(Received for publication, June 5, 1995; and in revised form, January 10, 1996)
From the
We employed neutrophils and enucleate neutrophil cytoplasts to
study the activation of the mitogen-activated protein kinases (MAPKs)
p44 and p42
in
neutrophils by inflammatory agonists that engage G protein-linked
receptors. Formyl-methionyl-leucylphenylalanine (FMLP) rapidly and
transiently activated MAPK in neutrophils and cytoplasts, consistent
with a role in signaling for neutrophil functions. FMLP stimulated
p21
activation in neutrophils and Raf-1
translocation from cytosol to plasma membrane in cytoplasts, with
kinetics consistent with events upstream of MAPK activation. Insulin, a
protein tyrosine kinase receptor (PTKR) agonist, stimulated neutrophil
MAPK activation, demonstrating an intact system of PTKR signaling in
these post-mitotic cells. FMLP- and insulin-stimulated MAPK activation
in cytoplasts were inhibited by Bt
cAMP, consistent with
signaling through Raf-1 and suggesting a mechanism for cAMP inhibition
of neutrophil function. However, Bt
cAMP had no effect on
FMLP-stimulated MAPK activation in neutrophils. The extent of MAPK
activation by various chemoattractants correlated with their capacity
to stimulate neutrophil and cytoplast homotypic aggregation. Consistent
with its effects on MAPK, Bt
cAMP inhibited FMLP-stimulated
aggregation in cytoplasts but not neutrophils. Insulin had no
independent effect but primed neutrophils for aggregation in response
to FMLP. Our studies support a p21
-,
Raf-1-dependent pathway for MAPK activation in neutrophils and suggest
that neutrophil adhesion may be regulated, in part, by MAPK.
The mitogen-activated protein kinases (MAPKs) ()p44
and p42
are serine/threonine kinases that participate in cell
signaling for growth and differentiation(1) . The most
completely elucidated pathway for p44
and
p42
activation utilizes protein tyrosine kinase
receptors (PTKR) to activate MAPK through p21
and Raf-1. In this pathway, ligation of the PTKR results in
interaction of the receptor with a complex of the adaptor protein Grb2 (2) and SOS, a guanine nucleotide exchange factor(3) .
These interactions bring SOS into proximity of
p21
(4, 5) , stimulating GTP/GDP
exchange(6) . Activated p21
recruits the
serine/threonine kinase Raf-1 to the plasma membrane (PM) where it is
activated(7, 8, 9) . Activated Raf-1
phosphorylates a dual threonine/tyrosine kinase, MAPK or Erk
kinase(10) , which in turn phosphorylates and activates
p44
and
p42
(11) . Raf-1 appears to play a
pivotal role in this pathway because its activation can be negatively
regulated by cAMP-dependent protein kinase A
(PKA)(12, 13) . Among other signaling pathways leading
to MAPK activation are those activated by G protein-linked receptors,
including receptors for lysophosphatidic acid (LPA)(14) ,
acetylcholine (15) , and thrombin(16) . Whereas LPA and
acetylcholine activate MAPK via
p21
(14, 16) , the thrombin
receptor can activate MAPK through a
p21
/Raf-1-independent
pathway(17, 18) .
Circulating neutrophils are
terminally differentiated, post-mitotic phagocytes that constitute the
first line of host defense against microorganisms. In contrast to
dividing cells that respond slowly to mitogens, neutrophils respond
rapidly to inflammatory stimuli. One class of neutrophil agonists, the
chemoattractants, engage seven transmembrane-spanning domain receptors
that activate G proteins. The only well-documented effector
downstream of neutrophil G
is phospholipase
C
, a regulatory enzyme not directly linked to the MAPK
pathway. Nevertheless, the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP) has been shown
to activate MAPK (19) and to stimulate MAPK autophosphorylation
in neutrophils in a pertussis toxin-sensitive fashion(20) .
MAPKs thus represent candidate effectors for the signaling pathway(s)
leading from G protein activation to rapid neutrophil responses.
Because currently understood mechanisms of neutrophil activation fail
to explain the observation that agents that elevate intracellular cAMP
inhibit some chemoattractant-stimulated neutrophil responses, the
possibility that chemoattractants activate MAPKs in a
p21
/Raf-1-dependent fashion is an attractive
hypothesis.
In the present study we utilize intact neutrophils and
neutrophil cytoplasts (enucleate, granule-poor, metabolically active
cell fragments) to demonstrate that MAPK activation by FMLP is
associated with activation of p21 and
translocation of Raf-1 to the PM and that cAMP acts via PKA to inhibit
FMLP-stimulated MAPK activation in cytoplasts but not neutrophils. We
also show that insulin, known to activate MAPK via p21
and Raf-1 in mitotic cells, activates MAPK in neutrophils
and cytoplasts. Finally, we observed a strong correlation between MAPK
activation and cell-cell adhesion in neutrophils and cytoplasts
suggesting a new regulatory role for MAPK in a process critical for
inflammation.
Figure 1: FMLP stimulates MAPK activation in neutrophils and enucleate neutrophil cytoplasts. Neutrophils (A) and cytoplasts (B) were incubated for 1 min at 37 °C in the absence or presence of 100 nM FMLP and analyzed for MAP kinase activity by gel renaturation MAP kinase activity assay as described under ``Experimental Procedures.'' Neutrophils (C) and cytoplasts (D) were incubated for 1 min at 37 °C in the absence or presence of 100 nM FMLP, lysed with 1% Nonidet P-40, and analyzed for kinase activity toward an MBP-derived peptide substrate specific for MAPK (MBPp). Results shown are representative of eight (A and B), or are the mean ± S.E. (C and D) of three, experiments for each condition.
To establish that the FMLP-sensitive MBP kinase activity observed in the gel renaturation assay corresponded to MAPK-type phosphorylation of the MBP molecule, which has numerous non-MAPK phosphorylation sites, we tested the ability of lysates of FMLP-stimulated cells or cytoplasts to phosphorylate MBPp, a synthetic peptide containing only the MBP amino acid sequence specifically phosphorylated on threonine by Erk (PRTP)(26) . Neutrophil and cytoplast lysates both contained MBPp kinase activity that was markedly stimulated by FMLP (Fig. 1, C and D). A peptide in which valine was substituted for threonine gave only background counts. The fold-increase stimulated by FMLP was greater in neutrophils than cytoplasts, consistent with the results obtained in the gel renaturation assay.
We confirmed the identity of cytoplast
FMLP-sensitive MBP kinases as p44 and p42
by immunoprecipitating p44
and p42
from unstimulated or FMLP-stimulated cytoplasts and analyzing the
precipitates by immunoblot and gel renaturation MBP kinase assays.
Coimmunoprecipitation of p44
and p42
followed by immunoblot using a third antiserum recognizing both
Erks revealed two polypeptides of expected molecular weight that were
unaffected by FMLP stimulation (Fig. 2A). Neither
protein was precipitated by a control antiserum. In contrast,
p44
/p42
antisera precipitated MBP kinase
activity in the 42-44-kDa region of the gel only from lysates of
cytoplasts that had been stimulated with FMLP (Fig. 2B). The resolving power of the MBP-impregnated
gels in these experiments was inadequate to distinguish p44
from p42
kinase activity. However, when
p44
and p42
were immunoprecipitated
separately from FMLP-stimulated lysates each precipitate contained
42-44-kDa MBP kinase activity, although somewhat less than the
coprecipitate (Fig. 2B). FMLP-stimulated lysates
immunoprecipitated with control antisera contained no 42-44-kDa
MBP kinase activity, although higher molecular weight activities were
pulled down nonspecifically. Thus, the measurement of radioactivity in
immunoprecipitates in the absence of simultaneous assessment of the
molecular weight of the kinase is inadequate for measuring MAPK
activity specifically. Accordingly, these data are the clearest
demonstration to date that FMLP, acting through a G protein-linked
receptor, activates p44
and p42
in
neutrophils. Moreover, they demonstrate that the signaling pathway for
formyl peptide receptor-stimulated MAPK activation does not depend on
nuclear or granular elements since it is retained in neutrophil
cytoplasts.
Figure 2:
Anti-Erk antisera immunoprecipitate active
and inactive p44 and p42
from cytoplast lysates. A, cytoplasts (1.5
10
/condition) were incubated in the absence (lane
1) or presence of 100 nM FMLP (lanes 2 and 3) for 1 min at 37 °C, lysed, and immunoprecipitated with
antisera to p44
and p42
together (lanes 1 and 2) or control
antiserum (lane 3) as described under ``Experimental
Procedures'' and then analyzed by SDS-polyacrylamide gel
electrophoresis and Western blot with a third antibody directed against
both p44
and p42
. B, cytoplasts (1.5
10
/condition) were
incubated in the absence (lane 1) or presence of 100 nM FMLP (lanes 2-5) for 1 min at 37 °C, lysed, and
immunoprecipitated with antisera to p44
and
p42
together (lanes 1 and 2),
p44
alone (lane 3), p42
alone (lane 4), or control antiserum (lane
5) as described under ``Experimental Procedures'' and
then analyzed by gel renaturation MAP kinase assay. Results shown are
representative of four experiments.
Because neutrophils required post-stimulation processing
that made precise kinetic measurements difficult, we studied the
kinetics of FMLP-stimulated MAPK activation in cytoplasts that could be
rapidly lysed in SDS sample buffer without releasing nucleic acids and
granular proteases (Fig. 3A and Fig. 4).
FMLP-stimulated activation of p44 and p42
was transient, peaking at 1 to 2 min and returning to base line
by 10 min. Because MAPK activity is associated with tyrosine
phosphorylation of p44
and p42
, we
compared MBP kinase activities with tyrosine phosphorylation of
cytoplast proteins following stimulation with FMLP (Fig. 3B). Cytoplast lysates contained a prominent
42-kDa tyrosine phosphoprotein whose phosphorylation was stimulated by
FMLP with kinetics identical to those of the p42
kinase
activity. When the blot was stripped and reprobed with an antiserum
directed to p44
/p42
(Fig. 3C), the 42-kDa phosphoprotein aligned
precisely with p42
. The amount of p44
and
p42
detected by immunoblot did not change with FMLP
stimulation, confirming that the antiphosphotyrosine blot detected
changes in phosphotyrosine content, not amount of protein. Since the
anti-Erk antiserum used has greater affinity for p44
than p42
, the immunoblot results revealing a
darker p42
band suggest that more p42
than p44
is expressed in human neutrophils. This
could explain why the anti-phosphotyrosine antibody was apparently only
sensitive enough to detect phosphorylated p42
, a result
consistent with earlier studies(27) . These data confirm that
enucleate neutrophil cytoplasts retain the signaling molecules
necessary to respond to FMLP stimulation by phosphorylating and then
dephosphorylating Erk on tyrosine and transiently activating MAPK
activity.
Figure 3:
Kinetics of MAPK activation and
p42 phosphorylation/dephosphorylation in
FMLP-stimulated cytoplasts. Cytoplasts stimulated with 100 nM FMLP at 37 °C for the times indicated were analyzed for MAP
kinase activity by gel renaturation assay (A) and
immunoblotting for phosphotyrosine-containing proteins (B) as
described under ``Experimental Procedures.'' C, the
nitrocellulose from B was stripped (19) and reprobed
with an anti-p44
/p42
antiserum. Results shown are representative of two
experiments.
Figure 4: Insulin stimulates MAPK activity in cytoplasts and neutrophils. Cytoplasts or neutrophils were stimulated for the times indicated in the absence or presence of FMLP (100 nM) or insulin (200 nM) and analyzed for MAP kinase activity by gel renaturation assay quantitated by phosphorimaging. Results shown are the mean ± S.E. for 4 (FMLP, cytoplasts, insulin, and neutrophils) or 10 (insulin and cytoplasts) experiments.
To study the rapid
FMLP-stimulated p21 activation predicted by the kinetics
of MAPK activation, we studied FMLP-stimulated guanine nucleotide
exchange on p21
. Because isolated neutrophils had a bench
life (<6 h by lactate dehydrogenase release assay) inadequate to
ensure equilibrium labeling of nucleotide pools by metabolic labeling
with [
P]orthophosphate, we permeabilized
neutrophils by electroporation in the presence of
[
-
P]GTP to rapidly label intracellular
pools of GTP. In this system p21
activation is measured
as total [
-
P]guanine nucleotide loading
(guanine nucleotide exchange with or without GTPase activation).
Electroporated neutrophils were stimulated with FMLP (100 nM),
p21
was immunoprecipitated from cell lysates, and the
amount of [
-
P]guanine nucleotide associated
with p21
was determined by thin layer chromatography (Table 1). The amount of [
-
P]guanine
nucleotide associated with p21
following FMLP stimulation
was 164 ± 20% control at 30 s and remained stable for as long as
5 min, suggesting that FMLP-stimulated guanine nucleotide exchange on
p21
in neutrophils peaks no later than 30 s after
stimulation. Since hydrolysis of [
-
P]GTP on
p21
results in
p21
[
-
P]GDP, total
[
-
P]guanine nucleotide associated with
p21
cannot distinguish transient from persistent
p21
activation. However, the percentage of
[
-
P]guanine nucleotide associated with
p21
as [
-
P]GTP declined after
1 min, suggesting a GTPase-activating protein activity limiting
activation.
Membrane association of Raf-1 appears to be required for
its activation by PTKR(7, 8, 9) . Since Raf-1
is recruited to the PM of cells transfected with oncogenic, activated
p21(7, 9) , we tested whether FMLP, in
addition to activating p21
, can stimulate Raf-1
translocation from the CS to PM of cytoplasts. When unstimulated
cytoplasts were sonicated and separated by centrifugation into soluble
and insoluble fractions, 14 ± 3% of total immunodetected Raf-1
(supernatant + pellet) was associated with the pellet. This
analysis is likely to overestimate the true membrane-associated pool
since cytoplast disruption may not have been complete and vesiculated
cytoplast membrane is likely to sequester CS. FMLP stimulated a
2.2-fold increase in the amount of Raf-1 associated with cytoplast
membranes. Thus, despite the potential overestimation of basal
membrane-associated Raf-1, our system was sensitive enough to detect
membrane translocation of this molecule. Kinetic analysis (Fig. 5) revealed that Raf-1 translocation to the membrane
peaked at 30 s to 1 min and remained stable for at least 10 min
following FMLP stimulation. In contrast to Raf-1, we observed no
FMLP-stimulated translocation of p44
or p42
to PM in cytoplasts. Although neutrophil CS contained an abundant
supply of SOS, this molecule also did not translocate from cytoplast CS
to PM in response to FMLP. Thus, both FMLP-stimulated p21
activation and Raf-1 translocation preceded MAPK activation,
consistent with a role for both events upstream of Erk activation in
the FMLP-stimulated pathway.
Figure 5: FMLP stimulates translocation of Raf-1 from CS to PM in cytoplasts. Cytoplasts were incubated at 37 °C in the absence or presence of 100 nM FMLP for the times indicated, sonicated, and separated into membrane and soluble fractions as described under ``Experimental Procedures.'' Fractions were assayed for Raf-1 by immunoblot quantitated by phosphorimaging. Results shown are the mean ± S.E. for four experiments.
PTKR activation of MAPK via p21 and Raf-1 may be down-regulated by cAMP-dependent, PKA-mediated
phosphorylation of Raf-1, resulting in impaired interactions between
p21
and Raf-1(12, 13) . We therefore
tested whether FMLP-stimulated MAPK activity in neutrophil cytoplasts
can be similarly inhibited by cAMP. The membrane-permeable,
phosphodiesterase-resistant cAMP analog dibutyryl cAMP
(Bt
cAMP) (1 mM) completely inhibited
insulin-stimulated cytoplast MAPK activity and inhibited
FMLP-stimulated cytoplast MAPK activity by 46.4 ± 11.7% (Fig. 6A). Agents that raise intracellular cAMP by
indirect mechanisms, including isobutrylmethylxanthine (50
µM), forskolin (50 µM), isoproterenol (10
µM), and the adenosine A
receptor agonist NECA
(10 µM) also inhibited FMLP-stimulated MAPK activity by
approximately 50% (Fig. 6B). To confirm that cAMP
inhibition of FMLP-stimulated MAPK in cytoplasts is PKA-dependent, we
tested the effect of KT5720 that, at concentrations below 2
µM, is a specific inhibitor of PKA (29) . KT5720
(1 µM) reversed the inhibitory effect of
Bt
cAMP on FMLP-stimulated MAPK activation (Fig. 6C). These data demonstrate that FMLP-stimulated
MAPK activity in cytoplasts is inhibited by cAMP in a PKA-dependent
manner and support a requirement for p21
/Raf-1
interactions in FMLP-stimulated MAPK activation.
Figure 6:
cAMP inhibits FMLP-stimulated MAPK
activity in cytoplasts but not neutrophils. A, cytoplasts were
incubated for 5 min in the absence or presence of 1 mM BtcAMP, stimulated (100 nM FMLP for 1 min or
200 nM insulin for 10 min), and analyzed for MAP kinase
activity by gel renaturation assay quantitated by phosphorimaging. B, cytoplasts were incubated for 5 min in the presence of
isobutyrylmethylxanthine (50 µM), forskolin (50
µM), isoproterenol (10 µM), or NECA (10
µM) and stimulated for 1 min with 100 nM FMLP and
analyzed by gel renaturation assay and phosphorimaging. C, cytoplasts were incubated for 5 min in the absence or presence of
the specific PKA inhibitor KT5720 (1 µM), followed by
5-min incubation with Bt
cAMP (1 mM) and 1-min
stimulation with 100 nM FMLP, and analyzed by gel renaturation
assay and phosphorimaging. D, neutrophils or cytoplasts were
incubated for 10 min with 1 mM Bt
cAMP, stimulated
with 100 nM FMLP for 1 min, and analyzed for MBPp kinase
activity. Results are expressed as the percent of stimulated MAPK
activity in the absence of drugs and are given as the mean ±
S.E. of three experiments.
Yu et al.(37) have recently reported that cAMP failed to inhibit
FMLP-stimulated MAPK activity in human neutrophils. To explore this
discrepancy we compared the effect of BtcAMP on
FMLP-stimulated MAPK activity in neutrophils and cytoplasts (Fig. 6D). As measured by the MBPp kinase activity
assay, 1 mM Bt
cAMP inhibited FMLP-stimulated
cytoplast MAPK activity by 53 ± 13% but had no effect on
FMLP-stimulated MAPK activity in neutrophils. Thus, a regulatory role
for cAMP in MAPK activation can be observed in vitro in
lysates from cytoplasts but not from intact neutrophils suggesting that
a factor(s) derived from the nucleus and/or cytoplasmic granules masks
the effect.
Figure 7:
Chemoattractant-induced homotypic
aggregation and MAPK activation in neutrophils and cytoplasts.
Neutrophils (10/ml) (A) and cytoplasts (5
10
/ml) (B) prepared from the same donor were
analyzed for homotypic aggregation in response to FMLP (100
nM), LTB
(300 nM), C5a (100 nM),
and Il-8 (100 nM). The correlations between MAPK activation
(abscissae) and aggregation (ordinate) in neutrophils (C) and
cytoplasts (D) in response to various chemoattractants were
plotted, and regression coefficients were calculated. Results shown are
representative (A and B) or the means (C and D) of three experiments.
Figure 8:
BtcAMP inhibits
FMLP-stimulated homotypic aggregation in cytoplasts but not in
neutrophils. Neutrophils or cytoplasts were incubated for 15 min at 37
°C in the absence or presence of 1 mM Bt
cAMP
and then assayed for homotypic aggregation in response to 100 nM FMLP. Results shown are the means ± S.E. for three
experiments.
In contrast to the chemoattractants, insulin activated
neutrophil and cytoplast MAPK but failed to stimulate neutrophil
aggregation, suggesting that MAPK activation may be necessary but not
sufficient to support aggregation. We therefore tested whether insulin
could prime neutrophils for chemoattractant-stimulated functions.
Preincubation of neutrophils with insulin had little or no effect on
homotypic aggregation stimulated by concentrations of chemoattractants
inducing maximal aggregation responses. However, preincubation with
insulin for 10 min primed neutrophils for aggregation in response to
concentrations of FMLP and LTB that induced submaximal
aggregation responses (Fig. 9). Shorter incubations (i.e. times at which insulin failed to stimulate MAPK activity) had no
effect on aggregation. The priming effect of insulin on FMLP- and
LTB
-stimulated aggregation was dose-dependent, peaking at
200 nM. A trend toward insulin priming of neutrophils for
C5a-stimulated aggregation was observed but did not achieve statistical
significance. Insulin had no effect on Il-8-stimulated aggregation.
Thus, the effect of 200 nM insulin on neutrophil homotypic
aggregation by submaximal concentrations of chemoattractants was
proportional to the ability of these chemoattractants to stimulate MAPK
activity in neutrophils (FMLP>LTB
>C5a>Il-8).
Insulin potentiation of FMLP- and LTB
-stimulated homotypic
aggregation was glucose-independent. In contrast, insulin had no direct
effect on FMLP-induced O
generation and
-glucuronidase release and potentiated these responses only in the
presence of glucose, presumably by increasing glucose transport and
affecting metabolism.
Figure 9:
Insulin primes neutrophils for
chemoattractant-stimulated homotypic aggregation. Neutrophils were
incubated in the presence of the indicated concentrations of insulin
for 10 min at 37 °C, stimulated with FMLP (10 nM),
LTB (3 nM), C5a (10 nM), or IL-8 (10
nM) at concentrations determined to induce submaximal
neutrophil aggregation and analyzed for homotypic aggregation. Results
shown are the means ± S.E. for three or more
experiments.
Although well established, the link between
chemoattractant-stimulated G protein signaling pathways and the MAPK
cascade is poorly elucidated. Neutrophils are a good system in which to
study G protein-mediated signaling because the cellular responses are
rapid and easily quantitated. Enucleate, granule-depleted neutrophil
cytoplasts retain the capacity to respond to chemoattractants (30) and thus represent a simplified system useful in studying
chemoattractant signaling through G proteins. We employed neutrophils
and cytoplasts to study the kinetics of chemoattractant-stimulated
activation of p21, Raf-1, and MAPK and observed an
association between MAPK activation and cell-cell adhesion.
The
analysis of MAPK activity in cytoplast lysates by an MBP kinase gel
renaturation assay offered distinct advantages over similar studies of
lysates of intact neutrophils, including resolution of two MAPKs in
cytoplast lysates, identified by immunoprecipitation as p44 and p42
. Moreover, the ability to terminate
stimulation by direct addition of SDS sample buffer permitted more
accurate kinetic analysis of cytoplasts than of neutrophils.
FMLP-stimulated cytoplast MAPK activation was rapid and transient,
consistent with a role for MAPK in signaling pathways for neutrophil
functions such as O
generation,
degranulation, and cell-cell adhesion but slower than the previously
reported kinetics of neutrophil MAPK
activation(19, 27, 31) . The greater
precision afforded by kinetic analysis of MAPK in cytoplasts thus
allowed comparison with the kinetics of activation of other putative
elements in the FMLP-stimulated MAPK cascade, such as p21
and Raf-1. The use of cytoplasts also permitted observation of
MAPK signaling in the absence of nuclear or granular elements. Thus,
phosphorylation and dephosphorylation on tyrosine residues of
p42
in cytoplasts, with kinetics paralleling those of
MAPK activation, indicate that the molecular machinery required for
regulating MAPK activity by Erk kinase and phosphatase activities is
retained in cytoplasts and so independent of any nuclear factors that
may regulate MAPK. This observation may distinguish neutrophils from
proliferating cells, in which activated MAPK translocates to the
nucleus where it is down-regulated by dual
phosphothreonine/phosphotyrosine phosphatases such as
PAC-1(32) .
Although a wide variety of G protein-linked and
non-G protein-linked receptors have been demonstrated on neutrophils,
none of the classical PTKRs have been reported. Insulin, however, has
been shown to bind to human neutrophils(33) , stimulate
chemokinesis(34) , and prime for chemotaxis to
FMLP(35) , indicating that PTKRs for insulin are expressed on
these cells. Our observation that insulin activated MAPK in neutrophils
and cytoplasts suggests that a p21/Raf-1 pathway is
functionally intact and can be engaged by at least one PTKR. Thus, the
neutrophil formyl peptide receptor may also activate MAPK through the
p21
/Raf-1 pathway. However, the longer latency for
insulin- than for FMLP-stimulated MAPK activation indicates that the
pathways to p21
activation may be distinct.
Our
observation that FMLP activated p21 in neutrophils
supports p21
/Raf-1 signaling. Although a previous study
came to the same conclusion using neutrophils metabolically labeled
with [
P]orthophosphate(36) , we found
the bench life of neutrophils insufficient to label nucleotide pools to
equilibrium, an absolute requirement for interpreting GTP/GDP ratios of
GTPase-bound nucleotide as an indicator of p21
activation. We therefore analyzed total labeled guanine
nucleotide associated with immunoprecipitated p21
from
lysates of cells electroporated in the presence of
[
-
P]GTP and found maximal increase after 30
s of exposure to FMLP (i.e. preceding peak MAPK activity).
Concordant with a prior report(36) , the proportion of
[
-
P]GTP associated with p21
declined by 5 min, suggesting sequential guanine nucleotide
exchange factor and GTPase-activating protein activities following FMLP
stimulation.
Raf-1 has been shown to translocate from CS to PM in
cultured cells exposed to serum(7) . However, Raf-1
translocation in response to ligation of neither a specific PTKR nor a
G protein-linked receptor has been demonstrated in any cell type. We
have shown that cytoplasts are an ideal system with which to assay PM
translocation of cytosolic proteins(24) . Using this system, we
now report FMLP-stimulated translocation of Raf-1 to the PM. These data
complement those of Worthen et al.(36) who reported
FMLP-stimulated Raf-1 kinase activity. Like p21 activation, FMLP-stimulated Raf-1 translocation preceded MAPK
activation. Our failure to observe SOS translocation in response to
FMLP suggests that SOS may not participate in G protein activation of
p21
. Alternatively, translocation of SOS may not be
necessary for its activity, or the kinetics of SOS translocation may be
too rapid to have been appreciated in our assay.
Our observation
that MAPK activity in cytoplasts was inhibited by agents that raise
intracellular cAMP and that a PKA antagonist reversed this inhibition
is also consistent with p21/Raf-1-dependent signaling
since cAMP has been shown to down-regulate MAPK activation by
PKA-dependent phosphorylation of Raf-1, inhibiting
p21
/Raf-1 interactions(12, 13) .
Bt
cAMP has been shown to inhibit neutrophil Raf-1 kinase
activity(36) , supporting an effect of cAMP in neutrophils at
the level of Raf-1. However, Bt
cAMP inhibition of
FMLP-stimulated MAPK activation has not previously been demonstrated.
Indeed, Yu et al.(37) reported that
Bt
cAMP does not inhibit FMLP-stimulated MAPK in
cytochalasin B-treated neutrophils. Our data confirm this observation
in intact neutrophils but show that cytoplasts express a cAMP-sensitive
pathway. The exposure of a cAMP-sensitive pathway in cytoplasts may be
explained by increased phosphodiesterase or Raf-1 phosphatase
activities in detergent lysates of granule-replete, nucleated
neutrophils. Alternatively, neutrophils may possess both cAMP-sensitive
and -insensitive G protein-linked pathways of MAPK activation, the
latter preferentially inactivated during cytoplast preparation. Indeed,
Faure and Bourne (38) have recently shown that cell lines in
which stimulation of MAPK activity by LPA is cAMP-insensitive
nevertheless demonstrate cAMP inhibition of Raf, suggesting a
Raf-independent pathway of MAPK activation.
Several groups have
proposed a role for MAPK in neutrophil O generation(39) . However, Yu et al.(37) have recently demonstrated that MAPK activation and
O
generation can be dissociated in
neutrophils. In contrast, we observed a good correlation between MAPK
activation and cell-cell adhesion in both neutrophils and cytoplasts.
The extent of MAPK activation correlated closely with the degree of
homotypic aggregation stimulated by each of four chemoattractants in
both cells and cytoplasts. Interestingly, both cell-cell adhesion and
MAPK responsiveness to LTB
, C5a, and Il-8 were
preferentially lost in the process of preparing cytoplasts relative to
their responsiveness toward FMLP. Whereas Yu et al.(37) observed a discordance between the marked inhibition
of O
generation by
Bt
cAMP(37, 40) and its failure to inhibit
MAPK activation in FMLP-stimulated neutrophils(37) , in our
studies Bt
cAMP inhibited neither FMLP-stimulated MAPK
activation nor FMLP-stimulated homotypic aggregation in neutrophils but
significantly inhibited both of these responses in cytoplasts. Thus the
effect of Bt
cAMP on FMLP-stimulated MAPK activity in both
neutrophils and cytoplasts correlated with its effect on cell-cell
adhesion. Our discovery that insulin both activated MAPK in human
neutrophils and primed these cells for homotypic aggregation in
response to chemoattractants demonstrates a further correlation between
MAPK activation and cell-cell adhesion. The inability of insulin to
directly stimulate aggregation suggests that MAPK may be necessary but
not sufficient to directly or indirectly regulate adhesion molecules.
The failure of insulin to stimulate or prime neutrophils for
O
generation or degranulation in the
absence of extracellular glucose supports the hypothesis that MAPK
regulates some but not all neutrophil functions. Thus, although
O
generation can be dissociated from
MAPK activation, our studies of neutrophils and cytoplasts support a
role for MAPK activation in cell-cell adhesion.
Neutrophil homotypic
aggregation is mediated by activation of the integrin
CD11b/CD18(25) . The activation states of integrins appear to
be regulated by interactions of the cytoplasmic domains of these
heterodimeric transmembrane glycoproteins with the actin cytoskeleton
through focal adhesion plaques(41) . Thus, MAPK might regulate
cell-cell adhesion through phosphorylation of molecules regulating
focal adhesion plaques. In addition to a hypothetical role in
regulating the actin cytoskeleton, MAPKs have a well-established role
in regulating the microtubule cytoskeleton by associating with and
phosphorylating microtubule-associated proteins(42) . The
relationship between MAPK activation and leukocyte adhesion suggests
new targets for anti-inflammatory drugs since leukocyte adhesion to
vascular endothelium is the first committed step in the inflammatory
response. Furthermore, insulin stimulation of neutrophil MAPK and
priming for chemoattractant-stimulated adhesion suggest a molecular
mechanism for impaired neutrophil function in type I diabetes, a state
of insulin deficiency associated with increased susceptibility to
bacterial infection.
Since neutrophils are terminally
differentiated, non-mitotic cells, the effects of MAPK on transcription
factors related to growth and differentiation are unlikely to be
relevant. Our studies with enucleate cytoplasts support this view.
Marshall (43) has recently proposed that the outcome of MAPK
signaling is dependent largely on its duration of activation. If so,
rapid MAPK activation in neutrophils may represent a distinct category
of signaling. Alternatively, differentiated cells might also be
distinguished by their complement of MAPK substrates. The only
well-defined MAPK substrate also implicated in neutrophil activation is
cytoplasmic phospholipase A(44) . Further studies
are likely to identify other MAPK substrates involved in rapid
neutrophil responses.