH oxidase (30,
36-38). As shown in Fig. 1A, preincubating HL-60
cells with BtcAMP (1 mM) for 10 min had a
significant (84%) inhibitory effect on the fMLP-induced oxidative
burst. In contrast, it did not affect the total amount of
PMA-stimulated O production (Fig. 1B). In addition, neither the rate nor the extent
of O
generation were altered by
Bt
cAMP treatment as determined by continuous assay for
O production (data not shown). To confirm
that the Bt
cAMP effect was due to raised levels of
intracellular cAMP, HL-60 cells were incubated for 15 min with or
without IBMX or forskolin. IBMX enhances intracellular cAMP
accumulation by inhibiting cAMP phosphodiesterase and blocking
inhibitory adenosine receptors linked to
G
(39, 40) . Forskolin directly activates the
catalytic subunit of adenylate cyclase and thus increases cAMP
accumulation intracellularly(41) . Incubation with IBMX or
forskolin generated superoxide profiles consistent with those obtained
with BtcAMP (Fig. 1, A and B).
These results, obtained by using three agents that raise intracellular
cAMP concentration via independent mechanisms, demonstrate that cAMP
selectively inhibits the fMLP-induced oxidative burst in HL-60 cells.
Figure 1:
Effect of cAMP on superoxide formation
in MeSO-differentiated HL-60 cells. Six days after growth
in medium supplemented with 1.3% MeSO, differentiated HL-60
cells were incubated for 10 min in Hanks' buffered salt solution
containing 1% MeSO (vehicle) or 1 mM BtcAMP or for 15 min in 0.5 mM IBMX or 0.05
mM forskolin (forsk). Each of these treatments was
divided into three parts, one as control, one stimulated with
10M fMLP, and another one stimulated with
100 ng/ml PMA. O
production was
determined by superoxide assays as described under ``Materials and
Methods.'' The relative values (control = 1) are means of
triplicates with standard error bars. This experiment was repeated
three times with similar results. A, fMLP-induced superoxide
formation. B, PMA-induced superoxide
formation.
cAMP Inhibits PMA- but Not fMLP-induced MAPK
Phosphorylation and Activation in HL-60 Cells
Recent studies
have shown that increased cAMP inhibits the platelet-derived growth
factor- and epidermal growth factor-activated MAPK signaling
pathway(25, 26, 27) . It has also been noted
that PMA-stimulated MAPK activation can be partially blocked
(approximately 50%) by BtcAMP in Rat1 cells(27) . We
investigated whether enhanced accumulation of cAMP in HL-60 cells could
affect fMLP- and PMA-induced MAPK activation by examining MAPK
phosphorylation using Western blot analysis.
MeSO-differentiated HL-60 cells were incubated in the
presence or absence of BtcAMP and stimulated with either
fMLP or PMA. All of the treatments and stimulations were carried out
under conditions identical to the superoxide assays, and often the two
kinds of experiments were performed in parallel. The polyclonal
anti-MAPK antiserum 642 recognizes both Erk1 and Erk2 isoforms of MAPK
in unstimulated HL-60 cells (Fig. 2, A and B).
Stimulation with fMLP or PMA resulted in the appearance of two slower
migrating bands that were reactive with the antiserum, indicating that
a fraction of Erk1 and Erk2 were phosphorylated (Erk1* and Erk2*). In Fig. 2A, some Erk1* could also be detected in control
cells, but in stimulated cells the level of Erk1* was increased. A time
course experiment shows that increased levels of Erk1* and Erk2* appear
as early as 3 min after fMLP stimulation and increased further at 5 min (Fig. 2A). At 10 min, the abundance of Erk1* and Erk2*
decreased. For PMA-stimulated cells, Erk1* and Erk2* were apparent at
10 min and remained activated at 20 min (although at levels lower than
that observed at 10 min) (Fig. 2A). These results show
that activation of MAPK and release of O (detectable 10 min after addition of agonists) occur within a
similar time frame in HL-60 cells.
Figure 2:
Time course of, and BtcAMP
effect on, fMLP- and PMA-stimulated MAPK phosphorylation in HL-60
cells. HL-60 cells, differentiated and pretreated similarly as for the
superoxide assays described in the Fig. 1 legend, were lysed by boiling
in SDS-sample buffer. Total cell lysate (10
cells/sample) was run on a 10% SDS-polyacrylamide gel,
transferred to nitrocellulose paper, and probed with 642 anti-Erk
antiserum. A, kinetics of fMLP and PMA simulation. Erk1* and
Erk2* denote the phosphorylated p44 and p42 isoforms of MAPK,
respectively, with shifted electrophoretic mobilities. B,
effect of BtcAMP on MAPK phosphorylation. HL-60 cells were
lysed 5 min and 10 min after fMLP and PMA stimulation, respectively.
This experiment was repeated three times with similar
results.
As shown in Fig. 2B, BtcAMP pretreatment had no effect
on fMLP-induced Erk1* and Erk2*. In contrast, PMA-induced Erk1* and
Erk2* were reduced by a preincubation with BtcAMP.
Incubation with IBMX or forskolin had similar effects on Erk1* and
Erk2* appearance as did BtcAMP (data not shown). The
results indicate that while an increase in cAMP concentration reduced
fMLP-induced O release, it had no
influence on MAPK phosphorylation in HL-60 cells. Conversely, in
PMA-stimulated HL-60 cells, enhanced cAMP accumulation did partially
inhibit MAPK phosphorylation but had no effect on the oxidative burst.
cAMP
pretreatment (Fig. 3A). Stimulation with fMLP (5 min)
increased Erk2* kinase activity about 7-fold, and PMA (10 min)
increased the kinase activity 12-fold (Fig. 3, A and B). While BtcAMP inhibited PMA-induced Erk2
activation (60% reduction), it did not affect fMLP-induced Erk2
activity (Fig. 3, A and B). To ensure
equivalent immunoprecipitation of Erk2, the same membrane was
immunoblotted with anti-MAPK antiserum 642 (data not shown). In the
case of fMLP-stimulated HL-60 cells, therefore, our results demonstrate
that an increase in cAMP concentration blocks the signal transduction
pathway to O formation even though MAPK
activity remains high. By contrast, in PMA-stimulated cells, although
cAMP inhibits the MAPK pathway, it does not interfere with the
oxidative burst.
Figure 3:
BtcAMP effect on MAPK (Erk2)
activation as determined by immunocomplex kinase assays. HL-60 cells
were differentiated, pretreated, and stimulated under the same
conditions as for the superoxide and electrophoretic mobility shift
assays in Figs. 1 and 2. Cells were lysed as described under
``Materials and Methods,'' and cell lysates were incubated
with TR10 anti-Erk2 antiserum and then precipitated by protein
A-Sepharose. After extensive washing, the beads were added to kinase
assay buffer containing MBP and [P]ATP. A, kinase assay products were resolved on a 15%
SDS-polyacrylamide gel and transferred to a nitrocellulose membrane.
The data shown here represent results of one of the three independent
experiments with similar results. B, phosphorylation of MBP
substrate was quantified by direct counting of
[
P] in an AMBIS 4000 radioisotope analyzer using
the same membrane shown in A. The values presented are net
counts incorporated
10
.
MAPK Activation Also Can Be Dissociated from the
Oxidative Burst in Human PMNs
Fig. 4illustrates the
effect of BtcAMP and IBMX on the oxidative burst in human
PMNs. As with HL-60 cells, both BtcAMP and IBMX had an
inhibitory effect on the fMLP-induced oxidative burst (Fig. 4A). Also like HL-60 cells, neither
BtcAMP nor IBMX blocked the PMA-induced oxidative burst (Fig. 4B). Significantly, the levels of oxidant release
induced by PMA at 10 ng/ml and 100 ng/ml were similar (Fig. 4B).
Figure 4:
Effect of increased intracellular cAMP
concentration on fMLP- and PMA-induced oxidative burst in PMN. PMNs
were pretreated with either 1 mM BtcAMP (15 min)
or 0.5 mM IBMX (15 min) and then incubated with cytochalasin B
for 3 min before adding 10M fMLP. For PMA
stimulation, both 10 and 100 ng/ml PMA were used. The superoxide assays
were performed in duplicate, and two independent experiments were
carried out for Bt
cAMP with nearly identical results. A, fMLP stimulation; B, PMA stimulation. Values
presented are means (control = 1) with standard errors
shown.
A time course experiment was performed to
determine the optimal stimulation time with fMLP and PMA for MAPK
phosphorylation in PMNs (Fig. 5A). The 5-min time point
was used for both fMLP and PMA stimulation to evaluate the cAMP effects
on MAPK phosphorylation (Fig. 5B). As in HL-60 cells,
neither BtcAMP nor IBMX had a noticeable inhibitory effect
on fMLP-induced MAPK phosphorylation in PMNs. However, PMA-induced MAPK
phosphorylation was not affected by BtcAMP or IBMX, unlike
what was found in HL-60 cells. Moreover, 10 ng/ml PMA, which induced
the oxidative burst to a similar extent as 100 ng/ml PMA (Fig. 4B), failed to activate either Erk1 or Erk2
significantly (Fig. 5B). Therefore, in PMNs the
PMA-induced oxidative burst could occur in the absence of detectable
MAPK activation. Conversely, the fMLP-induced oxidative burst was
greatly inhibited by cAMP despite the high level of MAPK activation. To
determine whether MAPK activation correlated with the oxidative burst
when other activation pathways, such as protein kinase C, were shut
down, PMNs were pretreated with the protein kinase C inhibitor
staurosporine (100 nM) prior to activation with fMLP. The
fMLP-stimulated O generation was blocked
(from 35.7 ± 0.5 to 0.5 ± 0.5 nmol of
O
/10
cells/5 min), while MAPK
activation was not affected (Fig. 6). Taken together, our results
demonstrate that O generation is
independent of MAPK activation.
Figure 5:
Activation of MAPK in fMLP- and
PMA-stimulated PMNs. Pretreatment and stimulation of PMNs in these
experiments paralleled those done for the superoxide assays shown in
Fig. 4. A, time course of MAPK activation. Cells were lysed at
the times indicated, and cell lysates were analyzed by Western blotting
with 642 anti-Erk antiserum. B, effects of BtcAMP
and PMA concentration on MAPK activation. Cells were lysed in
SDS-sample buffer 5 min after receiving either fMLP or PMA. Results
shown for BtcAMP-pretreated cells were repeated with
similar results.
Figure 6:
Effect of staurosporine on fMLP-induced
MAPK activation in PMNs. PMNs were incubated with 100 nM staurosporine for 5 min at 37 °C prior to fMLP stimulation.
Conditions for fMLP stimulation and Western blotting were the same as
described in the legend to Fig. 5.
generation without affecting MAPK
activation. Moreover, in PMNs, even though 10 ng/ml PMA was sufficient
to induce nearly maximum O
release, 100
ng/ml PMA was required to stimulate significant levels of MAPK
activation. These results establish that MAPK activation can be
dissociated from the oxidative burst in neutrophils.
release in fMLP-stimulated PMNs, Raf-1
kinase remains implicated in the pathway leading to NADPH oxidase
activation. Therefore, Raf-1 kinase may phosphorylate a substrate other
than MEK that is involved in mediating the oxidative burst in PMNs. In
HL-60 cells, but not in PMNs, we found that PMA-induced MAPK activity
was reduced approximately 60% by cAMP. This partial inhibition is
comparable with the effect of cAMP on PMA-induced MAPK activity in Rat1
fibroblasts(27) . As in fibroblasts, it is possible that the
partial inhibition in HL-60 cells reflects a block in Raf-1 activation
in PMA-stimulated cells. Thus, in HL-60 cells, MAPK may be activated by
both Raf-dependent and Raf-independent pathways in response to PMA
stimulation of protein kinase C. There is other evidence consistent
with a role for Raf-1 in the activation of MEK by protein kinase C,
since Raf-1 kinase has been found to be activated in cells stimulated
with phorbol esters(16, 19, 29, 42) . On
the other hand, because we found that Bt
cAMP had no effect
on MAPK activation induced by PMA in PMNs, it is likely that PMA
activates MAPK in a completely Raf-independent manner in these cells.
The basis for this difference between HL-60 cells and PMNs in response
to PMA stimulation is not clear. However, this effect may be downstream
of protein kinase C, since inhibition of protein kinase C with
staurosporine did not alter MAPK activation in fMLP-treated PMNs.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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R. El Bekay, M. Alvarez, M. Carballo, J. Martin-Nieto, J. Monteseirin, E. Pintado, F. J. Bedoya, and F. Sobrino Activation of phagocytic cell NADPH oxidase by norfloxacin: a potential mechanism to explain its bactericidal action J. Leukoc. Biol., February 1, 2002; 71(2): 255 - 261. [Abstract] [Full Text] [PDF]
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 C. FERNANDEZ-PATRON, C. ZOUKI, R. WHITTAL, J. S. D. CHAN, S. T. DAVIDGE, and J. G. FILEP Matrix metalloproteinases regulate neutrophil-endothelial cell adhesion through generation of endothelin-1[1-32] FASEB J, October 1, 2001; 15(12): 2230 - 2240. [Abstract] [Full Text] [PDF]
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E. Mollapour, D. C. Linch, and P. J. Roberts Activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate oxidase and phospholipase A2 are dissociated by inhibitors of the kinases p42ERK2 and p38SAPK and by methyl arachidonyl fluorophosphonate, the dual inhibitor of cytosolic and calcium-independent phospholipase A2 Blood, April 15, 2001; 97(8): 2469 - 2477. [Abstract] [Full Text] [PDF]
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 C. A. Harrison, M. J. Raftery, J. Walsh, P. Alewood, S. E. Iismaa, S. Thliveris, and C. L. Geczy Oxidation Regulates the Inflammatory Properties of the Murine S100 Protein S100A8 J. Biol. Chem., March 26, 1999; 274(13): 8561 - 8569. [Abstract] [Full Text] [PDF]
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K. Suzuki, M. Hino, F. Hato, N. Tatsumi, and S. Kitagawa Cytokine-Specific Activation of Distinct Mitogen-Activated Protein Kinase Subtype Cascades in Human Neutrophils Stimulated by Granulocyte Colony-Stimulating Factor, Granulocyte-Macrophage Colony-Stimulating Factor, and Tumor Necrosis Factor-alpha Blood, January 1, 1999; 93(1): 341 - 349. [Abstract] [Full Text] [PDF]
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M. Tardif, M.-J. Rabiet, T. Christophe, M.-D. Milcent, and F. Boulay Isolation and Characterization of a Variant HL60 Cell Line Defective in the Activation of the NADPH Oxidase by Phorbol Myristate Acetate J. Immunol., December 15, 1998; 161(12): 6885 - 6895. [Abstract] [Full Text] [PDF]
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C. Capodici, S. Hanft, M. Feoktistov, and M. H. Pillinger Phosphatidylinositol 3-Kinase Mediates Chemoattractant-Stimulated, CD11b/CD18-Dependent Cell-Cell Adhesion of Human Neutrophils: Evidence for an ERK-Independent Pathway J. Immunol., February 15, 1998; 160(4): 1901 - 1909. [Abstract] [Full Text] [PDF]
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G. P. Downey, J. R. Butler, H. Tapper, L. Fialkow, A. R. Saltiel, B. B. Rubin, and S. Grinstein Importance of MEK in Neutrophil Microbicidal Responsiveness J. Immunol., January 1, 1998; 160(1): 434 - 443. [Abstract] [Full Text] [PDF]
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J.-P. Mira, T. Dubois, J.-P. Oudinet, S. Lukowski, F. Russo-Marie, and B. Geny Inhibition of Cytosolic Phospholipase A2 by Annexin V in Differentiated Permeabilized HL-60 Cells. EVIDENCE OF CRUCIAL IMPORTANCE OF DOMAIN I TYPE II Ca2+-BINDING SITE IN THE MECHANISM OF INHIBITION J. Biol. Chem., April 18, 1997; 272(16): 10474 - 10482. [Abstract] [Full Text] [PDF]
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M. H. Pillinger, A. S. Feoktistov, C. Capodici, B. Solitar, J. Levy, T. T. Oei, and M. R. Philips Mitogen-activated Protein Kinase in Neutrophils and Enucleate Neutrophil Cytoplasts J. Biol. Chem., May 17, 1996; 271(20): 12049 - 12056. [Abstract] [Full Text] [PDF]
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