Am J Physiol Cell Physiol 286: C55–C64, 2004.
First published September 3, 2003; 10.1152/ajpcell.00131.2003.
Actin reorganization and morphological changes in human neutrophils
stimulated by TNF, GM-CSF, and G-CSF: the role of MAP kinases
Haruo Kutsuna,1,2 Kenichi Suzuki,1 Noriko Kamata,1 Takayuki Kato,1 Fumihiko Hato,1
Kensaku Mizuno,3 Hiromi Kobayashi,2 Masamitsu Ishii,2 and Seiichi Kitagawa1
1
Department of Physiology and 2Department of Dermatology, Osaka City University Medical School,
Asahi-machi, Abeno-ku, Osaka 545-8585; and 3Department of Biomolecular Sciences, Graduate
School of Life Sciences, Tohoku University, Aramaki-aza-aoba, Aoba-ku, Sendai 980-8578, Japan
Submitted 7 April 2003; accepted in final form 30 August 2003
Address for reprint requests and other correspondence: S. Kitagawa, Dept.
of Physiology, Osaka City Univ. Medical School, Asahi-machi, Abeno-ku,
Osaka 545-8585, Japan (E-mail: kitagawas@med.osaka-cu.ac.jp).
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.
neutrophil; actin reorganization; cytokines; mitogen-activated protein
kinase; tumor necrosis factor-␣; granulocyte-macrophage colony
stimulating factor
VARIOUS FUNCTIONS OF HUMAN
http://www.ajpcell.org
0363-6143/04 $5.00 Copyright © 2004 the American Physiological Society
C55
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017
neutrophils are activated by chemoattractants or inflammatory cytokines, such as tumor necrosis factor-␣ (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte CSF (G-CSF) (16, 41,
42). Some functions such as superoxide anion (O⫺
2 ) release and
adherence appear to be similarly activated by chemoattractants
and cytokines, whereas some functions such as locomotion and
morphological changes may be differentially affected by these
stimuli. For example, chemoattractants such as N-formyl-methionyl-leucyl-phenylalanine (FMLP) and interleukin-8 induce
neutrophil chemotaxis, whereas TNF, GM-CSF, and G-CSF
inhibit neutrophil chemotaxis (6, 13, 17, 28, 38). Polarization, a morphological change closely associated with neutrophil chemotaxis and actin polymerization, is induced by
chemoattractants but not by TNF (2). These findings suggest
that cytokines regulate actin reorganization in human neutrophils
in a different way from chemoattractants. It is well known that
chemoattractants induce an overall increase in F-actin content
and actin polymerization, although the precise mechanisms for
chemoattractant-induced actin reorganization remain to be elucidated (13, 28). On the other hand, actin reorganization induced by
inflammatory cytokines is largely unknown.
A major signaling system activated in human neutrophils
stimulated by cytokines is the mitogen-activated protein
kinase (MAPK) cascade (31, 32). In mammalian cells, there
are at least three MAPK subtypes, i.e., extracellular signalregulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase. Each MAPK subtype is activated by phosphorylation on threonine and tyrosine residues by upstream
dual-specificity kinases, such as MAPK/ERK kinase
(MEK), MAPK kinase-3 or -6 (MKK3/6), and MKK4/7.
Activation of the distinct MAPK subtype cascade is dependent on the types of cells and the stimuli used, and the
functional role of each MAPK subtype may be different
according to the types of cells (27, 39). Our recent studies
with human neutrophils show that distinct MAPK subtype
cascades are activated in a cytokine-specific manner and that
the MEK-ERK and MKK3/6-p38 MAPK cascades play an
important role in cytokine-induced activation of human
neutrophil functions such as O⫺
2 release, adherence, and
upregulation of 2-integrin (30, 31, 32).
In this study, we investigated actin reorganization and morphological changes in human neutrophils stimulated by TNF,
GM-CSF, and G-CSF, and the role of MAPKs in these responses. The results show that in contrast to a chemotactic
peptide FMLP, TNF, GM-CSF, and G-CSF induce an overall
decrease in F-actin content (actin depolymerization) and concomitant morphological changes in human neutrophils and
suggest that activation of ERK and p38 MAPK plays a critical
role in these responses.
Kutsuna, Haruo, Kenichi Suzuki, Noriko Kamata, Takayuki
Kato, Fumihiko Hato, Kensaku Mizuno, Hiromi Kobayashi,
Masamitsu Ishii, and Seiichi Kitagawa. Actin reorganization and
morphological changes in human neutrophils stimulated by TNF,
GM-CSF, and G-CSF: the role of MAP kinases. Am J Physiol Cell
Physiol 286: C55–C64, 2004. First published September 3, 2003;
10.1152/ajpcell.00131.2003.—Stimulation of human neutrophils with
tumor necrosis factor-␣ (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) resulted in
decreased fluorescence intensity of FITC-phalloidin (actin depolymerization) and morphological changes. Cytokine-induced actin depolymerization was dependent on the concentration of cytokines used as
stimuli. The maximal changes were detected at 10 min after stimulation with TNF or GM-CSF and at 20 min after stimulation with
G-CSF. Cytokine-induced actin depolymerization was sustained for at
least 30 min after stimulation. In contrast, N-formyl-methionyl-leucylphenylalanine (FMLP) rapidly (within 45 s) induced an increase in the
fluorescence intensity of FITC-phalloidin (actin polymerization) and
morphological changes. TNF- and GM-CSF-induced actin depolymerization and morphological changes, but not FMLP-induced responses, were partially inhibited by either PD-98059, an inhibitor of
mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase, or SB-203580, an inhibitor of p38 MAPK,
and were almost completely abolished by these inhibitors in combination. G-CSF-induced responses were almost completely abolished
by PD-98059 and were unaffected by SB-203580. These findings are
consistent with the ability of these cytokines to activate the distinct
MAPK subtype cascade in human neutrophils. Phosphorylated ERK
and p38 MAPK were not colocalized with F-actin in neutrophils
stimulated by cytokines or FMLP. Furthermore, FMLP-induced polarization and actin polymerization were prevented by cytokine pretreatment. These findings suggest that TNF, GM-CSF, and G-CSF
induce actin depolymerization and morphological changes through
activation of ERK and/or p38 MAPK and that cytokine-induced actin
reorganization may be partly responsible for the inhibitory effect of
these cytokines on neutrophil chemotaxis.
C56
ACTIN REORGANIZATION AND MAP KINASES
MATERIALS AND METHODS
AJP-Cell Physiol • VOL
RESULTS
TNF, GM-CSF, and G-CSF induce an overall decrease in
F-actin content in neutrophils. The F-actin content in human
neutrophils was analyzed using FITC-phalloidin. As shown in
Fig. 1, stimulation of neutrophils with FMLP resulted in rapid
increase in the fluorescence intensity, which reflects actin
Fig. 1. Effects of tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and N-formylmethionyl-leucyl-phenylalanine (FMLP) on F-actin content in neutrophils.
Neutrophils were stimulated with TNF (100 U/ml) or GM-CSF (5 ng/ml) for
10 min, G-CSF (50 ng/ml) for 20 min, or FMLP (10⫺7 M) for 45 s at 37°C.
F-actin content was measured by flow cytometry using FITC-phalloidin.
Dotted line and shaded area represent the profiles of unstimulated control cells
and unstained cells, respectively. Results shown are representative of 7
independent experiments.
286 • JANUARY 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017
Reagents. Highly purified recombinant human G-CSF, GM-CSF,
and TNF produced by Escherichia coli were provided by Kirin
Brewery (Tokyo, Japan), Schering-Plough (Osaka, Japan), and Dainippon Pharmaceutical (Osaka, Japan), respectively. The specific
activity of TNF was 3 ⫻ 106 U/mg protein. Endotoxin contamination
of each preparation was ⬍100 pg/mg protein. SB-203580 (p38 MAPK
inhibitor) was purchased from Calbiochem (California, CA). FMLP
and FITC-conjugated phalloidin were purchased from Sigma Chemical (St. Louis, MO). Conray was purchased from Mallinckrodt (St.
Louis, MO). Ficoll and the enhanced chemiluminescence (ECL)
Western blotting system were purchased from Amersham Pharmacia
Biotech (Buckinghamshire, England). PD-98059 (MEK inhibitor) and
rabbit polyclonal antibodies against ERK1/2, Thr202/Tyr204-phosphorylated ERK1/2, p38 MAPK, and Thr180/Tyr182-phosphorylated
p38 MAPK were purchased from Cell Signaling Technology (Beverley, MA). Alexa Fluor 568-labeled goat anti-rabbit IgG antibody was
purchased from Molecular Probes (Eugene, OR). Rabbit polyclonal
antibodies against cofilin and Ser3-phosphorylated cofilin were prepared as described previously (3, 37).
Preparation of cells. Human neutrophils were prepared from
healthy adult donors as described previously (31), using dextran
sedimentation, centrifugation with Conray-Ficoll, and hypotonic lysis
of contaminated erythrocytes. Neutrophil fractions contained ⬎98%
neutrophils. Cells were suspended in Hanks’ balanced salt solution
(HBSS) containing 10 mM N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid (HEPES) (pH 7.4).
Determination of actin reorganization and immunofluorescence.
Actin reorganization was analyzed using flow cytometry and confocal
laser scanning microscopy. Neutrophils (1 ⫻ 107/ml, 50 l) suspended in HBSS were stimulated with TNF, GM-CSF, G-CSF, or
FMLP for indicated periods at 37°C. When required, cells were
pretreated with PD-98059 (50 M) or SB-203580 (10 M) for 20 min
at 37°C before stimulation with each agent. The reactions were
performed in polystyrene tubes (Falcon 2054; BD Labware) precoated
with fetal calf serum (FCS) to prevent strong adhesive interaction
between cytokine-stimulated neutrophils and polystyrene surface,
which could affect actin reorganization. After incubation with each
agent, cells were fixed with IntraPrep Reagent 1 (Immunotech, Marseille, France) for 15 min at room temperature. After being washed
with phosphate-buffered saline (PBS), cells were permeabilized with
IntraPrep Reagent 2 (Immunotech) for 5 min at room temperature and
thereafter incubated with FITC-phalloidin (5 ng/ml) in the dark for 15
min at room temperature. Cells were washed with PBS, resuspended
in PBS containing 0.5% paraformaldehyde, and analyzed by flow
cytometry with FACS Calibur (Beckton Dickinson, Mountain View,
CA) using CellQuest analysis software or by confocal laser scanning
microscopy (Zeiss LSM510, Welwyn, Garden City, UK).
For indirect immunofluorescence, cells were similarly processed as
described above, and phospho-ERK1/2 or phospho-p38 MAPK antibody was added with FITC-phalloidin after permeabilization with
IntraPrep Reagent 2. After incubation in the dark for 15 min at room
temperature, cells were washed with PBS and stained with Alexa
Fluor 568-labeled goat anti-rabbit IgG antibody in the dark for 15 min
at room temperature. Cells were washed with PBS and resuspended in
PBS containing 0.5% paraformaldehyde. Fluorescence images were
photographed with a confocal laser scanning microscope.
Western blotting. Neutrophils (1 ⫻ 107/ml) suspended in HBSS
were prewarmed for 10 min at 37°C and thereafter stimulated with
TNF (100 U/ml), GM-CSF (5 ng/ml), or G-CSF (50 ng/ml) for 10 min
at 37°C. When required, cells were pretreated with PD-98059 (50
M) or SB-203580 (10 M) for 20 min at 37°C. The reactions were
terminated by rapid centrifugation, and the pellets were frozen in
liquid nitrogen, resuspended in ice-cold extraction buffer containing
50 mM HEPES (pH 7.4), 1% Triton X-100, 2 mM sodium orthovana-
date, 100 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 100 g/ml aprotinin, and 10 g/ml
leupeptin, and lysed for 10 min at 4°C. After rapid centrifugation, the
supernatant was mixed 1:1 with 2⫻ sample buffer [4% sodium
dodecyl sulfate (SDS), 20% glycerol, 10% mercaptoethanol, and a
trace amount of bromophenol blue dye in 125 mM Tris䡠HCl, pH 6.8],
heated at 100°C for 5 min and then frozen at ⫺80°C until use.
Samples were subjected to 10% SDS gel electrophoresis. After electrophoresis, proteins were electrophoretically transferred from the gel
onto a nitrocellulose membrane in a buffer containing 25 mM Tris,
192 mM glycine, and 20% methanol at 2 mA/cm2 for 1.5 h at 25°C.
Residual binding sites on the membrane were blocked by incubating
the membrane in Tris-buffered saline (pH 7.6) containing 0.1%
Tween 20 and 5% nonfat dry milk for 2 h at 25°C. The blots were
washed in Tris-buffered saline containing 0.1% Tween 20 and then
incubated with appropriate antibody overnight at 4°C. After being
washed, the membrane was incubated with anti-rabbit IgG antibody
conjugated with horseradish peroxidase, and the antibody complexes
were visualized by the ECL detection system as directed by the
manufacturer. Immunoreactive bands were quantified by a National
Institutes of Health (NIH) Image program on a Macintosh computer.
Kinase assay of p38 MAPK. The kinase activity of p38 MAPK was
determined by using a nonradioactive immunoprecipitation kinase
assay kit (Cell Signaling Technology) according to the manufacturer’s
instructions. Phosphorylated p38 MAPK was immunoprecipitated by
using immobilized monoclonal antibody against Thr180/Tyr182-phosphorylated p38 MAPK. The resulting immunoprecipitates were incubated with GST-ATF-2 fusion protein in the presence of ATP.
Phosphorylation of ATF-2 was determined by Western blotting using
antibody against Thr71-phosphorylated ATF-2. When required, PD98059 (50 M) and/or SB-203580 (1 or 10 M) were added to the
reaction mixture.
Statistical analysis. Student’s t-test was used to determine statistical significance.
ACTIN REORGANIZATION AND MAP KINASES
polymerization (13, 28). The maximal change was detected at
45 s after stimulation, and the increased fluorescence intensity
returned to the basal level within 5 min (Fig. 2A). In contrast
to FMLP, stimulation of neutrophils with TNF, GM-CSF, or
AJP-Cell Physiol • VOL
G-CSF resulted in decreased fluorescence intensity, which may
reflect actin depolymerization (Fig. 1). The maximal change in
the fluorescence intensity was detected at 10 min after stimulation with TNF or GM-CSF and at 20 min after stimulation
with G-CSF, respectively (Fig. 2A). No significant change was
detected at early time points (within 5 min) after stimulation
with each cytokine. The decreased fluorescence intensity induced by these cytokines was sustained at least for 30 min after
stimulation. The effect of TNF, GM-CSF, and G-CSF on the
changes in fluorescence intensity was dependent on the concentration of cytokines used as stimuli (Fig. 2B). Significant
effect was obtained at 1 U/ml TNF, 0.5 ng/ml GM-CSF, and 50
ng/ml G-CSF, respectively.
To further confirm that these cytokines induced actin depolymerization, cells were analyzed with confocal microscopy.
As shown in Fig. 3, confocal microscopy revealed that control
cells showed diffuse distribution of F-actin and round shape.
Stimulation of cells with TNF, GM-CSF, or G-CSF resulted in
an overall decrease in F-actin content with redistribution of
F-actin and concomitant morphological changes. In contrast,
FMLP induced an overall increase in F-actin content with
concomitant morphological changes. Typical polarization induced by FMLP was not detected at this early time point (28)
(Fig. 3, see also Fig. 6).
Involvement of ERK and p38 MAPK in actin depolymerization and morphological changes induced by TNF, GM-CSF, or
G-CSF. A major signaling system activated in human neutrophils in response to stimulation with TNF, GM-CSF, or G-CSF
is the MAPK cascades; specifically, the ERK and p38 MAPK
cascades (31). Our recent studies show that ERK and p38
MAPK play a critical role in cytokine-induced activation of
human neutrophil functions such as O⫺
2 release, adherence, and
upregulation of 2-integrin (30, 31, 32). Then, possible involvement of the MAPK cascades in cytokine-induced actin
depolymerization and morphological changes was explored
using PD-98059 (MEK inhibitor) (1) and SB-203580 (p38
MAPK inhibitor) (18). As shown in Fig. 4, pretreatment of
cells with PD-98059 (50 M) significantly and partially inhibited actin depolymerization induced by TNF or GM-CSF and
almost completely abolished actin depolymerization induced
by G-CSF. Similarly, pretreatment of cells with SB-203580 (10
M) also significantly and partially inhibited actin depolymerization induced by TNF or GM-CSF but not by G-CSF. These
findings are consistent with the ability of these cytokines to
activate the distinct MAPK subtype cascade in human neutrophils; i.e., ERK is activated by TNF, GM-CSF, and G-CSF,
whereas p38 MAPK is activated by TNF and GM-CSF, but not
by, or very weakly by, G-CSF (31) (see also Fig. 9A). When
PD-98059 (50 M) and SB-203580 (10 M) were used in
combination, TNF- or GM-CSF-induced actin depolymerization was almost completely abolished. G-CSF-induced actin
depolymerization was almost completely abolished by PD98059 alone. In contrast, neither PD-98059 nor SB-203580
affected FMLP-induced actin polymerization, in agreement
with the previous report (9) (Fig. 4). These inhibitors alone or
in combination also did not affect the rate of actin depolymerization (a “falling” phase of actin polymerization), which
followed rapid actin polymerization in response to FMLP (Fig.
4C, the data obtained with each inhibitor alone are not shown).
These findings obtained by flow cytometry were confirmed
by confocal microscopy. As shown in Fig. 3, TNF- or GM-
286 • JANUARY 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017
Fig. 2. Effects of TNF, GM-CSF, G-CSF, and FMLP on F-actin content in
neutrophils. A: neutrophils were stimulated with TNF (100 U/ml), GM-CSF (5
ng/ml), G-CSF (50 ng/ml), or FMLP (10⫺7 M) for indicated times at 37°C, and
then F-actin content was measured by flow cytometry. The change in F-actin
content is expressed as a relative fold change in the mean fluorescence intensity,
and the fluorescence intensity at time 0 is expressed as 1. Data are expressed as
means ⫾ SD of 3–6 experiments. *Significantly decreased compared with unstimulated control cells (P ⬍ 0.01). †Significantly increased compared with
unstimulated control cells (P ⬍ 0.01). B: neutrophils were stimulated with
indicated concentrations of TNF or GM-CSF for 10 min or G-CSF for 20 min at
37°C, and then F-actin content was measured by flow cytometry. The change in
F-actin content is expressed as a relative fold change in the mean fluorescence
intensity as described in A. Data are expressed as means ⫾ SD of 3 experiments.
*Significantly decreased compared with unstimulated control cells (P ⬍ 0.01).
C57
C58
ACTIN REORGANIZATION AND MAP KINASES
CSF-induced actin depolymerization as well as morphological
changes was partially inhibited by pretreatment of cells with
PD-98059 or SB-203580. When PD-98059 and SB-203580
were used in combination, TNF- or GM-CSF-induced actin
depolymerization as well as morphological changes was almost
completely abolished, and these cells showed apparently similar profiles to control cells (Fig. 3, A and B). On the other
hand, G-CSF-induced actin depolymerization as well as morphological changes was almost completely abolished by pretreatment of cells with PD-98059 and was unaffected by
SB-203580 (Fig. 3C). In contrast, neither PD-98059 nor SB203580 affected FMLP-induced actin polymerization and morphological changes (Fig. 3D).
FMLP-induced polarization and actin polymerization are
prevented by cytokine pretreatment. The results depicted in
Figs. 1–4 show that actin reorganization is differentially regulated by cytokines and FMLP, suggesting that FMLP-induced
polarization and actin polymerization might be affected by
cytokine pretreatment. To explore this possibility, neutrophils
were pretreated with each cytokine for 10 or 20 min and
AJP-Cell Physiol • VOL
thereafter challenged with FMLP. As shown in Fig. 5, later
challenge with FMLP caused significant actin polymerization
in TNF-, GM-CSF-, or G-CSF-pretreated neutrophils when
analyzed at 45 s after FMLP stimulation. However, the magnitude of FMLP-induced actin polymerization in cytokinepretreated cells was always less than polymerization that was
apparent in cells treated with FMLP alone (P ⬍ 0.05 for all
cytokines, n ⫽ 4). Confocal microscopy revealed that FMLPinduced polarization, which was detected at 10 min after
stimulation, was prevented by pretreatment of cells with TNF,
GM-CSF, or G-CSF (Fig. 6). Prevention or attenuation of
FMLP-induced actin polymerization by cytokine pretreatment
was also confirmed by confocal microscopy.
Phosphorylated ERK and p38 MAPK are localized separately from F-actin. The results described above indicate a
close relationship between actin depolymerization and activation of ERK and p38 MAPK. If activation of ERK and p38
MAPK is actually involved in actin depolymerization in human
neutrophils, it would be expected that phosphorylated ERK and
p38 MAPK are localized separately from F-actin and are not
286 • JANUARY 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017
Fig. 3. Analysis with confocal microscopy. Neutrophils were stimulated with TNF (100 U/ml) (A) or GM-CSF (5 ng/ml) (B) for
10 min, G-CSF (50 ng/ml) (C) for 20 min, or FMLP (10⫺7 M) (D) for 45 s at 37°C. When required, cells were pretreated with
PD-98059 (PD, 50 M) and/or SB-203580 (SB, 10 M) for 20 min before stimulation with TNF, GM-CSF, G-CSF, or FMLP. As
a control, FMLP-induced actin polymerization is shown in A, B, and C using the same cell preparations. F-actin distribution and
morphological changes were analyzed with confocal microscopy using FITC-phalloidin. Results shown are representative of 7
independent experiments.
ACTIN REORGANIZATION AND MAP KINASES
C59
Fig. 4. Effects of PD-98059 and SB-203580 on F-actin content in neutrophils
stimulated by TNF, GM-CSF, G-CSF, or FMLP. Neutrophils were pretreated
with PD-98059 (PD, 50 M) and/or SB-203580 (SB, 10 M) for 20 min at
37°C. Thereafter, cells were stimulated with TNF (100 U/ml) or GM-CSF (5
ng/ml) for 10 min, G-CSF (50 ng/ml) for 20 min, or FMLP (10⫺7 M) for 45 s
at 37°C. F-actin content was measured by flow cytometry. A: profiles of
changes in FITC-phalloidin fluorescence intensity. In each column, dotted and
solid lines represent unstimulated control cells and cells stimulated with each
agonist, respectively. Bold line represents the profile obtained in the presence
of PD-98059 and/or SB-203580. The results shown are representative of 5
independent experiments. B: the change in F-actin content is expressed as a
relative fold change in the mean fluorescence intensity, and the fluorescence
intensity of unstimulated control cells is expressed as 1. Data are expressed as
means ⫾ SD of 3–7 experiments. *Significantly decreased by stimulation with
TNF, GM-CSF, or G-CSF (P ⬍ 0.01). #Significantly increased by stimulation
with FMLP (P ⬍ 0.01). ⫹,†Cytokine-induced decrease in F-actin content was
significantly inhibited by PD-98059 and/or SB-203580 (⫹P ⬍ 0.01, †P ⬍ 0.05).
C: neutrophils were pretreated with PD-98059 (50 M) and SB-203580 (10 M)
for 20 min at 37°C. Thereafter, cells were stimulated with FMLP (10⫺7 M) for
indicated times at 37°C, and then F-actin content was measured by flow cytometry.
The change in F-actin content is expressed as a relative fold change in the mean
fluorescence intensity, and the fluorescence intensity at time 0 is expressed as 1.
Data are expressed as means ⫾ SD of 6 experiments. FMLP-induced change in
F-actin content was unaffected by pretreatment with PD-98059 and SB-203580.
AJP-Cell Physiol • VOL
colocalized with F-actin, because depolymerization of the
surrounding F-actin by activated ERK or p38 MAPK could
cause the spatial separation of remaining F-actin from phosphorylated ERK or p38 MAPK. As shown in Fig. 7, in
unstimulated control neutrophils F-actin was diffusely distributed in the cytoplasm with no or negligible staining for phosphorylated ERK and p38 MAPK. In neutrophils stimulated by
TNF for 10 min, phosphorylated ERK and p38 MAPK were
strongly stained in the cytoplasm, whereas F-actin was markedly decreased and residual F-actin was primarily localized at
the periphery with uneven distribution. It should be noted that
phosphorylated ERK and p38 MAPK are localized separately
from F-actin in TNF-stimulated cells (Fig. 7). Essentially, the
similar results were obtained for neutrophils stimulated by
GM-CSF or G-CSF for 10 min. Phosphorylated ERK and p38
MAPK were strongly stained in the cytoplasm in GM-CSFstimulated cells, and both molecules were localized separately
from F-actin. In G-CSF-stimulated cells, phosphorylated ERK
was strongly stained in the cytoplasm with negligible staining
for phosphorylated p38 MAPK, and it was localized separately
from F-actin (Fig. 7). In neutrophils stimulated by FMLP for
45 s, increased F-actin was primarily localized at the periphery,
and phosphorylated ERK and p38 MAPK were primarily
stained in the central region and were localized separately from
F-actin (Fig. 7). At 10 min after stimulation with FMLP,
F-actin was primarily localized in the polarized portion, and
phosphorylated ERK was still localized separately from F-
286 • JANUARY 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017
Fig. 5. Effect of FMLP on F-actin content in cytokine-pretreated neutrophils.
Neutrophils were stimulated with TNF (100 U/ml) or GM-CSF (5 ng/ml) for
10 min, G-CSF (50 ng/ml) for 20 min, or FMLP (10⫺7 M) for 45 s at 37°C.
When required, TNF-, GM-CSF-, or G-CSF-stimulated cells were further
challenged with FMLP (10⫺7 M) for 45 s. F-actin content was measured by
flow cytometry. Results shown are representative of 4 independent experiments.
C60
ACTIN REORGANIZATION AND MAP KINASES
Fig. 6. Effect of cytokine pretreatment on FMLP-induced polarization and actin polymerization in neutrophils. Neutrophils
were pretreated with TNF (100 U/ml) or GM-CSF (5 ng/ml) for
10 min, or G-CSF (50 ng/ml) for 20 min and thereafter stimulated with FMLP (10⫺7 M) for 10 min at 37°C. F-actin distribution and morphological changes were analyzed with confocal
microscopy using FITC-phalloidin. The results shown are representative of 4 independent experiments.
AJP-Cell Physiol • VOL
of p38 MAPK but not MKK3/6. Then, the effects of PD-98059
and SB-203580 on p38 MAPK kinase activity were analyzed
using immunoprecipitated p38 MAPK obtained from TNFstimulated neutrophils. Consistent with increased phosphorylation of p38 MAPK, the kinase activity of p38 MAPK was
increased by stimulation with TNF when the kinase activity
was determined using ATF-2 as a substrate (Fig. 9C). The
kinase activity of p38 MAPK was markedly inhibited by
SB-203580 (1 and 10 M) but unaffected by PD-98059 (50
M). SB-203580 (1 and 10 M) and PD-98059 (50 M) in
combination caused essentially the same effect as SB-203580
(1 and 10 M) alone (Fig. 9C). These findings indicate that the
concentrations of PD-98059 and SB-203580 used in the
present experiments are sufficient for obtaining significant
inhibition of ERK and/or p38 MAPK activity and that PD98059 and SB-203580 may inhibit cytokine-induced actin
depolymerization and morphological changes through inhibiting the distinct MAPK subtype cascades.
DISCUSSION
The present experiments show that stimulation of human
neutrophils with cytokines (TNF, GM-CSF, and G-CSF) results in an overall decrease in F-actin content (actin depolymerization) with concomitant morphological changes. Actin
depolymerization induced by these cytokines is detected at
10–20 min after stimulation and is apparently preceded by
activation of ERK and p38 MAPK, which is detectable within
3 min after stimulation with these cytokines (31). In addition,
cytokine-induced actin depolymerization and morphological
changes were inhibited by PD-98059 and SB-203580 in a
cytokine-specific manner. The cell responses induced by TNF
and GM-CSF, which activate ERK and p38 MAPK, were
partially inhibited by either PD-98059 or SB-203580 and were
almost completely abolished by these inhibitors in combination. On the other hand, the cell responses induced by G-CSF,
which primarily activates ERK, was almost completely abolished by PD-98059 but not inhibited by SB-203580. Furthermore, phosphorylated ERK and p38 MAPK are localized
286 • JANUARY 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017
actin. Staining for phosphorylated p38 MAPK became much
weaker at this time point. These findings obtained with staining
for phosphorylated ERK and p38 MAPK in control and stimulated neutrophils were consistent with those obtained with
immunoblotting for these molecules (see Fig. 9) (data not
shown for FMLP-stimulated cells) (31). The studies with
confocal microscopy reveal that phosphorylated ERK and p38
MAPK are localized separately from F-actin in neutrophils
stimulated by FMLP as well as cytokines.
Cofilin was dephosphorylated by stimulation with FMLP but
not with cytokines. Cofilin is a major actin-depolymerizing
protein, being active in the dephosphorylated form (4). To
explore the possible participation of cofilin in cytokine-induced
depolymerization in neutrophils, the level of phosphorylated
cofilin was analyzed using antibody against Ser3-phosphorylated cofilin. As shown in Fig. 8, cofilin was rapidly (within
45 s) dephosphorylated by stimulation with FMLP, in agreement with the previous report (11). The increased dephosphorylation of cofilin was sustained for at least 10 min after
stimulation with FMLP. On the other hand, the level of
phosphorylated cofilin was unaltered by stimulation with TNF,
GM-CSF or G-CSF for 5–20 min (Fig. 8). These findings
indicate that cytokine-induced actin depolymerization is unlikely to be ascribed to increased level of dephosphorylated
cofilin.
Effects of PD-98059 and SB-203580 on the activation of
ERK and activity of p38 MAPK. To determine the specificity of
the effects of PD-98059 and SB-203580 on cytokine-induced
actin depolymerization and morphological changes in neutrophils, the effects of these inhibitors on activation of ERK and
activity of p38 MAPK were analyzed. As shown in Fig. 9B,
TNF-, GM-CSF-, or G-CSF-induced phosphorylation of ERK
was markedly inhibited by PD-98059 (50 M) but unaffected
by SB-203580 (10 M). PD-98059 (50 M) and SB-203580
(10 M) in combination caused essentially the same effect as
PD-98059 (50 M) alone. On the other hand, TNF- or GMCSF-induced phosphorylation of p38 MAPK was neither affected by PD-98059 (50 M) nor SB-203580 (10 M) as
expected (data not shown), because SB-203580 is an inhibitor
ACTIN REORGANIZATION AND MAP KINASES
C61
separately from F-actin in neutrophils stimulated by these
cytokines. These findings indicate that TNF-, GM-CSF-, and
G-CSF-induced actin depolymerization and morphological
changes may be mediated through activation of ERK and/or
p38 MAPK. The cell responses induced by these cytokines
show remarkable contrast to the responses provoked by a
chemotactic peptide FMLP, which stimulates an overall increase in F-actin content (actin polymerization) with concomitant morphological changes (13, 28), and FMLP-induced responses are not affected by PD-98059 nor SB-203580 (9). It
should be noted that phosphorylated ERK and p38 MAPK are
also localized separately from F-actin even in neutrophils
AJP-Cell Physiol • VOL
stimulated by FMLP, suggesting that activation of ERK and
p38 MAPK is associated with actin depolymerization even in
neutrophils stimulated by FMLP. On the other hand, a “falling”
phase of FMLP-induced actin polymerization was unaffected
by MAPK inhibitors, suggesting that this phase is primarily
regulated by other molecules than MAPKs. The present experiments also show that morphological changes are accompanied
with actin depolymerization (an overall decrease in F-actin
content), as well as polymerization.
Accumulated evidence indicates that the MAPK cascades
play an important role in various neutrophil functions. It has
been demonstrated that ERK is involved in cytokine (TNF and
286 • JANUARY 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017
Fig. 7. Phosphorylated ERK and p38 MAPK
are localized separately from F-actin. Neutrophils were stimulated with TNF (100
U/ml), GM-CSF (5 ng/ml), or G-CSF (50
ng/ml) for 10 min or with FMLP (10⫺7 M)
for 45 s or 10 min at 37°C. Localization of
phosphorylated ERK (red color in upper
panel for each set) and phosphorylated p38
MAPK (red color in lower panel for each
set) was analyzed with indirect immunofluorescence using phospho-ERK1/2 or phospho-p38 MAPK antibody and Alexa Fluor
568-labeled goat anti-rabbit IgG antibody.
F-actin distribution (green color for each set)
was analyzed using FITC-phalloidin. Left
panels: double staining for F-actin and phosphorylated ERK or p38 MAPK. Right panels: Nomarski images. Control cells were
incubated for 10 min at 37°C in the absence
of any stimuli. Control cells in B were
stained with phospho-ERK1/2 (upper panels) or phospho-p38 MAPK (lower panels)
antibody, whereas control cells in A were
stained with isotype-matched control antibody. Results shown are representative of 4
independent experiments.
C62
ACTIN REORGANIZATION AND MAP KINASES
this possible adhesive interaction, the reactions were performed in FCS-precoated tubes in the present experiments.
Possible involvement of MAPKs in actin reorganization has
been reported in several cell types. However, the precise
mechanisms by which MAPKs regulate actin reorganization
remain to be determined. In endothelial cells, p38 MAPK is
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017
Fig. 8. Cofilin is dephosphorylated by stimulation with FMLP but not with
cytokines. Neutrophils were stimulated with TNF (100 U/ml), GM-CSF (5
ng/ml), G-CSF (50 ng/ml), or FMLP (10⫺7 M) for indicated times at 37°C.
Western blotting was performed using antibodies against phosphorylated and
nonphosphorylated forms of cofilin. The ratio of phosphorylated cofilin to total
cofilin band intensity was calculated, and the ratio at time 0 is expressed as 1
(lower panel). Results shown are representative of 3 independent experiments.
GM-CSF)-induced O⫺
2 release and adherence, Fc␥ receptor
(Fc␥R) IIa-mediated phagocytosis and activation of myosin
light chain kinase, and Fc␥R-mediated actin polymerization (7,
9, 21, 30, 31, 32). On the other hand, p38 MAPK is reported to
be involved in chemotaxis, LPS-induced activation of NF-B
and synthesis of TNF, Fc␥RIIIb-mediated H2O2 production,
TGF-1-induced actin polymerization, and cytokine (TNF,
GM-CSF, and IL-1)-induced O⫺
2 release, adherence, and
upregulation of 2 integrin (7, 8, 10, 23, 24, 30, 31, 32, 35).
The results presented here show that both ERK and p38 MAPK
play a critical role in cytokine-induced actin depolymerization
and morphological changes in human neutrophils.
The physiological role of sustained actin depolymerization
induced by cytokines remains to be determined. One possible
role is that sustained strong actin depolymerization might
inhibit neutrophil chemotaxis by preventing coordinated polymerization, depolymerization, and redistribution of actin cytoskeleton, which is crucial for directed cell motility (6, 13, 28,
29, 38). In fact, FMLP-induced polarization was prevented by
cytokine pretreatment, and the extent of FMLP-induced actin
polymerization in cytokine-pretreated neutrophils was less than
that in control cells unexposed to cytokines. Consistent with
this is the findings that neutrophil chemotaxis is impaired by
preexposure of cells with TNF, GM-CSF, or G-CSF (6, 13, 17,
28, 38). It is conceivable that cytokines may partly contribute
to accumulation of neutrophils to the inflammatory sites by
preventing neutrophils from leaving there, where a lot of
cytokines are produced. It has been reported that TNF induces
a slight increase in F-actin content in human neutrophils (5), in
contrast to our present experiments. The reason for this discrepancy is unknown. One possible reason is that strong
adhesive interaction between TNF-stimulated neutrophils and
plastic surface might affect actin reorganization. To prevent
AJP-Cell Physiol • VOL
Fig. 9. Effects of PD-98059 and SB-203580 on activation of ERK and activity
of p38 MAPK. A: neutrophils were stimulated with TNF (100 U/ml), GM-CSF
(5 ng/ml), or G-CSF (50 ng/ml) for 10 min at 37°C. Western blotting was
performed using antibodies against phosphorylated (upper and middle panels)
and nonphosphorylated (lower panel) forms of ERK1/2 and p38 MAPK. B:
neutrophils were pretreated with PD-98059 (PD, 50 M) and/or SB-203580
(SB, 10 M) for 20 min at 37°C and thereafter stimulated with TNF (100
U/ml), GM-CSF (5 ng/ml), or G-CSF (50 ng/ml) for 10 min at 37°C. Western
blotting was performed using antibodies against phosphorylated (upper panel)
and nonphosphorylated (lower panel) forms of ERK1/2. C: neutrophils were
stimulated with TNF (100 U/ml) for 10 min at 37°C. Phosphorylated p38
MAPK was immunoprecipitated with antibody against the phosphorylated
form of p38 MAPK. In vitro kinase assay was then performed using GSTATF-2 fusion protein as a substrate. When required, PD-98059 (PD, 50 M)
and/or SB-203580 (SB, 1 or 10 M) were added to the reaction mixture.
Phosphorylation of ATF-2 was determined by immunoblotting using antibody
against phosphorylated ATF-2. The cell lysates equivalent to 6 ⫻ 106 cells
were loaded onto each lane. Although two bands were detected by this method,
an upper band was identified as phosphorylated ATF-2 on a basis of molecular
weight. Results shown are representative of 3 independent experiments.
286 • JANUARY 2004 •
www.ajpcell.org
ACTIN REORGANIZATION AND MAP KINASES
AJP-Cell Physiol • VOL
apparent after a lag time of 5–10 min. By contrast, cofilin was
rapidly dephosphorylated by FMLP stimulation, which might
partly contribute to actin depolymerization (a “falling” phase
of actin polymerization) after rapid actin polymerization in
response to FMLP. It is also possible that other molecules such
as WASP (Wiskott-Aldrich syndrome protein) may be involved in actin depolymerization in neutrophils stimulated by
cytokines or FMLP (34). Although the precise mechanisms for
cytokine-induced actin depolymerization and morphological
changes in human neutrophils remain to be elucidated, the
present experiments demonstrate that ERK and p38 MAPK
play a critical role in these responses, as well as cytokineinduced O⫺
2 release and adherence.
GRANTS
This work was supported by Grant-in-Aid for Scientific Research, Japan
13770467.
REFERENCES
1. Alessi DR, Cuenda A, Cohen P, Dudley DT, and Saltiel AR. PD098059
is a specific inhibitor of the activation of mitogen-activated protein kinase
kinase in vitro and in vivo. J Biol Chem 270: 27489–27494, 1995.
2. Alonso-Lebrero JL, Serrador JM, Dominguez-Jimenez C, Barrerio O,
Luque A, del Pozo MA, Snapp K, Kansas G, Schwartz-Albeiz R,
Furthmayr H, Lozano F, and Sanchez-Madrid F. Polarization and
interaction of adhesion molecules P-selectin glycoprotein ligand 1 and
intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells.
Blood 95: 2413–2419, 2000.
3. Amano T, Kaji N, Ohashi K, and Mizuno K. Mitosis-specific activation
of LIM-kinase and roles of cofilin phosphorylation and dephosphorylation
in mitosis. J Biol Chem 277: 22093–22102, 2002.
4. Benndorf R, Hayess K, Ryazantsev S, Wieske M, Behlke J, and Lutsch
G. Phosphorylation and supramolecular organization of murine small heat
shock protein HSP25 abolish its actin polymerization-inhibiting activity.
J Biol Chem 269: 20780–20784, 1994.
5. Berkow RL, Wang D, Larrick JW, Dodson RW, and Howard TH.
Enhancement of neutrophil superoxide production by preincubation with
recombinant human tumor necrosis factor. J Immunol 139: 3783–3791,
1987.
6. Binder R, Kress A, Kan G, Herrmann K, and Kirschfink M. Neutrophil priming by cytokines and vitamin D binding protein (Gc-globulin):
impact on C5a-mediated chemotaxis, degranulation and respiratory burst.
Mol Immunol 36: 885–892, 1999.
7. Coxon PY, Rane MJ, Powell DW, Klein JB, and McLeish KR.
Differential mitogen-activated protein kinase stimulation by Fc␥ receptor
IIa and Fc␥ receptor IIIb determines the activation phenotype of human
neutrophils. J Immunol 164: 6530–6537, 2000.
8. Detmers PA, Zhou D, Polizzi E, Thieringer R, Hanlon WA, Vaidya S,
and Bansal V. Role of stress-activated mitogen-activated protein kinase
(p38) in 2-integrin-dependent neutrophil adhesion and the adhesiondependent oxidative burst. J Immunol 161: 1921–1929, 1998.
9. Downey GP, Butler JR, Tapper H, Fialkow L, Saltiel AR, Rubin BB,
and Grinstein S. Importance of MEK in neutrophil microbicidal responsiveness. J Immunol 160: 434–443, 1998.
10. Hannigan M, Zhan L, Ai Y, and Huang CK. The role of p38 MAP
kinase in TGF-1-induced signal transduction in human neutrophils.
Biochem Biophys Res Commun 246: 55–58, 1998.
11. Heyworth PG, Robinson JM, Din J, Ellis BA, and Badwey JA. Cofilin
undergoes rapid dephosphorylation in stimulated neutrophils and translocates to ruffled membranes enriched in products of the NADPH oxidase
complex. Evidence for a novel cycle of phosphorylation and dephosphorylation. Histochem Cell Biol 108: 221–233, 1997.
12. Howard T, Li Y, Torres M, Guerrero A, and Coates T. The 47-kDa
protein increased in neutrophil actin dysfunction with 47- and 89-kDa
protein abnormalities is lymphocyte-specific protein. Blood 83: 231–241,
1994.
13. Howard TH and Meyer WH. Chemotactic peptide modulation of actin
assembly and locomotion in neutrophils. J Cell Biol 98: 1265–1271, 1984.
14. Huang CK, Zhan L, Ai Y, and Jongstra J. LSP1 is the major substrate
for mitogen-activated protein kinase-activated protein kinase 2 in human
neutrophils. J Biol Chem 272: 17–19, 1997.
286 • JANUARY 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017
reported to activate MAPK-activated protein (MAPKAP) kinase 2, which phosphorylates heat shock protein of 27 kDa
(HSP27), an F-actin cap binding protein. Phosphorylation of
HSP27 would release its binding to actin, allowing polymerization and presumably depolymerization, as well, according to
the signals concomitantly generated (4, 15). In murine macrophage cell lines, TNF induces actin depolymerization through
undefined mechanisms and TNF inhibits Cdc42-induced filopodium formation through activation of p38 MAPK (26). In
human neutrophils, MAPKAP kinase 2 is activated by FMLP,
and MAPKAP kinase 2 phosphorylates HSP27 and lymphocyte-specific protein 1 (LSP1), an F-actin-bundling protein (14,
19). Neutrophils with increased expression of LSP1 exhibit
defective locomotion and enhanced O⫺
2 release (12). A recent
study indicates that an increased level of LSP1 induces actin
reorganization and formation of surface projection without
affecting F-actin content in U-937 cells (19), indicating that
actin reorganization and morphological changes are not necessarily accompanied with the overall changes in F-actin content.
Another possible molecule responsible for enhanced actin
depolymerization may be cofilin. Cofilin is a major actindepolymerizing protein and promotes rapid actin filament turnover (36). Its activity is regulated by phosphorylation, being
inactive in the phosphorylated form (4). It has been reported
that ⬃50% of cofilin exists in the phosphorylated form in
resting neutrophils, and cofilin is rapidly dephosphorylated by
stimulation with opsonized zymosan, FMLP, arachidonic acid,
the Ca2⫹ ionophore A-23187, or phorbol myristate acetate (11,
25, 33). In other types of cells, cofilin is phosphorylated by
LIM-kinase through activation of Rac and Rho, resulting in
stabilization and reorganization of actin filaments (20). It
should be noted that multiple signals are generated by receptor
activation according to the stimuli used. In fact, concomitant
generation of the signals promoting actin polymerization and
depolymerization has been demonstrated in murine macrophage cell lines using mutated TNF receptors (26). Furthermore, many stimulants, including FMLP, phorbol myristate
acetate, and opsonized zymosan, induce actin polymerization
despite concomitant dephosphorylation of cofilin (11, 25, 33),
which could lead to actin depolymerization. It is likely that in
neutrophils stimulated by such an agonist, the signals promoting actin polymerization may be dominant and overwhelm the
signals promoting actin depolymerization such as dephosphorylation of cofilin, resulting in an overall increase in F-actin
content. In contrast, it is likely that in neutrophils stimulated by
cytokines (TNF, GM-CSF, and G-CSF), the signals promoting
actin depolymerization may be generated to a greater extent
than the signals promoting actin polymerization. The present
experiments show that cytokine-induced actin depolymerization is not caused by an increased level of dephosphorylated
cofilin because cofilin was neither phosphorylated nor dephosphorylated by stimulation with cytokines. This finding, however, does not exclude the possible role of cofilin in cytokineinduced actin depolymerization. Cofilin might play some role
in cytokine-induced actin depolymerization because cofilinmediated actin depolymerization is accelerated by increased
pH (22) and sustained increase in cytoplasmic pH in neutrophils is induced by stimulation with cytokines (TNF, GM-CSF,
and G-CSF) (40). Cytokine-induced increase in cytoplasmic
pH is apparent after a lag time of 3–5 min (40) and precedes the
onset of cytokine-induced actin depolymerization, which was
C63
C64
ACTIN REORGANIZATION AND MAP KINASES
AJP-Cell Physiol • VOL
30. Suzuki K, Hasegawa T, Sakamoto C, Zhou Y, Hato F, Hino M,
Tatsumi N, and Kitagawa S. Cleavage of mitogen-activated protein
kinases in human neutrophils undergoing apoptosis: role in decreased
responsiveness to inflammatory cytokines. J Immunol 166: 1185–1192,
2001.
31. Suzuki K, Hino M, Hato F, Tatsumi N, and Kitagawa S. Cytokinespecific activation of distinct mitogen-activated protein kinase subtype
cascade in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor
necrosis factor-␣. Blood 93: 341–349, 1999.
32. Suzuki K, Hino M, Kutsuna H, Hato F, Sakamoto C, Takahashi T,
Tatsumi N, and Kitagawa S. Selective activation of p38 mitogenactivated protein kinase cascade in human neutrophils stimulated by
IL-1. J Immunol 167: 5940–5947, 2001.
33. Suzuki K, Yamaguchi T, Tanaka T, Kawanishi T, Nishiwaki-Nogami
T, Yamamoto K, Tsuji T, Irimura T, Hayakawa T, and Takahashi A.
Activation induces dephosphorylation of cofilin and its translocation to
plasma membranes in neutrophil-like differentiated HL-60 cells. J Biol
Chem 270: 19551–19556, 1995.
34. Takenawa T and Miki H. WASP and WAVE family proteins: key
molecules for rapid rearrangement of cortical actin filaments and cell
movement. J Cell Sci 114: 1801–1809, 2001.
35. Tandon R, Sha’afi RI, and Thrall RS. Neutrophil 2-integrin upregulation is blocked by a p38 MAP kinase inhibitor. Biochem Biophys Res
Commun 270: 858–862, 2000.
36. Theriot JA. Accelerating on a treadmill: ADF/cofilin promotes rapid actin
filament turnover in the dynamic cytoskeleton. J Cell Biol 136: 1165–
1168, 1997.
37. Toshima J, Toshima JY, Amano T, Yang N, Narumiya S, and Mizuno
K. Cofilin phosphorylation by protein kinase TESK1 and its role in
integrin-mediated actin reorganization and focal adhesion formation. Mol
Biol Cell 12: 1131–1145, 2001.
38. Vollmer KL, Alberts JS, Carper HT, and Mandell GL. Tumor necrosis
factor-␣ decreases neutrophil chemotaxis to N-formyl-L-methionyl-Lleucyl-L-phenylalanine: analysis of single cell movement. J Leukoc Biol
52: 630–636, 1992.
39. Widmann C, Gibson S, Jarpe MB, and Johnson GL. Mitogen-activated
protein kinase: conservation of a three-kinase module from yeast to
human. Physiol Rev 79: 143–180, 1999.
40. Yuo A, Kitagawa S, Azuma E, Natori Y, Togawa A, Saito M, and
Takaku F. Tyrosine phosphorylation and intracellular alkalinization are
early events in human neutrophils stimulated by tumor necrosis factor,
granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor. Biochim Biophys Acta 1156: 197–203, 1993.
41. Yuo A, Kitagawa S, Ohsaka A, Saito M, and Takaku F. Stimulation
and priming of human neutrophils by granulocyte colony-stimulating
factor and granulocyte-macrophage colony-stimulating factor: qualitative
and quantitative differences. Biochem Biophys Res Commun 171: 491–
497, 1990.
42. Yuo A, Kitagawa S, Suzuki I, Urabe A, Okabe T, Saito M, and
Takaku F. Tumor necrosis factor as an activator of human granulocytes:
potentiation of the metabolisms triggered by the Ca2⫹-mobilizing agonists. J Immunol 142: 1678–1684, 1989.
286 • JANUARY 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017
15. Huot J, Houle F, Rousseau S, Deschesnes RG, Shah GM, and Landry
J. SAPK2/p38-dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis. J Cell Biol 143: 1361–
1373, 1998.
16. Kitagawa S, Takaku F, and Sakamoto S. A comparison of the superoxide-releasing response in human polymorphonuclear leukocytes and
monocytes. J Immunol 125: 359–364, 1980.
17. Leavey PJ, Sellins KS, Thurman G, Elzi D, Hiester A, Silliman CC,
Zerbe G, Cohen JJ, and Ambruso DR. In vivo treatment with granulocyte colony-stimulating factor results in divergent effects on neutrophil
functions measured in vitro. Blood 92: 4366–4374, 1998.
18. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green
D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, Strickler
JE, McLaughlin MM, Siemens IR, Fisher SM, Livi GP, White JR,
Adams JL, and Young PR. A protein kinase involved in the regulation
of inflammatory cytokine biosynthesis. Nature 372: 739–746, 1994.
19. Li Y, Zhang Q, Aaron R, Hilliard L, and Howard TH. LSP1 modulates
the locomotion of monocyte-differentiated U937 cells. Blood 96: 1100–
1105, 2000.
20. Maekawa M, Ishizaki T, Boku S, Watanabe N, Fujita A, Iwamatsu A,
Obinata T, Ohashi K, Mizuno K, and Narumiya S. Signaling from Rho
to the actin cytoskeleton through protein kinases ROCK and LIM-kinase.
Science 285: 895–898, 1999.
21. Mansfield PJ, Shayman JA, and Boxer LA. Regulation of polymorphonuclear leukocyte phagocytosis by myosin light chain kinase after activation of mitogen-activated protein kinase. Blood 95: 2407–2412, 2000.
22. Moon A and Drubin DG. The ADF/cofilin proteins: stimulus-responsive
modulators of actin dynamics. Mol Biol Cell 6: 1423–1431, 1995.
23. Nick JA, Avdi NJ, Young SK, Knall C, Gerwins P, Johnson GL, and
Worthen GS. Common and distinct intracellular signaling pathways in
human neutrophils utilized by platelet activating factor and FMLP. J Clin
Invest 99: 975–986, 1997.
24. Nick JA, Avdi NJ, Young SK, Lehman LA, McDonald PP, Frasch SC,
Billstrom MA, Henson PM, Johnson GL, and Worthen GS. Selective
activation and functional significance of p38␣ mitogen-activated protein
kinase in lipopolysaccharide-stimulated neutrophils. J Clin Invest 103:
851–858, 1999.
25. Okada K, Takano-Ohmuro H, Obinata T, and Abe H. Dephosphorylation of cofilin in polymorphonuclear leukocytes derived from peripheral
blood. Exp Cell Res 227: 116–122, 1996.
26. Peppelenbosch M, Boone E, Jones GE, van Deventer SJH, Haegeman
G, Fiers W, Grooten J, and Ridley AJ. Multiple signal transduction
pathways regulate TNF-induced actin reorganization in macrophages:
inhibition of Cdc-42-mediated filopodium formation by TNF. J Immunol
162: 837–845, 1999.
27. Schaeffer HJ and Weber MJ. Mitogen-activated protein kinases: specific
messages from ubiquitous messengers. Mol Cell Biol 19: 2435–2444,
1999.
28. Sham RL, Phatak PD, Ihne TP, Abboud CN, and Packman CH. Signal
pathway regulation of interleukin-8-induced actin polymerization in neutrophils. Blood 82: 2546–2551, 1993.
29. Stossel TP. From signal to pseudopod. How cells control cytoplasmic
actin assembly. J Biol Chem 264: 18261–18264, 1989.