C-reactive Protein Inhibits Chemotactic Peptide-induced p38
Mitogen-activated Protein Kinase Activity and Human Neutrophil
Movement*
Rita M.
Heuertz
§¶,
Sally M.
Tricomi
,
Uthayashanker R.
Ezekiel
, and
Robert O.
Webster
§
From the Departments of
Internal Medicine and
§ Molecular Microbiology and Immunology, Saint Louis
University School of Medicine and the
Department of
Neurology, Washington University School of Medicine,
St. Louis, Missouri 63110
 |
ABSTRACT |
Serum levels of the acute-phase reactant,
C-reactive protein (CRP), increase dramatically during acute
inflammatory episodes. CRP inhibits migration of neutrophils toward the
chemoattractant, f-Met-Leu-Phe (fMLP) and therefore acts as an
anti-inflammatory agent. Since tyrosine kinases are involved in
neutrophil migration and CRP has been shown to decrease phosphorylation
of some neutrophil proteins, we hypothesized that CRP inhibits
neutrophil chemotaxis via inhibition of MAP kinase activity. The
importance of p38 MAP kinase in neutrophil movement was determined by
use of the specific p38 MAP kinase inhibitor, SB203580. CRP and
SB203580 both blocked random and fMLP-directed neutrophil movement in a
concentration-dependent manner. Additionally, extracellular
signal-regulated MAP kinase (ERK) was not involved in fMLP-induced
neutrophil movement as determined by use of the MEK-specific inhibitor,
PD98059. Blockade of ERK with PD98059 did not inhibit chemotaxis nor
did it alter the ability of CRP or SB203580 to inhibit fMLP-induced
chemotaxis. More importantly, CRP inhibited fMLP-induced p38 MAP kinase
activity in a concentration-dependent manner as measured by
an in vitro kinase assay. Impressively, CRP-mediated
inhibition of p38 MAP kinase activity correlated with CRP-mediated
inhibition of fMLP-induced chemotaxis (r =
0.7144).
These data show that signal transduction through p38 MAP kinase is
necessary for neutrophil chemotaxis and that CRP intercedes through
this pathway in inhibiting neutrophil movement.
 |
INTRODUCTION |
Upon the onset of inflammation, there is an increase in the
concentration of a number of plasma proteins which are collectively termed acute-phase reactants. C-reactive protein
(CRP),1 the classic
acute-phase reactant in man, has a normal serum concentration of less
than 1 µg/ml; however, within 24 h after onset of inflammation, levels can increase as much as 1000-fold (1). This dramatic increase in
CRP suggests an important role for CRP in inflammation and the fact
that CRP is highly conserved throughout evolution indicates an
important biological role for CRP. Besides this humoral acute-phase
response, there are also cellular components of acute inflammation.
Neutrophils are among the first leukocytic cells to migrate into
tissues in response to invading pathogens or other initiators of
inflammatory injury. One of the first steps of neutrophil involvement
in acute inflammation is chemotaxis, directed movement toward
chemotactic agents, such as complement fragments (C5a and C5a des-Arg),
cytokines (interleukin-8), and bacteria-derived peptides such as
formyl-methionine-leucine-phenylalanine (fMLP).
CRP is present at sites of inflammation in association with
neutrophils. Selective localization of CRP to sites of inflammation in vivo has been shown by double staining rabbit muscle
tissue sections to visualize necrotic muscle associated with CRP (2). Du Clos et al. (3) induced allergic encephalomyelitis in
rabbits and found increased neutrophil numbers in the spinal cord
sections that correlated with elevated spinal fluid CRP levels. Parish showed that serum CRP levels in patients with chronic neutrophil vasculitis lesions were double that seen in patients with chronic mononuclear cell vasculitis lesions (4). In humans, neutrophils have
been implicated in the pathogenesis of several acute inflammatory diseases such as acute myocardial infarction, bacterial infections, and
acute respiratory distress syndrome (5-7). Acute respiratory distress
syndrome is a noncardiogenic pulmonary edema with characteristics of an
acute inflammatory reaction located primarily in the lung causing gross
alterations in lung structure and function. In addition to massive
influx of neutrophils into the alveolar space, significant elevations
of CRP have been found in serum (8) and bronchoalveolar lavage fluid
(9) from acute respiratory distress syndrome patients. CRP plays a role
in modulating inflammatory and immune responses as a result of its
ability to activate the classical complement pathway (10, 11), to
modify platelet-activating factor-induced platelet aggregation (12), to
bind T lymphocytes and suppress certain lymphocyte functions (13), and
to enhance phagocytosis (14, 15). We have shown that CRP inhibits
neutrophil chemotaxis in vitro (8) and neutrophil alveolitis
in vivo (16-18).
Little is known about CRP-induced signal transduction in the
neutrophil. A specific CRP receptor is present on the surface of
neutrophils (19, 20) but the identity of the receptor remains elusive.
CRP pretreatment of human neutrophils stimulated with fMLP results in a
significant reduction in the degree of phosphorylation of several
neutrophil proteins (21). Stimulation of the fMLP receptor on the
neutrophil results in activation of two known members of the MAP kinase
family, extracellular signal-regulated kinase (ERK) and p38 MAP kinase
(22, 23). Activation of p38 MAP kinase, but not ERK, is indispensable
for fMLP-induced chemotaxis of human neutrophils (22). Inhibition of
p38 MAP kinase blocks transforming growth factor-
-induced neutrophil
chemotaxis and actin polymerization (24) and
2-integrin
mobilization from cytoplasmic granules to the plasma membrane (25).
Activation of p38 MAP kinase leads to the activation of
mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2
(26) which phosphorylates the small heat shock protein, hsp27 (27, 28). Hsp27 has chaperone-like properties and acts as an inhibitor of actin
polymerization (29, 30). Phosphorylation of hsp27 stimulates the
polymerization of actin (31) and so help to repair the actin microfilament network disrupted during cellular stress thereby maintaining cellular integrity and aiding cell survival during stress.
In the current study, we hypothesized that CRP inhibits neutrophil
movement by blocking the p38 MAP kinase signal transduction pathway.
Here we show that inhibition of p38 MAP kinase with the specific
inhibitor, SB203580, inhibits directed and random movement of
neutrophils similar to that seen with CRP. Additionally, we show that
CRP inhibits p38 MAP kinase activity and that inhibition of p38 MAP
kinase activity correlates with inhibition of chemotaxis.
 |
EXPERIMENTAL PROCEDURES |
Human C-reactive Protein--
Human CRP was obtained from a
commercial source (Biodesign International, Kennebunk, ME). SDS-PAGE of
the human CRP followed by silver stain revealed a single band at 23 kDa
and was ascertained as pure by densitometry (Molecular Dynamics,
Sunnyvale, CA). The concentration of endotoxin in the purified CRP was
132 endotoxin units/ml (11 ng/ml) as determined by the
Limulus amebocyte assay (Associates of Cape Cod, Woods Hole,
MA). Immunoblotting for serum amyloid P, a common contaminant found in
CRP preparations, was negative. Concentration of the purified CRP was
determined by the BCA protein assay (Pierce Chemical Co., Rockford,
IL). In all experiments, CRP concentrations were expressed as
micrograms/ml. Using 125 kDa as the molecular mass of native human CRP,
100 µg/ml corresponded to 0.8 µM native molecule.
Isolation of Neutrophils--
Venous blood obtained from normal
healthy human volunteers was treated with EDTA (0.15%) to prevent
coagulation and layered onto NIMTM isolation media
(Cardinal Associates, Santa Fe, NM) using a ratio of 1 part
NIMTM to 1.4 parts whole blood. After centrifugation
(630 × g, 22 °C, 40 min), the granulocyte-rich
fraction was collected and recentrifuged (1000 × g,
4 °C, 10 min). The cell pellet was resuspended in sterile phosphate-buffered (pH 7.3) saline and hypotonic lysis performed to
remove contaminating erythrocytes. Neutrophils were pelleted and washed
once with sterile phosphate-buffered saline. Neutrophil yields were
routinely >95% with >98% viability.
Neutrophil Chemotaxis--
Chemotaxis was quantified by the
leading front method of Zigmond and Hirsch (32) utilizing a 48-well
microchemotaxis chamber (Neuroprobe, Cabin John, MD) and nitrocellulose
filters with a pore size of 3 µm (Neuroprobe). All samples were
prepared in chemotaxis buffer consisting of Hanks' balanced salt
solution containing 10 mM Hepes (pH 7.4, Sigma) and 1%
bovine serum albumin (Sigma). Neutrophils were pretreated with the p38
MAP kinase inhibitor, SB203580 (Calbiochem, San Diego, CA), the
MEK1/MEK2 inhibitor, PD98059 (Calbiochem) at a concentration (100 µM) that blocks ERK1 and ERK2 activities (33), human CRP
or normal human plasma with or without addition of purified human CRP
for 10 min at 23 °C. Buffer or the chemoattractant fMLP (1 × 10
7 M) was added in duplicate to the lower
chambers and neutrophils (200,000/well) were added to the upper
chambers. After incubation for 35 min at 37 °C, the filters were
removed, fixed in isopropyl alcohol, and stained with Harris acid
hematoxylin. Either net neutrophil migration (distance moved in
response to treatment minus distance moved in response to buffer
(µm/35 min)) or percent inhibition of chemotaxis ([1
net
migration of treatment group divided by net migration of fMLP] × 100)
was reported. In order to study mechanisms by which CRP inhibits
neutrophil chemotaxis, an optimal concentration of fMLP (0.1 µM) was used. At this concentration of fMLP,
concentrations of SB203580 severalfold higher than the IC50
were used.
Neutrophil Treatment and Preparation of Whole Cell
Lysates--
Purified neutrophils were diluted to 1 × 108/ml in Hank's balanced salt solution with 0.25%
bovine serum albumin (HBSA: Sigma) and 2 × 107 cells
were pretreated with CRP by incubation for 10 min at 23 °C. In some
experiments, cells were treated with 50% normal human plasma with or
without the addition of purified human CRP. Cells were activated by the
addition of fMLP (1 × 10
7 M) followed
by incubation at 37 °C for 1 min after which all tubes were
immediately centrifuged (200 × g, 10 min, 4 °C) to pellet the neutrophils. Supernatants were aspirated and pellets resuspended in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, 1 mM Na3VO4,
1 µg/ml leupeptin) containing 1% protease inhibitor mixture (Sigma).
Reaction mixtures were sonicated on wet ice at 35% power for ten 1-s
pulses using a Sonic Dismembrator 300 (Fisher Scientific, Pittsburgh,
PA). Reactions were then centrifuged (15,000 × g, 2 min, 4 °C) and supernatants quantitated for protein content using
the BCA assay (Pierce Chemical Co.). Neutrophil cell lysates were
frozen at
70 °C until assayed.
Immunoprecipitation of p38 MAP Kinase and Phosphorylation of p38
MAP Kinase Substrate, ATF2--
Neutrophil lysates (200 µg) were
incubated overnight at 4 °C with 4 µl of rabbit anti-human p38 MAP
kinase polyclonal antibody (New England Biolabs, Beverly, MA) followed
by adsorption with protein A-Sepharose (1 h, 4 °C: Pharmacia Biotech
Inc., Piscataway, NJ). Samples were centrifuged (15,000 × g, 1 min, 4 °C), washed twice with lysis buffer, and
twice with kinase reaction buffer (25 mM Tris, pH 7.5, 5 mM
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM
MgCl2). Each pellet was resuspended and incubated (30 min,
30 °C) in kinase buffer containing 200 µM ATP (New
England Biolabs) and 2 µg of ATF-2 fusion protein (New England
Biolabs). Reactions were stopped by addition of 6 × Laemmli
buffer and boiling for 5 min. Cell lysates (50 µg of protein/lane)
were resolved on 12.75% SDS-PAGE gels. Proteins were then transferred
to nitrocellulose membranes (0.45 µm pore size: Micron Separations
Inc., Westboro, MA) using a semidry transfer unit (Fisher Scientific).
Each membrane was stained with Ponceau S (Sigma) to verify efficient
and equal transfer of protein samples. Membranes were blocked with 5%
nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST:
1 h, 23 °C) followed by overnight incubation at 4 °C with
rabbit anti-human phospho-ATF2 (Thr71) peptide (New England
Biolabs) in TBST with 5% bovine serum albumin (1:1000). The secondary
antibody was horseradish peroxidase-conjugated anti-rabbit IgG diluted
1:2000. To detect the phosphorylated ATF2-antibody-peroxidase complex,
enhanced chemiluminescence was used (NEN Life Science Products, Boston,
MA). Band intensity was quantitated by densitometry (Molecular
Dynamics, Sunnyvale, CA). Results were expressed as the ratio of ATF-2
intensity units to IgG intensity units (treatment/load control) or as
the percent of the positive control (activity of treatment group
divided by activity of fMLP group × 100).
p38 MAP Kinase-induced MAPKAP Kinase 2 Phosphorylation of
Hsp27--
Neutrophils were treated with CRP or HBSA buffer for 10 min
at 23 °C followed by exposure to fMLP (1 × 10
7
M) or HBSA buffer for 1 min at 37 °C. Cells were
centrifuged (200 × g, 4 °C, 10 min) and pellets
suspended vigorously in lysis buffer (20 mM Tris-HCl, pH
7.5, 150 mM NaCl, 1 mM EGTA, 1 mM
EDTA, 1% Triton X-100, 0.5% Igepal CA-630, 20 mM sodium
fluoride, 2 mM Na3VO4, 5 mM phenylmethylsulfonyl fluoride, and 100 µl of
antiprotease mixture (Sigma)). Reaction mixtures were centrifuged
(3000 × g, 4 °C, 10 min) and equal volumes of
supernatant and kinase buffer (30 mM HEPES, pH 7.3, 20 mM MgCl2, 2 mM EGTA, 5 µM okadaic acid, 30 µM H-7 protein kinase C
inhibitor, 10 µM Na3VO4, 4 mM dithiothreitol, 30 µg/ml hsp27, and 60 µCi/ml
[
-32P]ATP (NEN Life Science Products Inc.)) were
mixed. Reactions were incubated (30 °C, 10 min) and stopped by the
addition of 6 × Laemmli buffer and boiling (100 °C, 5 min).
Reactants were separated by SDS-PAGE and protein bands stained with
Coomassie Blue to ensure equal protein loading in each lane. Gels were
dried and band intensity quantitated using a PhosphorImager (Molecular Dynamics). Results were expressed as percent of unstimulated control.
Statistical Analysis--
Data are reported as the mean ± S.E. Comparisons of sample means were analyzed using a repeated
measures ANOVA followed by a Tukey multiple range test. To determine if
a difference existed between treatments (CRP or SB203580 ± PD98059), a two-way ANOVA was performed. A correlation coefficient was
obtained by linear regression of percent inhibition of neutrophil
chemotaxis on p38 MAP kinase activity displayed as percent of positive
control. Differences with p < 0.05 were considered significant.
 |
RESULTS |
CRP Inhibits Neutrophil Movement--
Since chemotaxis of
neutrophils is an indicator of the cellular inflammatory response, the
effect of purified human CRP on neutrophil chemotaxis to the bacterial
chemotactic peptide, fMLP, was examined. To gain insight into
mechanistic aspects of CRP on neutrophil motility, random movement was
also assessed. CRP inhibited fMLP-induced chemotaxis and random
movement of neutrophils in a concentration-dependent manner
(Fig. 1) similar to what has been
reported (8, 18). CRP significantly inhibited neutrophil chemotaxis
from 100 to 500 µg/ml and random movement at 100 and 200 µg/ml. The
50% inhibitory concentration (IC50) for neutrophil chemotaxis to fMLP was 90 µg/ml and for random movement was 80 µg/ml.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of C-reactive protein on random and
fMLP-induced neutrophil movement. Human neutrophils were
pretreated with CRP or buffer for 10 min at 23 °C followed by
evaluation of movement toward fMLP (chemotaxis, ) or buffer (random
movement, ). Buffer or chemoattractant (fMLP, 1 × 10 7 M) was added in duplicate to the lower
chambers and neutrophils (200,000/well) were added to the upper
chambers. Movement was reported as net neutrophil migration (distance
moved in response to treatment minus distance moved in response to
buffer (µm/35 min)). n = 3. *, p < 0.05 versus no CRP control.
|
|
Blockade of p38 MAP Kinase Inhibits Neutrophil Movement--
Since
it has been reported that activation of p38 MAP kinase is necessary for
fMLP-induced chemotaxis of human neutrophils (22), we wanted to
determine whether random movement also required p38 MAP kinase and to
verify that the p38 MAP kinase specific inhibitor, SB203580, blocked
fMLP-induced chemotactic movement. As shown in Fig.
2, blockade of p38 MAP kinase inhibited
fMLP-induced chemotaxis and random movement of neutrophils in a
concentration-dependent manner. SB203580 effectively
inhibited fMLP-induced chemotaxis from 39.5 ± 5.3 µm/35
min (fMLP) to 31.3 ± 7.2, 15.3 ± 6.7, and 6.0 ± 6.6 µm/35 min (30, 50, and 100 µM SB203580,
respectively) with the maximal degree of inhibition being 85%.
Additionally, SB203580 blocked random movement concentration
dependently from 0 to
22.3 ± 7.0 µm/35 min (100 µM SB203580). These results indicate p38 MAP kinase is
involved in both random and fMLP-induced neutrophil movement. The
IC50 for chemotaxis to fMLP was 45 µM.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of p38 MAP kinase on random and
fMLP-induced neutrophil movement. Human neutrophils were isolated
and pretreated with the p38 MAP kinase specific inhibitor, SB203580, or
buffer for 10 min at 23 °C followed by evaluation of movement toward
fMLP (1 × 10 7 M) (chemotaxis, ) or
buffer (random movement, ) as described in the legend of Fig. 1.
n = 4-6. *, p < 0.05 versus the buffer control.
|
|
Co-Addition of CRP and SB203580 Does Not Augment the Inhibitory
Effect--
To aid in understanding whether CRP and SB203580 inhibit
neutrophil chemotaxis through similar signal transduction pathways, the
effect of co-addition of CRP and SB203580 on fMLP-induced movement was
assessed. When CRP and SB203580 were both added at their
IC50 concentrations, no additive nor synergistic
augmentation of inhibition of fMLP-induced neutrophil chemotaxis was
observed over that seen with CRP alone (Fig.
3). These results suggest that both CRP
and SB203580 inhibit neutrophil movement via the p38 MAP kinase
pathway.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of co-addition of CRP and SB203580 on
neutrophil chemotaxis. Human neutrophils were isolated and
pretreated with IC50 amounts of CRP (90 µg/ml), SB203580
(45 µM), or CRP (90 µg/ml) plus SB203580 (45 µM) for 10 min at 23 °C followed by evaluation of
movement toward fMLP (1 × 10 7 M).
n = 4. *, p < 0.001 versus
fMLP control.
|
|
ERK Is Not Necessary for fMLP-induced Neutrophil Movement--
It
has been reported that p38 MAP kinase, but not ERK is required for
chemotaxis of neutrophils (22). We sought to determine whether blockade
of ERK1 and ERK2 affected the ability of CRP and SB203580 to inhibit
chemotaxis. PD98059 specifically inhibits ERK1 and ERK2 by blocking
their upstream activators, MEK1 and MEK2 (34). We confirmed the
findings of Zu et al. (22) that PD98059 did not block
fMLP-induced chemotaxis at any concentration tested (Fig.
4A). Additionally, inhibition
of ERK with PD98059 at a concentration (100 µM) that
completely blocks MEK-induced activation of ERK in neutrophils (33) did
not affect the ability of CRP (Fig. 4B) or SB203580 (Fig.
4C) to inhibit fMLP-induced neutrophil chemotaxis. These
results suggest that CRP and blockade of p38 MAP kinase inhibit
chemotaxis through a pathway separate from ERK.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of PD98059 on fMLP-induced neutrophil
chemotaxis. Human neutrophils were isolated and pretreated with
PD98059 alone (A), with PD98059 and CRP (B) or
with PD98059 and SB203580 (C) for 10 min at 23 °C
followed by evaluation of movement toward fMLP (1 × 10 7 M). The concentration of PD98059 used in
the combination experiments was 100 µM. Filled
squares, PD98059 added; Open squares, no PD98059.
n = 4.
|
|
Inhibition of fMLP-induced p38 MAP Kinase Activity by CRP--
In
view of the above results, a likely mechanism that explains the
inhibitory action of CRP on neutrophil chemotaxis is that CRP inhibits
p38 MAP kinase. For this reason, the effect of CRP on p38 MAP kinase
activity was examined. When p38 MAP kinase was immunoprecipitated and
an in vitro kinase assay performed to detect p38 MAP kinase
phosphorylation of its substrate, activating transcription factor-2
(ATF2), CRP was found to inhibit fMLP-induced p38 MAP kinase activity
of neutrophils, especially at concentrations
100 µg/ml (Fig.
5A). The presence of two
predominant bands was noted, one of the phosphorylated ATF2 substrate
and the other of the immunoprecipitating antibody (IgG) reacting with
the anti-IgG secondary antibody. Stimulation of neutrophils with fMLP
significantly activated p38 MAP kinase over unstimulated (vehicle)
cells as previously reported (23, 35) (Fig. 5B). Analysis of
CRP inhibition of p38 MAP kinase activity revealed that CRP inhibited
substrate phosphorylation by p38 MAP kinase in a
concentration-dependent fashion with significance observed at
100 and 200 µg/ml CRP.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of CRP on fMLP-induced p38 MAP
kinase activity (phosphorylation of activating transcription
factor-2). Purified human neutrophils were pretreated with CRP or
vehicle (Veh) for 10 min at 23 °C. Cells were then
activated by incubation with fMLP (1 × 10 7
M, 37 °C, 1 min), centrifuged, and cell pellets lysed.
Neutrophil lysates (200 µg) were incubated with anti-p38 MAP kinase
antibody followed by adsorption with protein A-Sepharose. An in
vitro kinase activity assay was performed on immunoprecipitated
p38 MAP kinase using ATF2 as substrate. Reactants were separated by
SDS-PAGE, immunoblotted using anti-phosphorylated-ATF-2 antibody, and
visualized by enhanced chemiluminescence. The presence of two bands was
noted: phosphorylated ATF2 substrate and immunoprecipitating antibody
(IgG) reacting with the anti-IgG secondary antibody. A representative
immunoblot is shown in A. Band intensity was quantitated by
densitometry and results were expressed in B as percent of
the positive control (activity of treatment group divided by activity
of fMLP group × 100) after normalization of the data as the ratio
of ATF-2 intensity units to IgG intensity units (treatment/load
control). n = 7. *, p < 0.05 versus the fMLP control. **, p < 0.01 versus the fMLP control.
|
|
CRP also inhibited p38 MAP kinase activity in an in vitro
kinase activity assay assessing MAPKAP kinase-2 phosphorylation of
hsp27. MAPKAP kinase-2 is a physiologic target of p38 MAP kinase (26)
and phosphorylates hsp27 (28). In neutrophils, fMLP activates MAPKAP
kinase-2 which phosphorylates and activates hsp27 (36). CRP inhibited
phosphorylation of hsp27 by MAPKAP kinase-2 in a concentration-dependent manner with significant inhibition
evident at 500 µg/ml (Fig. 6). This
result confirms that CRP inhibits p38 MAP kinase activity.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of CRP on fMLP-induced p38 MAP
kinase activity (phosphorylation of hsp27 by MAPKAP kinase-2 after
substrate phosphorylation by p38 MAP kinase). Purified human
neutrophils were pretreated with CRP or vehicle for 10 min at 23 °C.
Cells were activated with fMLP (1 × 10 7
M, 37 °C, 1 min), centrifuged, and cell pellets lysed.
Cell supernatants were reacted with hsp27 and
[ -32P]ATP. Reactions were stopped by boiling and
reactants separated by SDS-PAGE. Gels were dried and band intensity
quantitated using a PhosphorImager (Molecular Dynamics). Results were
expressed as percent of unstimulated control. n = 5. *,
p < 0.01 versus fMLP control.
|
|
Correlation of Inhibition of Neutrophil Chemotaxis with Inhibition
of p38 MAP Kinase Activity--
Interestingly, CRP concentrations
inhibitory for fMLP-induced p38 MAP kinase activity were also
inhibitory for fMLP-induced chemotaxis (compare Figs. 1 and
5B). As shown in Fig. 7,
CRP-mediated inhibition of fMLP-induced p38 MAP kinase activity
correlated with CRP-mediated inhibition of fMLP-induced chemotaxis with
a correlation coefficient of
0.7144.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Comparison of the effect of CRP on
fMLP-induced p38 MAP kinase activity with the percent inhibition of
fMLP-induced chemotaxis by CRP. Percent inhibition of fMLP-induced
chemotaxis was regressed onto p38 MAP kinase activity (percent positive
control). Concentrations of human CRP (µg/ml) are shown next to each
point on the regression line. The correlation coefficient was obtained
by linear regression of percent inhibition of neutrophil chemotaxis on
p38 MAP kinase activity. n = 7 and r = 0.7144.
|
|
Inhibition of fMLP-induced Neutrophil Chemotaxis and p38 MAP Kinase
Activity in Normal Plasma Spiked with CRP--
To examine the
physiological relevance of CRP inhibition of p38 MAP kinase activity
and correlative inhibition of neutrophil movement, the effects of
normal human plasma spiked with purified human CRP on neutrophil
chemotaxis and p38 MAP kinase activity were assessed. CRP addition to
normal plasma resulted in a concentration-dependent inhibition of fMLP-directed movement of neutrophils with significant inhibition seen at 200 µg/ml CRP (Fig.
8A). Similarly, CRP addition to normal plasma resulted in inhibition of fMLP-induced p38 MAP kinase
activity with significance observed at 200 µg/ml CRP (Fig. 8B). These results indicate that in the presence of normal
plasma, CRP inhibits both fMLP-directed neutrophil movement and p38 MAP kinase activity.

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of purified CRP addition to normal
plasma on fMLP-induced neutrophil movement and p38 MAP kinase
activity. Human neutrophils were pretreated with different
concentrations of buffer or CRP in 50% normal plasma for 10 min at
23 °C (chemotaxis) or 1 min at 37 °C (p38 activity assay)
followed by evaluation of fMLP (1 × 10 7
M)-induced movement (A) or p38 MAP kinase
activity (B). Assays were performed as described in the
legend to Figs. 1 and 5. Control, normal human plasma
diluted 1:2 with chemotaxis buffer; CRP (50, 100, or 200),
normal human plasma diluted 1:2 with chemotaxis buffer containing
purified human CRP (50, 100, or 200 µg/ml). n = 3. *,
p < 0.05 versus normal plasma
control.
|
|
 |
DISCUSSION |
Stimulation of neutrophils by chemotactic peptides leads to the
phosphorylation of several intracellular proteins believed to
participate in activation of the microbicidal function of these cells.
In the present study we described the inhibition of p38 MAP kinase by
CRP and correlated inhibition of kinase with arrest of movement. The
specific p38 MAP kinase inhibitor, SB203580, inhibited both
fMLP-directed and random movement of neutrophils similar to that seen
with CRP. Additionally, inhibition of ERK1 and ERK2 with the specific
inhibitor, PD98059, revealed no significant difference from control in
chemotaxis thereby supporting the hypothesis that ERK1 and ERK2 are not
involved in neutrophil movement. The role of ERK in neutrophil
migration remains unclear, however, since it has been reported that
inhibition of ERK with PD98059 does not inhibit fMLP-induced chemotaxis
(22, 37, 38) and that PD98059 potentiates the chemotactic response of
neutrophils (39). Finally, addition of purified CRP to normal plasma
resulted in inhibition of neutrophil chemotaxis and p38 MAP kinase
activity indicating that these results have physiological significance.
To our knowledge, this is the first report of an endogenous serum
protein which inhibits cell movement by blockade of an intracellular signal transduction pathway. The precise kinase inhibited by CRP in
this cascade remains to be elucidated. Since CRP inhibits
phosphorylation of p38 MAP kinase substrates, the expectation is that
the site of CRP action is upstream of or at the point of p38 MAP kinase activation. Additionally, the precise mechanism by which CRP inhibits p38 MAP kinase (or an upstream activator) remains undetermined. It is
known that CRP binds a specific receptor on neutrophils (19, 20) which
to date has not been cloned. After binding, CRP may activate its own
signal transduction pathway which intercepts and alters the
fMLP-induced signaling cascade. Alternatively, CRP may directly
intercept the signaling pathway of fMLP. Conceivably, CRP in its native
or one of its altered forms could enter the cell and act directly on a
member of the p38 MAP kinase cascade. Finally, CRP may inhibit signal
transduction through receptors with similar structural motifs, for
example, the seven transmembrane-spanning receptors of chemoattractants
such as fMLP, complement fragment (C5a), and interleukin-8 (reviewed in
Ref. 40). It has been reported that fMLP and C5a receptors are highly
conserved in the transmembrane and cytoplasmic domains while being
maximally divergent in the extracellular loops where ligand binds (41).
It is interesting to speculate that the ability of CRP to inhibit
neutrophil movement to several different chemoattractants (16-18) may
be by alteration of signaling through the conserved cytoplasmic domains.
It has been proposed that a specific signaling pathway for
chemoattractant-induced neutrophil movement exists (42). This hypothesis has been suggested based upon the observation that both
respiratory burst and migration of neutrophils require tyrosine phosphorylation in the signaling pathway; however, the former needs
phospholipase D activation and the latter does not (42). Signal
transduction therefore appears to be similar for respiratory burst and
migration until the pathway reaches phospholipase D, at which point
there is a split in the signaling paths. CRP-induced signal
transduction may intercept fMLP-induced signaling at a point after
phospholipase D activation and before p38 MAP kinase substrate
phosphorylation. In addition, SB203580 inhibits neutrophil chemotaxis
but not superoxide production whereas PD98059 potentiates superoxide
and has no effect on chemotaxis (22) indicating that p38 MAP kinase is
necessary for movement and ERK may be an important pathway for the
respiratory burst response.
Cell movement is of the utmost importance in phagocytic cells such as
neutrophils since migration to the site of infection is an important
early step in host defense against microbial invasion. The recruitment
of neutrophils and their accumulation at extravascular sites of
inflammation is stimulated by soluble chemotactic factors and is
accompanied by several biochemical events related to activation of the
cells. As is seen in many cell types, phosphorylation of p38 MAP kinase
initiates a cascade of activation events associated with the
cytoskeleton. Phosphorylation of p38 MAP kinase in neutrophils results
in the activation of MAPKAP kinase-2 which phosphorylates hsp27 and
lymphocyte-specific protein 1 (LSP1), both of which are F-actin-binding
proteins (31, 43). Additionally, LSP1 is reported to be the major
substrate for MAPKAP kinase-2 in human neutrophils (36, 43).
Neutrophils isolated from patients with neutrophil actin dysfunction
are defective in motility and fMLP-induced actin polymerization and
overexpress LSP1 (44). It is conceivable that inhibition of p38 MAP
kinase by CRP leads to decreased phosphorylation of LSP1 with resultant
loss of actin polymerization and cell motility.
It has also been shown that transient increases in intracellular
calcium are required for cellular release from previous sites of
attachment but does not prevent cell polarization, ruffling, or other
processes associated with motility (45). Interestingly, CRP has no
effect on transient calcium increases in stimulated neutrophils2 thereby
suggesting that CRP is not acting at the point of cellular release from
attachment sites but rather at the step of ruffling or polarization.
Phosphorylation of hsp27 by MAPKAP kinase-2 may play a role in membrane
ruffling since a nonphosphorylatable hsp27 mutant inhibits membrane
ruffles (reviewed in Ref. 46). CRP inhibition of p38 MAP kinase may be
eliciting its inhibitory effect on neutrophil movement by inhibiting
the p38 MAP kinase substrate, MAPKAP kinase-2, and its substrate,
hsp27. By inhibiting phosphorylation of hsp27, CRP may be preventing
the formation of membrane ruffles necessary for movement. Finally,
microtubules are necessary for neutrophil polarization and microtubule
inhibitors decrease neutrophil motility (47), revealing the importance
of polarization to the movement process. Activation of p38 MAP kinase
may play an important role in proper arrangement of microtubules and
CRP may be inhibiting movement at this step.
The basis for the current study was the observation that CRP
pretreatment of fMLP-stimulated human neutrophils resulted in a
significant reduction in the degree of phosphorylation of several neutrophil proteins (21). These included proteins that were not
identified but had molecular weights similar to p38 MAP kinase and
other members of the MAP kinase family. Since ERK1 and ERK2 are not
involved in F-actin polymerization of neutrophils (38) nor are they
required for the activation of MAPKAP kinase-2 or hsp27 (27) or other
actin-binding proteins, a likely candidate was p38 MAP kinase.
Of importance to this discussion is the report that inhibition of p38
MAP kinase prevents the progression of inflammation-mediated arthritis
in an animal model (48). Arthritis patients have increased serum CRP
levels (49) and interestingly, transgenic mice expressing a rabbit CRP
transgene had mild or no inflammation compared with animals not
expressing the transgene in an antigen-induced arthritis model (50).
Also, overexpression of rabbit CRP by these transgenic mice prevented
neutrophil movement into chemoattractant-instilled lungs in an acute
lung injury animal model (16). Therefore, elucidation of the mechanism
of inhibition of neutrophil movement is of great importance in models
of inflammation. The current findings support inhibition of p38 MAP
kinase as a mechanism by which CRP inhibits neutrophil movement.
Additionally, CRP may play important roles in regulation of other
neutrophil functions, thereby halting the acute inflammation process.
Future work will focus on determining the precise mechanism by which
CRP inhibits p38 MAP kinase.
 |
ACKNOWLEDGEMENTS |
We thank Tradina R. Pichon and Deborah A. Williams for technical assistance.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant HL51199 (to R. O. W.) and the American Heart
Association, Missouri Affiliate (to R. M. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Pulmonary
Medicine, Saint Louis University School of Medicine, 3635 Vista Ave. at
Grand Blvd., St. Louis, MO 63110. Tel.: 314-577-8732; Fax: 314-577-8859; E-mail: heuertzr{at}wpogate.slu.edu.
2
R. M. Heuertz, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
CRP, C-reactive
protein;
fMLP, f-Met-Leu-Phe;
MAP, mitogen-activated protein;
ERK, extracellular signal-regulated kinase;
MAPKAP, mitogen-activated
protein kinase-activated protein;
PAGE, polyacrylamide gel
electrophoresis;
ATF, activating transcription factor.
 |
REFERENCES |
-
Kushner, I.,
Gewurz, H.,
and Benson, M.
(1981)
J. Lab. Clin. Med.
97,
739-749[Medline]
[Order article via Infotrieve]
-
Kushner, I.,
and Kaplan, M. H.
(1961)
J. Exp. Med.
114,
961-973
-
Du Clos, T. W.,
Mold, C.,
Paterson, P. Y.,
Alroy, J.,
and Gewurz, H.
(1981)
Clin. Exp. Immunol.
43,
565-573[Medline]
[Order article via Infotrieve]
-
Parish, W. E.
(1976)
Clin. Allergy
6,
543-550[Medline]
[Order article via Infotrieve]
-
Ban, K.,
Ikeda, U.,
Takahashi, M.,
Kanbe, T.,
Kasahara, T.,
and Shimada, K.
(1994)
Cardiovasc. Res.
28,
1258-1262[Medline]
[Order article via Infotrieve]
-
Fowler, A. A.,
Hyers, T. M.,
Fisher, B. J.,
Bechard, D. E.,
Centor, R. M.,
and Webster, R. O.
(1987)
Am. Rev. Respir. Dis.
136,
1225-1231[Medline]
[Order article via Infotrieve]
-
Weiland, J. E.,
Davis, W. B.,
Holter, J. F.,
Mohammed, J. R.,
Dorinsky, P. M.,
and Gadek, J. E.
(1986)
Am. Rev. Respir. Dis.
133,
218-225[Medline]
[Order article via Infotrieve]
-
Kew, R. R.,
Hyers, T. M.,
and Webster, R. O.
(1990)
J. Lab. Clin. Med.
115,
339-345[Medline]
[Order article via Infotrieve]
-
Li, J. J.,
Sanders, R. L.,
McAdam, K. P. W. J.,
Hales, C. A.,
Thompson, B. T.,
Gelfand, J. A.,
and Burke, J. F.
(1989)
J. Trauma
29,
1690-1697[Medline]
[Order article via Infotrieve]
-
Kaplan, M. H.,
and Volanakis, J. E.
(1974)
J. Immunol.
112,
2135-2147[Medline]
[Order article via Infotrieve]
-
Claus, D. R.,
Siegel, J.,
Petras, K.,
Osmand, A. P.,
and Gewurz, H.
(1977)
J. Immunol.
119,
187-192[Medline]
[Order article via Infotrieve]
-
Vigo, C.
(1985)
J. Biol. Chem.
260,
3418-3422[Abstract]
-
Mortensen, R. F.,
Braun, D.,
and Gewurz, H.
(1977)
Cell Immunol.
28,
59-68[Medline]
[Order article via Infotrieve]
-
Ganrot, P. O.,
and Kindmark, C.-O.
(1969)
Scand. J. Clin. Lab. Invest.
24,
215-219[Medline]
[Order article via Infotrieve]
-
Mortensen, R. F.,
and Duszkiewicz, J. A.
(1977)
J. Immunol.
119,
1611-1616[Abstract]
-
Heuertz, R. M.,
Xia, D.,
Samols, D.,
and Webster, R. O.
(1994)
Am. J. Physiol.
266,
L649-L654[Abstract/Free Full Text]
-
Heuertz, R. M.,
Piquette, C. A.,
and Webster, R. O.
(1993)
Am. J. Pathol.
142,
319-328[Abstract]
-
Ahmed, N.,
Thorley, R.,
Xia, D.,
Samols, D.,
and Webster, R. O.
(1996)
Am. J. Respir. Crit. Care Med.
153,
1141-1147[Abstract]
-
Buchta, R.,
Pontet, M.,
and Fridkin, M.
(1987)
FEBS Lett.
211,
165-168[CrossRef][Medline]
[Order article via Infotrieve]
-
Dobrinich, R.,
and Spagnuolo, P. J.
(1991)
Arthritis Rheum.
34,
1031-1038[Medline]
[Order article via Infotrieve]
-
Buchta, R.,
Gennaro, R.,
Pontet, M.,
Fridkin, M.,
and Romeo, D.
(1988)
FEBS Lett.
237,
173-177[CrossRef][Medline]
[Order article via Infotrieve]
-
Zu, Y. L.,
Qi, J.,
Gilchrist, A.,
Fernandez, G. A.,
Vazquez-Abad, D.,
Kreutzer, D. L.,
Huang, C.-K.,
and Sha'afi, R. I.
(1998)
J. Immunol.
160,
1982-1989[Abstract/Free Full Text]
-
Nick, J. A.,
Avdi, N. J.,
Young, S. K.,
Knall, C.,
Gerwins, P.,
Johnson, G. L.,
and Worthen, G. S.
(1997)
J. Clin. Invest.
99,
975-986[Abstract/Free Full Text]
-
Hannigan, M.,
Zhan, L. J.,
Ai, Y. X.,
and Huang, C. K.
(1998)
Biochem. Biophys. Res. Commun.
246,
55-58[CrossRef][Medline]
[Order article via Infotrieve]
-
Schnyder, B.,
Meunier, P. C.,
and Car, B. D.
(1998)
Biochem. J.
331,
489-495[Medline]
[Order article via Infotrieve]
-
Rouse, J.,
Cohen, P.,
Trigon, S.,
Morange, M.,
Alonso-Llamazares, A.,
Zamanillo, D.,
Hunt, T.,
and Nebreda, A. R.
(1994)
Cell
78,
1027-1037[Medline]
[Order article via Infotrieve]
-
Cuenda, A.,
Rouse, J.,
Doza, Y. N.,
Meier, R.,
Cohen, P.,
Gallagher, T. F.,
Young, P. R.,
and Lee, J. C.
(1995)
FEBS Lett.
364,
229-233[CrossRef][Medline]
[Order article via Infotrieve]
-
Stokoe, D.,
Engel, K.,
Campbell, D. G.,
Cohen, P.,
and Gaestel, M.
(1992)
FEBS Lett.
313,
307-313[CrossRef][Medline]
[Order article via Infotrieve]
-
Miron, T.,
Vancompernolle, K.,
Vandekerckhove, J.,
Wilchek, M.,
and Geiger, B.
(1991)
J. Cell Biol.
114,
255-261[Abstract]
-
Knauf, U.,
Jakob, U.,
Engel, K.,
Buchner, J.,
and Gaestel, M.
(1994)
EMBO J.
13,
54-60[Abstract]
-
Lavoie, J. N.,
Lambert, H.,
Hickey, E.,
Weber, L. A.,
and Landry, J.
(1995)
Mol. Cell. Biol.
15,
505-516[Abstract]
-
Zigmond, S. H.,
and Hirsch, J. G.
(1983)
Am. Rev. Respir. Dis.
127,
290-300[Medline]
[Order article via Infotrieve]
-
Kuroki, M.,
and O'Flaherty, J. T.
(1997)
Biochem. Biophys. Res. Commun.
232,
474-477[CrossRef][Medline]
[Order article via Infotrieve]
-
Pang, L.,
Sawada, T.,
Decker, S. J.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
13585-13588[Abstract/Free Full Text]
-
Krump, E.,
Sanghera, J. S.,
Pelech, S. L.,
Furuya, W.,
and Grinstein, S.
(1997)
J. Biol. Chem.
272,
937-944[Abstract/Free Full Text]
-
Zu, Y. L.,
Ai, Y.,
Gilchrist, A.,
Labadia, M. E.,
Sha'afi, R. I.,
and Huang, C. K.
(1996)
Blood
87,
5287-5296[Abstract/Free Full Text]
-
Coffer, P. J.,
Geijsen, N.,
Mrabet, L.,
Schweizer, R. C.,
Maikoe, T.,
Raaijmakers, J. A. M.,
Lammers, J. W. J.,
and Koenderman, L.
(1998)
Biochem. J.
329,
121-130[Medline]
[Order article via Infotrieve]
-
Downey, G. P.,
Butler, J. R.,
Tapper, H.,
Fialkow, L.,
Saltiel, A. R.,
Rubin, B. B.,
and Grinstein, S.
(1998)
J. Immunol.
160,
434-443[Abstract/Free Full Text]
-
Zhong, W.,
Qin, Z.,
Tebo, J.,
Schlottmann, K.,
Coggeshall, M.,
and Mortensen, R. F.
(1998)
J. Immunol.
161,
2533-2540[Abstract/Free Full Text]
-
Murphy, P. M.
(1994)
Annu. Rev. Immunol.
12,
593-633[CrossRef][Medline]
[Order article via Infotrieve]
-
Alvarez, V.,
Coto, E.,
Setien, F.,
Gonzalez-Roces, S.,
and Lopez-Larrea, C.
(1996)
Immunogenetics
44,
446-452[CrossRef][Medline]
[Order article via Infotrieve]
-
Yasui, K.,
Yamazaki, M.,
Miyabayashi, M.,
Tsuno, T.,
and Komiyama, A.
(1994)
J. Immunol.
152,
5922-5929[Abstract/Free Full Text]
-
Huang, C.-K.,
Zhan, L.,
Ai, Y.,
and Jongstra, J.
(1997)
J. Biol. Chem.
272,
17-19[Abstract/Free Full Text]
-
Howard, T. H.,
Li, Y.,
Torres, M.,
Guerrero, A.,
and Coates, T.
(1994)
Blood
83,
231-241[Abstract/Free Full Text]
-
Marks, P. W.,
and Maxfield, F. R.
(1990)
J. Cell Biol.
110,
43-52[Abstract]
-
Ridley, A. J.
(1994)
BioEssays
16,
321-327[Medline]
[Order article via Infotrieve]
-
Malech, H. L.,
Root, R. K.,
and Gallin, J. I.
(1977)
J. Cell Biol.
75,
666-693[Abstract/Free Full Text]
-
Jackson, J. R.,
Bolognese, B.,
Hillegass, L.,
Kassis, S.,
Adams, J.,
Griswold, D. E.,
and Winkler, J. D.
(1998)
J. Pharmacol. Exp. Ther.
284,
687-692[Abstract/Free Full Text]
-
Morley, J. J.,
and Kushner, I.
(1982)
Ann. N. Y. Acad. Sci.
389,
406-417[Medline]
[Order article via Infotrieve]
-
Samols, D.,
Xia, D.,
Jiang, S.-L.,
Pizzuto, T.,
and Stevenson, S.
(1995)
Arthritis Rheum.
38,
S233 (abstr.)
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.