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p38 Mitogen-Activated Protein Kinase Activation Is Required
for Human Neutrophil Function Triggered by TNF-
a
or
FMLP Stimulation
1
You-Li Zu,
2
* Jiafan Qi,
†
Annette Gilchrist,
§
Gustavo A. Fernandez,* Dolores Vazquez-Abad,
‡
Donald L. Kreutzer,
†
Chi-Kuang Huang,
†
and Ramadan I. Sha’afi*
Mitogen-activated protein (MAP) kinase-mediated signal-transduction pathways convert extracellular stimulation into a variety
of cellular functions. However, the roles of MAP kinases in neutrophils are not well understood yet. Protein phosphorylation
analysis of cellular MAP kinases indicates that exposure of human neutrophils to chemotactic factor FMLP as well as granulocyte-
macrophage CSF, PMA, or ionomycin rapidly induced the activation of p38 and p44/42 MAP kinases, but stimulation with
inflammatory cytokine TNF-
a
triggered the activation of p38 MAP kinase only. To study the cellular functions of these MAP
kinases, the inhibitor SB20358, which specifically inhibited enzymatic activity of cellular p38 MAP kinase, and the inhibitor
PD98059, which specifically blocked the induced protein phosphorylation and activation of p44/42 MAP kinase in intact neutro-
phils, were utilized. Inhibition of the cellular p38 MAP kinase activation almost completely abolished the TNF-
a
-stimulated IL-8
production and superoxide generation of human neutrophils. In addition, the FMLP-induced neutrophil chemotaxis as well as
superoxide generation were suppressed markedly by inhibiting the activation of cellular p38 MAP kinase, but not p44/42 MAP
kinase. Moreover, RIA indicates that the activation of cellular p38 MAP kinase was required for the neutrophil IL-8 production
stimulated by granulocyte-macrophage CSF or LPS as well as TNF-
a
, but not for that induced by PMA or ionomycin. These
results demonstrate that the activation of cellular p38 MAP kinase is indispensable for the TNF-
a
- or FMLP-mediated cellular
functions in human neutrophils, and suggest that p38 MAP kinase may play a different role in response to distinct stimulation.
The Journal of Immunology, 1998, 160: 1982–1989.
Human neutrophils constitute the first line of defense
against invading microorganisms and are the major cel-
lular component of an acute inflammatory reaction (1–
3). In response to stimulation of chemotactic factors, cytokines, or
immune complexes, human neutrophils become rapidly activated
(4–7). One of the early intracellular events to occur during neu-
trophil activation is the rapid induction of protein phosphorylation,
which plays an essential role in the regulation of many neutrophil
functions (6–13) as well as being important in other cell types.
MAP
3
kinases have been demonstrated to play a central role in
mediating intracellular signal transduction and regulating cellular
functions in response to variety of extracellular stimuli (14–18).
Recently, three distinct mammalian MAP kinases have been iden-
tified, including extracellular signal-regulated kinases (ERKs or
p44/42 MAP kinase), the stress-activated protein kinases or c-Jun
kinases, and p38 MAP kinase (a mammalian homologue of HOG1,
also known as CSBP, RK, or mpk2), each with apparently unique
signaling pathways (19–21).
Studies have demonstrated that p38 MAP kinase is involved in
an intracellular kinase cascade that regulates stress-activated signal
transduction. In response to certain environmental stresses or
proinflammatory cytokines, p38 MAP kinase becomes rapidly ac-
tivated and subsequently stimulates MAP kinase-activated protein
(MAPKAP) kinase 2 and/or MAPKAP kinase 3, which in turn
induce the phosphorylation of small heat-shock protein (22–24). In
addition, activated p38 MAP kinase has been shown to phosphor-
ylate specific transcription factors in vitro and in intact cells, and
thus may regulate gene expression (25–27). Investigation of hu-
man neutrophils has revealed that in response to certain extracel-
lular stimulation, the kinase activities of p38 MAP kinase (28–30)
and MAPKAP kinase 2 (31) rapidly increased, suggesting this ki-
nase cascade may play a pivotal role in regulating neutrophil func-
tion. In this study, to understand the potential physiologic func-
tion(s) of MAP kinases in the human neutrophils, the cellular
kinase activities of p38 or p44/42 MAP kinases were modified by
using the specific kinase inhibitors (32–35). Effects of cellular p38
MAP kinase or p44/42 MAP kinase on neutrophil IL-8 production,
superoxide generation, or chemotaxis induced by TNF-
a
or FMLP
were examined.
Materials and Methods
Cell preparation
Neutrophils were isolated from whole human blood using Ficoll-Hypaque
gradients, and contaminating erythrocytes were lysed by a hypotonic shock
(31). The resulting neutrophils represented at least 97% of the cells. Cell
viability, estimated by trypan blue exclusion, was 98%. The isolated neu-
trophils were resuspended in HBSS (Life Technologies, Gaithersburg,
MD) containing 10 mM HEPES (pH 7.5) and 1 mM calcium. To modify
Departments of *Physiology,
†
Pathology, and
‡
Medicine, University of Connecticut
Health Center, Farmington, CT 06030; and
§
Institute for Neuroscience, Northwestern
University, Chicago, IL 60611
Received for publication August 18, 1997. Accepted for publication November
3, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Patrick and Catherine Weldon Donaghue Medical
Research Foundation (Grant DF95-053), RICE and ACSIR Grants from UCHC, and
National Institutes of Health Grants HL53786 and AI20943.
2
Address correspondence and reprint requests to Dr. You-Li Zu, Department of Phys-
iology, University of Connecticut Health Center, Farmington, CT 06030-3505. E-mail
address: youlizu@panda.uchc.edu
3
Abbreviations used in this paper: MAP, mitogen-activated protein; ECL, enhanced
chemiluminescence; GM-CSF, granulocyte-macrophage CSF; MAPKAP, mitogen-
activated protein kinase-activated protein; NF-
k
B, nuclear factor-
k
B.
Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00
cellular MAP kinase activity, compound SB20358 (32, 33), a specific in-
hibitor for p38 MAP kinase (IC
50
50.6
m
M in vitro), was obtained from
SmithKline Beecham Pharmaceuticals (King of Prussia, PA), or compound
PD 98059, which indirectly blocks the activation of p44/42 MAP kinase
via inhibition of MAP kinase kinase-1 activation by c-Raf with an IC
50
5
4
m
M in vitro (34, 35), was purchased from New England Biolabs (Bev-
erly, MA). In addition, compound H7 (Seikagaku Corp., Tokyo, Japan), a
protein kinase C inhibitor with K
i
56
m
M (36), was used as a control.
Neutrophils were preincubated with or without the kinase inhibitors, as
indicated in figures, at 4°C for 40 min. Cells were then stimulated with 25
ng/ml TNF-
a
(Sigma Chemical Co., St. Louis, MO), 10
28
M FMLP (Sig-
ma Chemical Co.), 1 ng/ml GM-CSF (R&D Systems, Minneapolis, MN),
20 ng/ml PMA (Sigma Chemical Co.), 1
m
M ionomycin (CalBiochem, La
Jolla, CA), or 50 ng/ml LPS (Sigma Chemical Co.) at 37°C for various
times in different experiments, as indicated in figures, and the induced
MAP kinase activation and cellular functional change were examined.
Western blot analysis of cellular MAP kinases
It has been well documented that three groups of MAP kinases are acti-
vated by distinct upstream kinases through phosphorylation of both threo-
nine and tyrosine in a regulatory Thr-Xaa-Tyr site found on each kinase.
Protein phosphorylation of these kinases has been shown to be an accurate
indicator of their activation (37, 38). To quantify the protein phosphory-
lation of MAP kinases, following each stimulation, 4 310
6
neutrophils
were harvested and resuspended in 140
m
l sample buffer (1% Triton X-100,
150 mM NaCl, 10 mM Tris, (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.5%
Nonidet P-40, 0.2 mM PMSF, 20 mM sodium orthovanadate, 10
m
Mp-
nitrophenyl phosphate, 1 mM diisopropylfluorophosphate, 0.7 mg/ml pep-
statin A, 10 mg/ml leupeptin, and 2 mg/ml aprotinin) and solubilized with
the addition of 140
m
lof23Laemmli solution (9% (w/v) SDS, 6% (v/v)
b
-mercaptoethanol, 10% (v/v) glycerol, and a trace amount of bromphenol
blue dye in 0.196 M Tris/HCl (pH 6.7)). The cellular proteins (50-
m
l sam-
ples) were electrophoresed through 10% SDS-PAGE and then transferred
to Immobilon-P membranes (Millipore Corp., Bedford, MA) using a semi-
dry transfer system (Bio-Rad, Hercules, CA). Prestained protein standards
(Bio-Rad) were run in each gel. The blots were blocked in Tris-buffered
saline/Tween-20 (TBS-T containing 20 mM Tris base, pH 7.6, 137 mM
NaC1, and 0.1% Tween-20) supplemented with 5% BSA for 1 h, incubated
with 1/1000 diluted primary rabbit Abs specifically against Tyr182-phos-
phorylated p38 MAP kinase (NEB) or against tyrosine-phosphorylated
p44/42 MAP kinase (NEB) for 2 h, and then with 1/5000 diluted secondary
Abs of horseradish peroxidase-conjugated anti-rabbit IgG (Boehringer
Mannheim Corp., Indianapolis, IN) for 30 min at room temperature. After
three washes of 7 min each, blots were treated with enhanced chemilumi-
nescence (ECL from Amersham, Arlington Heights, IL) reagents, and the
phosphorylated MAP kinases were detected by autoradiography for vari-
able lengths of time (15 s to 3 min) with Kodak X-Omat film.
To confirm that the same amount of cellular proteins was loaded on each
lane, the primary Ab/secondary Ab complex was removed by incubating
the blot in stripping buffer (100 mM
b
-mercaptoethanol, 2% SDS, and 62.5
mM Tris/HCl, pH 6.7) for 30 min at 50°C. Blots were then subjected to
autoradiography for confirmation that the Ab signal had been removed.
After this procedure, the blots were blocked with buffer containing 5%
BSA, and reprobed with rabbit Abs against p38 MAP kinase (Santa Cruz
Biotech., Santa Cruz, CA), followed by incubation with secondary horse-
radish peroxidase-conjugated Abs, as described above. Proteins were
detected by the ECL method.
Protein phosphorylation assay
To evaluate enzymatic activities of cellular MAPKAP kinase 2, neutrophils
(1 310
7
) were harvested in 200
m
l cold sample buffer and treated with
mild sonication for 10 s. After centrifugation at 3000 3gfor 10 min, the
kinase activity in the resulting supernatant was examined by an in vitro
protein phosphorylation assay using commercially available human rhsp27
(StressGen Biotechnologies Corp., Victoria, British Columbia, Canada) as
the specific substrate for MAPKAP kinase 2 (39). The reaction was initi-
ated by the addition of an equal volume (30
m
l) of freshly prepared phos-
phorylation reaction mix containing: 30 mM HEPES (pH 7.3), 20 mM
MgCl
2
, 2 mM EGTA, 10
m
M sodium orthovanadate, 5
m
M okadaic acid,
4 mM DTT, 30
m
M H-7, 0.4 mM [
g
-
32
P]ATP (10
5
cpm/pmol), and 0.5
m
g
hsp27, to 30
m
l cellular supernatant. The in vitro phosphorylation reaction
was conducted at 30°C for 10 min and stopped by addition of 60
m
l23
Laemmli solution. Proteins were then separated on 11% SDS-PAGE, and
the induced protein phosphorylation of hsp27 was detected by
autoradiography.
IL-8 radioimmunoassay
To detect cellular IL-8 production, neutrophils (5 310
6
cells/ml) were
suspended in RPMI 1640 (Life Technologies) containing 10% FCS (Sigma
Chemical Co.), preincubated with the protein kinase inhibitors at 4°C for
40 min. Cells were then stimulated with TNF-
a
, FMLP, GM-CSF, iono-
mycin, PMA, or LPS, as described above, and cultured at 37°C under 5%
CO
2
condition. Medium was harvested at the various times as indicated in
figures, separated from cells by centrifugation at 2000 3gfor 5 min, and
analyzed for IL-8 production by an RIA, as described previously, with
slight modifications (40). Briefly, 100
m
l neutrophil-conditioned medium
was incubated for1hat25°C with 100
m
l chicken anti-human IL-8 Ab
(40) diluted 1/2000 in PBS containing 1% BSA. Human
125
I-labeled IL-8
(DuPont NEN, Boston, MA) at 70,000 to 80,000 cpm/ml in PBS was added
(100
m
l), and the reaction mix was incubated for another hour at 25°C. The
reaction was stopped by addition of 500
m
l cold PBS containing 10 mg/ml
BSA, and the immune complexes were precipitated using saturated am-
monium sulfate at 50% final concentration. The samples were centrifuged
at 5000 3gfor 20 min at 4°C, and the amount of radioactivity in the
resultant pellets was counted. Samples were quantified by reference to a
standard curve constructed using rIL-8 standards (0.03–10 ng/ml).
Oxidative burst assay
To define the role of MAP kinases in neutrophil superoxide production,
cells were suspended in HBSS containing 10 mM HEPES (pH 7.5) and 1
mM calcium and preincubated with the MAP kinase inhibitors, as above.
To measure the respiratory burst response, preincubated cells (1 310
7
cells/ml) were resuspended in HBSS containing 145 mM cytochrome c,2
mM sodium azide, 2 mM CaCl
2
, and 2.4 mM MgCl
2
, and equilibrated to
37°C for 5 min. Cells (100
m
l) were dispensed into prewarmed microtiter
plates, and superoxide anion production was initiated by stimulating neu-
trophils with 25 ng/ml TNF-
a
,5310
28
M FMLP, or 20 ng/ml PMA,
respectively. Superoxide production was measured kinetically at 37°C for
various times as indicated in figures, by the reduction of cytochrome cat
550 nm (OD550) using a THERMO
max
kinetic microplate reader (Molec-
ular Devices, Menlo Park, CA). Data were analyzed using the SOFT
max
program.
Chemotactic assays
Neutrophil chemotaxis induced by FMLP was assayed by a modified Boy-
den technique (41) using a Boyden chamber, as described previously (40).
Briefly, isolated human neutrophils were preincubated with or without the
MAP kinase inhibitors, as indicated in figures, at 4°C for 40 min in Mod-
ified Dulbecco’s medium with 1% BSA, and loaded into upper wells of the
Boyden chamber, which was separated from the lower wells by 3-
m
m
cellulose nitrate filters (Millipore). To induce chemotaxis, 10
28
M FMLP
in Modified Dulbecco’s medium with 1% BSA was added to the lower
wells of the Boyden chamber. Neutrophil migration proceeded in a humid-
ified 5% CO
2
incubator at 37°C for 60 min, and the filters were removed,
fixed, stained, and air dried. Cell migration was quantitated by a micro-
sectioning technique (42) using an image analyzer (43). The number of
cells migrating through the filter was determined in three fields for each
sample, which was run in duplicate. Neutrophil migration was expressed as
a chemotactic/migratory index (number of cells 3distance migrated
through the filter).
Results
Activation of cellular MAP kinases in human neutrophils
To better understand the regulation of MAP kinases in neutrophil
activation, freshly isolated cells were exposed to different stimuli,
including cytokines (TNF-
a
and GM-CSF), chemotactic factors
(FMLP), protein kinase C activator (PMA), or calcium ionophore
(ionomycin). Following stimulation, the activation of cellular
MAP kinases was evaluated by using Western blot to detect the
induced protein phosphorylation, as described under Materials and
Methods. As shown in Figure 1, cellular p38 MAP kinase became
phosphorylated and hence activated in human neutrophils in re-
sponse to stimulation of TNF-
a
, GM-CSF, FMLP, PMA, or iono-
mycin (Fig. 1A). Cellular p44/42 MAP kinase was also activated
by exposure of neutrophils to GM-CSF, FMLP, PMA, and iono-
mycin, but not to TNF-
a
(Fig. 1B). The findings were confirmed
1983The Journal of Immunology
by examining total cellular p38 MAP kinase, and Figure 1Cindi-
cates that the differences observed for the induced protein phos-
phorylation of the cellular MAP kinases did not result from
differences in loading or from cellular protein digestion.
The kinase inhibitor SB20358 inhibits the neutrophil p38 MAP
kinase activation stimulated by TNF-
a
The finding that TNF-
a
could specifically activate p38 MAP ki-
nase, but not p44/42 MAP kinase (Fig. 1, Aand B), led us to use
this cytokine to study the role of p38 MAP kinase in human neu-
trophil activation. In addition, recent discovery of compound
SB20358, a specific kinase inhibitor for p38 MAP kinase with an
IC
50
50.6
m
M in vitro that has no apparent effect on other protein
kinases, including p44/42 MAP kinase or the stress-activated pro-
tein kinase/c-Jun kinase (36 and 37), provides a powerful tool for
this purpose. To regulate cellular p38 MAP kinase, neutrophils
were preincubated with 0.6
m
M SB20358 at 4°C for 40 min to
inhibit the kinase activation triggered by following stimulation.
Inhibitory effect of SB20358 on cellular p38 MAP kinase was
detected by evaluating the activation of cellular MAPKAP kinase
2, which has been demonstrated to be a specific cellular substrate
for p38 MAP kinase (22 and 23), and is responsible for the phos-
phorylation of small heat-shock proteins (hsp25/27) (39, 44, and
45). Enzymatic activity of cellular MAPKAP kinase 2 was exam-
ined with an in vitro protein phosphorylation assay using human
rhsp27 as a specific substrate, as described under Materials and
Methods, and the resultant autoradiograph is shown in Figure 2B.
TNF-
a
stimulation of neutrophils induced the cellular MAPKAP
kinase 2 activation (Fig. 2B,lane 3), which resulted from the p38
MAP kinase activation (Fig. 1A,lane 2). Pretreatment of neutro-
phils with SB20358 to down-regulate cellular p38 MAP kinase
completely inhibited the TNF-
a
-stimulated MAPKAP kinase 2 ac-
tivation (Fig. 2B,lane 4), as well as having an inhibitory effect on
basal cellular MAPKAP kinase 2 activity (Fig. 2B,lane 2). These
results confirm that SB20358 pretreatment of neutrophils inhibited
the activation of cellular p38 MAP kinase and blocked the down-
stream signaling of the kinase cascade.
Inhibition of cellular p38 MAP kinase by SB20358 specifically
reduces TNF-
a
-stimulated neutrophil IL-8 production
To investigate the physiologic function of p38 MAP kinase, neu-
trophils were preincubated with SB20358, as above, and then ex-
posed to 25 ng/ml TNF-
a
. Effect of cellular p38 MAP kinase on
the induced IL-8 production was evaluated by an RIA, as described
under Materials and Methods, and results are shown graphically.
FIGURE 1. The activation of MAP kinases in human neutrophils. A,
Protein phosphorylation of p38 MAP kinase. Human neutrophils were iso-
lated from healthy donors and stimulated with control buffer (lane 1), 25
ng/ml TNF-
a
(lane 2), 1 ng/ml GM-CSF (lane 3), 10
28
M FMLP (lane 4),
20 ng/ml PMA (lane 5), or 1
m
M ionomycin for 5 min at 37°C, as de-
scribed under Materials and Methods. Cellular proteins were separated on
10% SDS-PAGE and transferred to Immobilon-P membranes. Western
blotting was performed using Abs specific for phosphorylated p38 MAP
kinase (NEB) and the ECL method. Molecular weight standards are shown
on the left. The position of phosphorylated p38 MAP kinase is indicated on
the right.B, The induced protein phosphorylation of p44/42 MAP kinase.
Western blotting of p44/42 MAP kinase, using the same neutrophils as
described above, was conducted using Abs specific for phosphorylated
p44/42 MAP kinase (NEB). The positions of phosphorylated p44/42 MAP
kinase are indicated on the right.C, Western blotting analysis of neutrophil
p38 MAP kinase. To verify that any detected changes in the level of phos-
phorylated p38 MAP kinase were not a result of cellular protein digestion
or variable amounts of protein samples, the total amount of p38 MAP
kinase was examined by Western blotting using p38 MAP kinase Abs
(Santa Cruz Biotech). The position of p38 MAP kinase is indicated on the
right. These results are representative of three similar experiments.
FIGURE 2. Enzymatic activity assay of cellular MAPKAP kinase 2. To
confirm the inhibition of neutrophil p38 MAP kinase by SB20358, we
monitored the enzymatic activity of cellular MAPKAP kinase 2, which is
specific cellular substrate for p38 MAP kinase. Neutrophils were pretreated
with 0.6
m
M SB20358 (lanes 2,4, and 6) or without inhibitor (lanes 1,3,
and 5) for 40 min at 4°C, and then stimulated by 25 ng/ml TNF-
a
(lanes
3–6) for 5 min at 37°C, as indicated at the top of the figure. Cells were
lysed, and the kinase activity in each cell lysate was evaluated using an in
vitro protein phosphorylation assay with rhsp27 as the specific substrate for
MAPKAP kinase 2 (lanes 1– 4), as described under Materials and Meth-
ods. The reaction was stopped by addition of 23Laemmli solution, and
proteins were separated on 11% SDS-PAGE. A, Coomasie blue staining of
the proteins. Molecular weight standards are marked on the left. The po-
sition of rhsp27 is indicated on the right. An asterisk on the right indicates
a major neutrophil protein that was considered as an internal marker to
monitor that equal protein amounts were loaded in each lane. Lane 7 is
rhsp27 alone control. B, Autoradiograph of the induced protein phosphor-
ylation in A. The position of phosphorylated rhsp27 induced by neutrophil
MAPKAP kinase 2 is indicated on the right (phospho-rhsp27). These
results are representative of three similar experiments.
1984 p38 MAP KINASE AND NEUTROPHIL FUNCTION
Unstimulated human neutrophils produced a very low, but detect-
able, level of IL-8 (Fig. 3A,(2)/(2)), and SB20358 pretreatment
of cells had no effect on basal IL-8 production (Fig. 3A, SB20358/
(2)). Stimulation of neutrophils with TNF-
a
induced a 27-fold
increase in IL-8 production at 16 h (Fig. 3A,(2)/TNF-
a
). The
TNF-
a
-mediated increase in IL-8 production was abolished almost
completely by pretreatment of neutrophils with the p38 MAP ki-
nase inhibitor SB20358 (Fig. 3A, SB20358/TNF-
a
). Furthermore,
dose analysis indicates that the inhibitory effect of SB20358 on the
TNF-
a
-induced IL-8 production was concentration dependent and
reached a maximal inhibition at 0.15
m
M (Fig. 3B), a concentra-
tion that also inhibited the TNF-
a
-induced cellular p38 MAP
kinase activation in human neutrophils (data not shown).
To test the specificity of SB20358, compound PD98059, which
indirectly blocks the activation of p44/42 MAP kinase in intact
cells (34, 35), and compound H7, a protein kinase C inhibitor (36),
were utilized as controls in similar experiments. Figure 3Cshows
that pretreatment of human neutrophils with 4
m
M compound
PD98059, which completely inhibited the FMLP-stimulated phos-
phorylation and activation of cellular p44/42 MAP kinase (Fig. 5),
had no effect on the TNF-
a
-induced IL-8 production. Similarly, 6
m
M compound H7, which induced about 75% inhibition of the
PMA-stimulated IL-8 production (our unpublished observation),
could not inhibit the TNF-
a
-induced IL-8 production in human
neutrophils (Fig. 3C). Moreover, 0.6
m
M SB20358 had no inhib-
itory effect on PMA- or ionomycin-induced IL-8 production (Fig.
8), indicating that SB20358 was specific for cellular p38 MAP
kinase and not cytotoxic to human neutrophils, at least at concen-
tration used in this study. Taken together, these results demonstrate
that p38 MAP kinase plays a key role in mediating the TNF-
a
-
induced IL-8 production in human neutrophils.
SB20358 inhibits the TNF-
a
-induced superoxide generation of
human neutrophils
In addition to IL-8 production, another important cellular function
of the activated neutrophils is the generation of superoxide anions.
To investigate whether the p38 MAP kinase-mediated signaling
pathway is involved in the respiratory burst response, neutrophils
were pretreated with SB20358, as described above, to down-reg-
ulate cellular p38 MAP kinase. Cells were then activated by 25
ng/ml TNF-
a
or 20 ng/ml PMA stimulation, and the induced su-
peroxide generation was measured kinetically. In human neutro-
phils, a relatively low level (0.714 60.096 nmol superoxide/10
min/10
7
cells) of spontaneous superoxide generation was detect-
able (Fig. 4A), and stimulation of cells with TNF-
a
induced a
3.3-fold increase (2.381 60.274 nmol superoxide/10 min/10
7
cells) in superoxide generation (Fig. 4B). Down-regulation of cel-
lular p38 MAP kinase by SB20358 dramatically inhibited the
TNF-
a
-induced superoxide generation (0.476 60.038 nmol su-
peroxide/10 min/10
7
cells, Fig. 4B), and slightly decreased the
basal level (0.238 60.031 nmol superoxide/10 min/10
7
cells, Fig.
4A). However, SB20358 pretreatment of cells had no effect on the
PMA-induced respiratory burst response (Fig. 4C). In addition,
inhibition of cellular p44/42 MAP kinase with PD98059 had no
effect on the TNF-
a
-induced superoxide generation, as expected
(data not shown). These results indicate that the cellular p38 MAP
kinase activation is necessary for the neutrophil respiratory burst
stimulated by TNF-
a
, but not for that triggered by PMA.
Inhibition of the cellular MAP kinase activation stimulated by
FMLP
Exposure of human neutrophils to FMLP activated p38 and p44/42
MAP kinases (Fig. 1). Thus, it is interesting to know what is cel-
lular function of each kinase. To down-regulate cellular kinase
FIGURE 3. Effects of the kinase inhibitor SB20358 on the TNF-
a
-in-
duced neutrophil IL-8 production. A, Inhibition of the TNF-
a
-induced IL-8
production. To study cellular function of p38 MAP kinase in human neu-
trophils, cells were pretreated with 0.6
m
M SB20358 at 4°C for 40 min and
then stimulated with 25 ng/ml TNF-
a
. After being cultured for 0, 8, 16, or
24 h at 37°C, the medium was separated from the cells and used for an RIA
of IL-8, as described under Materials and Methods. The amount of de-
tected IL-8 is shown graphically as concentration of IL-8 in ng/10
7
neu-
trophils. Data presented are the mean 6SEM of three separate experi-
ments. B, Concentration-dependent effect of SB20358. Neutrophils were
pretreated with the listed concentrations of SB20358, as indicated at the
bottom of figure, and then stimulated with TNF-
a
at 37°C for 16 h. The
amount of IL-8 present in the media was evaluated by RIA, and the in-
hibitory effect of SB20358 is shown as a percentage of the maximal activity
of TNF-
a
-induced IL-8 production in the absence of SB20358, which was
arbitrarily set as 100%. The results are representative of three similar ex-
periments. C, Effect of different kinase inhibitors on the TNF-
a
-induced
neutrophil IL-8 production. To test the specificity of SB20358, neutrophils
were pretreated with 4
m
M of compound PD98059 (NEB), which specif-
ically inhibits p44/42 MAP kinase activation in intact cells, or 6
m
M H7,
a protein kinase C inhibitor at 4°C for 40 min. Cells were then stimulated
by 25 ng/ml TNF-
a
, and the induced IL-8 production after 16-h culture was
evaluated with RIA. The protein kinase inhibitors and TNF-
a
stimulation
used in each experiment are indicated at the bottom. The effect of kinase
inhibitors is shown as a percentage of activity of IL-8 production in a
graph, as described above. Data are representative of three similar
experiments.
1985The Journal of Immunology
activity, neutrophils were pretreated with the protein kinase inhib-
itors SB20358 or PD98059, respectively, as described above. Ef-
fect of the protein kinase inhibitors on the FMLP-stimulated acti-
vation of cellular p44/42 MAP kinase was detected by evaluating
the induced protein phosphorylation of the kinase, as described
under Materials and Methods. As shown in Figure 5B, the FMLP-
stimulated p44/42 MAP kinase activation (lane 2) was inhibited
completely by pretreatment of neutrophils with PD98059 (lane 4),
but not affected by SB20358 (lane 3). Activity of cellular p38
MAP kinase was detected indirectly by evaluating enzymatic ac-
tivity of cellular MAPKAP kinase 2 with an in vitro protein phos-
phorylation assay, as described above, since SB20358 specifically
binds to and inhibits p38 MAP kinase activity (32, 33), but has no
effect on the induced protein phosphorylation of p38 MAP kinase
(Fig. 5A,lane 3). Autoradiography analysis shows that exposure of
neutrophils to FMLP induced increase of cellular MAPKAP kinase
2 activity (Fig. 5C,lane 2), and this induced kinase activation was
inhibited completely by the p38 MAP kinase inhibitor SB20358
(Fig. 5C,lane 3), but not PD98059 (Fig. 5C,lane 4 ). Results
indicate that pretreatment of human neutrophils with SB20358 or
PD98059 could specifically block the FMLP-stimulated p38 or
p44/42 MAP kinase activation, respectively.
Down-regulation of cellular p38 MAP kinase by SB20358
inhibits the FMLP-induced neutrophil chemotaxis
To study the role of p38 and p44/42 MAP kinases in the FMLP-
activated neutrophils, the cellular kinases were down-regulated by
pretreatment of cells with the kinase inhibitors specific for each
kinase, as described above. Following FMLP stimulation, change
in the induced chemotaxis of human neutrophils was examined.
For this purpose, cellular migratory activity was evaluated by the
modified Boyden chamber assay and shown in a graph of chemo-
taxis index, as described under Materials and Methods. Chemo-
taxis assay demonstrates that exposure of neutrophils to FMLP
induced remarkable chemotaxis (17-fold higher chemotaxis index
than no stimulation control cells), and the FMLP-induced chemo-
taxis was suppressed dramatically by pretreatment of neutrophils
with the p38 MAP kinase inhibitor SB20358, but not influenced by
FIGURE 4. Effect of the kinase inhibitor SB20358 on neutrophil super-
oxide generation. Neutrophils were pretreated with 0.6
m
M SB20358 (SB/)
or without inhibitor ((2)/) for 40 min at 4°C. The respiratory burst re-
sponse was triggered by addition of 25 ng/ml TNF-
a
(/TNF-
a
)inB,20
ng/ml PMA (/PMA) in C, and no stimulus (/(2)) in A. The reaction was
allowed to proceed at 37°C for 10 min. Superoxide anion production was
measured kinetically by the reduction of cytochrome cat 550 nm (OD550)
using a THERMOmaxPRO microplate reader, as described under Materi-
als and Methods. The data shown are representative of five similar
experiments.
FIGURE 5. Inhibition of the FMLP-induced MAP kinase activation by
the specific kinase inhibitors. To regulate cellular MAP kinase activity,
neutrophils were preincubated with 0.6
m
M SB20358 (lane 3), a specific
p38 MAP kinase inhibitor, or 4
m
M PD98059 (lane 4), which specifically
blocks the p44/42 MAP kinase activation in intact cells, or without inhib-
itor (lanes 1 and 2) at 4°C for 40 min. Cells were then stimulated with 10
28
M FMLP (lanes 2– 4) or without stimulus as a control (lane 1) at 37°C for
5 min. The induced protein phosphorylation of cellular p38 or p44/42 MAP
kinases was examined, and Western blotting results are shown in Aand B,
respectively. Positions of the detected phosphorylated MAP kinases are
indicated by arrows on the right. The cellular p38 MAP kinase activity was
evaluated indirectly by examination of the in vitro rhsp27 phosphorylation
induced by cellular MAPKAP kinase 2, a specific intracellular substrate for
p38 MAP kinase, as described under Materials and Methods. The autora-
diograph is shown in C, and the position of phosphorylated rhsp27 is in-
dicated by an arrow on the right. These results are representative of three
similar experiments.
1986 p38 MAP KINASE AND NEUTROPHIL FUNCTION
the kinase inhibitor PD98059 for p44/42 MAP kinase (Fig. 6).
These findings indicate that the activation of cellular p38 MAP
kinase is indispensable for the FMLP-induced neutrophil
chemotaxis.
Inhibition of cellular p38 MAP kinase by SB20358 suppresses
the FMLP-stimulated superoxide production of human
neutrophils
To investigate the effect of each MAP kinase on neutrophil respi-
ratory burst, cells were pretreated with the kinase inhibitors
SB20358 or PD98059 to down-regulate the cellular p38 or p44/42
MAP kinases as above, respectively. Neutrophils were then ex-
posed to FMLP, and the induced superoxide generation was mea-
sured kinetically, as described under Materials and Methods.As
shown in Figure 7, stimulation of human neutrophils with FMLP
rapidly induced an 8.2-fold increase in superoxide generation at 3
min (9.285 61.538 nmol superoxide/3 min/10
7
cells, (2)/FMLP).
Down-regulation of cellular p38 MAP kinase by SB20358 com-
pletely inhibited the FMLP-induced superoxide generation
(0.762 60.104 nmol superoxide/3 min/10
7
cells, SB20358/
FMLP). However, inhibition of cellular p44/42 MAP kinase with
PD98059 had no inhibitory effect on the FMLP-induced respira-
tory burst (11.904 61.538 nmol superoxide/3 min/10
7
cells,
PD98059/FMLP). These results reveal that in human neutrophils,
the activation of cellular p38 MAP kinase is essential for the
respiratory burst response stimulated by FMLP.
Different effects of cellular p38 MAP kinase on neutrophil IL-8
production
The study shown in Figure 3 reveals that the p38 MAP kinase
activation was required for the neutrophil IL-8 production induced
by TNF-
a
, which stimulated cellular p38 MAP kinase only and
had no effect on p44/42 MAP kinase (Fig. 1). To understand the
role of p38 MAP kinase in IL-8 production of the activated neu-
trophils, in which both p38 and p44/42 MAP kinases are activated,
cells were pretreated with the p38 MAP kinase inhibitor SB20358
and stimulated with FMLP, GM-CSF, PMA, or ionomycin.
Changes in neutrophil IL-8 production were examined by RIA, and
results are shown graphically, as described under Materials and
Methods. Stimulation of cells with GM-CSF, a hemopoietic
growth factor that has been reported to potentiate neutrophil func-
tions (6), strongly induced IL-8 production (3.414 ng/10
7
cells vs
basal level 0.162 ng/10
7
cells), while down-regulation of cellular
p38 MAP kinase by SB20358 abolished the GM-CSF-stimulated
IL-8 production (Fig. 8). Unexpectedly, FMLP had little capacity
for inducing IL-8 production in human neutrophils (Fig. 8), even
though it stimulated both p38 and p44/42 MAP kinase activation
FIGURE 6. Effect of cellular MAP kinases on the FMLP-induced che-
motaxis. Neutrophils were pretreated with the kinase inhibitors SB20358 or
PD98059, or without inhibitor (2), as indicated in figure, and loaded into
the upper wells of the modified Boyden chamber, as described under Ma-
terials and Methods. Neutrophil chemotaxis was induced by the presence
of 10
28
M FMLP in the lower wells of the Boyden chamber at 37°C for 60
min. The number of the cells migrating through the filter, which was lo-
cated between the upper and lower wells, was counted, and the chemotac-
tic/migratory index is shown in figure, as described under Materials and
Methods. Number of chemotaxis index is shown on the left, and stimulation
of FMLP is indicated on the bottom. Data presented are the mean 6SEM
of three separate experiments.
FIGURE 7. Effect of cellular MAP kinases on the FMLP-stimulated
superoxide generation. Neutrophils were pretreated with the kinase inhib-
itors SB20358 (SB20358/) or PD98059 (PD98059/), or without inhibitor
((2)/), as described above. Cells were then stimulated with 5 310
28
M
FMLP (/FMLP) or without as a control (/(2)) at 37°C, and the induced
superoxide generation was detected by the reduction of cytochrome cat
550 nm (OD550) for 0 to 4 min, as described under Materials and Meth-
ods. The volume of the detected OD550 and the time are shown on the left
and the bottom, respectively. The used kinase inhibitors and stimulus are
indicated on the right. The data shown are representative of five similar
experiments.
FIGURE 8. Effects of p38 MAP kinase on neutrophil IL-8 production in
response to different stimuli. To examine the role of p38 MAP kinase in
IL-8 production of the activated neutrophils, in which multiple protein
kinases are stimulated, cells were pretreated with 0.6
m
M SB20358 or
without (2) at 4°C for 40 min, followed by exposure to 1 ng/ml GM-CSF,
10
28
M FMLP, 1
m
M ionomycin, or 50 ng/ml LPS at 37°C for 16 h, or 20
ng/ml PMA at 37°C for 8 h. At the end of the given time period, the media
were harvested and used to determine IL-8 production by RIA, as described
under Materials and Methods. Results are shown graphically as concen-
tration of IL-8 in ng/10
7
neutrophils. The stimulation used in each exper-
iment is indicated at the bottom. Data presented are the mean 6SEM of
three separate experiments.
1987The Journal of Immunology
(Fig. 5). PMA, a potential protein kinase C activator, and iono-
mycin, a calcium ionophore, activated both cellular p38 and
p44/42 MAP kinases (Fig. 1) and markedly stimulated neutrophil
IL-8 production, 6.552 and 13.11 ng/10
7
cells, respectively (Fig.
8). However, down-regulation of cellular p38 MAP kinase by pre-
treatment of cells with SB20358 had little inhibitory effect on
PMA- or ionomycin-induced IL-8 production (Fig. 8). Inhibition
of cellular protein kinase C activation by H7 resulted in about 75%
inhibition of the PMA-stimulated IL-8 production (data not
shown). In addition, exposure of neutrophils to LPS, a bacterial
product that primes human neutrophils and stimulates cellular p38
MAP kinase, but not p44/42 MAP kinase, as previously reported
(28 and 29), markedly induced IL-8 production (7.686 ng/10
7
cells), and inhibition of cellular p38 MAP kinase by SB20358
diminished the LPS-induced IL-8 production almost completely
(Fig. 8). These results indicate that multiple signaling pathways are
involved in regulating neutrophil IL-8 production, and that p38
MAP kinase may play different roles in regulating neutrophil func-
tion in response to distinct stimulation.
Discussion
It has been reported that stimulation of neutrophils with FMLP
activated cellular p44/42 MAP kinase via Ras- and Raf-mediated
pathway (46 and 47). Treatment of neutrophils with genistein, a
general tyrosine kinase inhibitor, reduced the tyrosine phosphory-
lation of cellular proteins, including p44/42 MAP kinase, and in-
hibited the neutrophil functional response to FMLP (48). In this
study, detailed analysis of cellular MAP kinases shows that expo-
sure of neutrophils to FMLP induced the tyrosine phosphorylation
and the activation of p38 MAP kinase as well as p44/42 MAP
kinase (Fig. 5). Cellular functional assay by using the specific
MAP kinase inhibitors indicates that the activation of cellular p38
MAP kinase, but not p44/42 MAP kinase, was indispensable for
the FMLP-induced chemotaxis and superoxide generation of hu-
man neutrophils (Figs. 6 and 7). In addition, our previous research
has demonstrated that inhibition of neutrophil MAPKAP kinase 2,
a cellular substrate specific for p38 MAP kinase, diminished the
FMLP-induced superoxide generation (31). These observations
strongly suggest that p38 MAP kinase-mediated signaling pathway
plays a central role in regulating neutrophil chemotaxis and super-
oxide generation stimulated by FMLP. Moreover, down-regulation
of p38 MAP kinase inhibited the IL-8 production stimulated by
TNF-
a
, GM-CSF, or LPS, and had no effect on that induced by
PMA or ionomycin (Fig. 8), suggesting that p38 MAP kinase may
play different role in human neutrophil response to distinct stim-
ulation, as p44/22 MAP kinase does (49). Our previous study re-
vealed that in human neutrophils, a 60-kDa cytosolic protein is a
major substrate for MAPKAP kinase 2 (31). This 60-kDa cytosolic
protein has been demonstrated to be lymphocyte-specific protein 1
(50), an F-actin-binding protein. Whether lymphocyte-specific
protein 1 is involved in the p38 MAP kinase/MAPKAP kinase
2-regulated neutrophil functional responses to TNF-
a
or FMLP
remains to be determined.
In this study, we first demonstrated that p38 MAP kinase-sig-
naling pathway is involved in regulating IL-8 production of human
neutrophils (Figs. 3 and 8). However, the mechanism of the p38
MAP kinase-mediated IL-8 production is unknown. Most recent
research indicates that stimulation of human neutrophils with
TNF-
a
induced the activation and nuclear translocation of tran-
scription factor NF-
k
B (51). Gene transcription studies have re-
vealed that the NF-
k
B-like binding sites located in the 59-flanking
region of the IL-8 gene are essential for the transcriptional acti-
vation response to stimulation of TNF-
a
(52 and 53). Considering
these findings, it seems likely that in human neutrophils, TNF-
a
stimulates the activation of cellular p38 MAP kinase, which in turn
activates NF-
k
B directly or indirectly, and results in IL-8 gene
transcription/expression. Thus, it will be interesting to address
whether p38 MAP kinase regulates the protein phosphorylation
and activity of cellular I
k
B
a
, a cytoplasmic protein that controls
the nuclear translocation and activation of NF-
k
B (54).
Acknowledgments
We thank Dr. John Lee (SmithKline Beecham) for providing p38 MAP
kinase inhibitor SB20358, Dr. Lawrence Rothfield (Microbiology Depart-
ment of University of Connecticut Health Center (UCHC)) for helpful loan
of equipment, and Dr. Elmer L. Becker (Pathology Department of UCHC)
for reviewing the manuscript.
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1989The Journal of Immunology
