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or FMLP Stimulation1
Departments of
*
Physiology,
Pathology, and
Medicine, University of Connecticut Health Center, Farmington, CT 06030; and
§
Institute for Neuroscience, Northwestern University, Chicago, IL 60611
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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 neutrophils, were utilized. Inhibition
of the cellular p38 MAP kinase activation almost completely abolished
the TNF--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-, 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--
or FMLP-mediated cellular functions in human neutrophils, and suggest
that p38 MAP kinase may play a different role in response to distinct
stimulation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 activated 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, 23, 24). In addition, activated p38 MAP
kinase has been shown to phosphorylate specific transcription factors
in vitro and in intact cells, and thus may regulate gene expression
(25, 26, 27). Investigation of human neutrophils has revealed that in
response to certain extracellular stimulation, the kinase activities of
p38 MAP kinase (28, 29, 30) and MAPKAP kinase 2 (31) rapidly increased,
suggesting this kinase cascade may play a pivotal role in regulating
neutrophil function. In this study, to understand the potential
physiologic function(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, 33, 34, 35). Effects of cellular
p38 MAP kinase or p44/42 MAP kinase on neutrophil IL-8 production,
superoxide generation, or chemotaxis induced by TNF- or FMLP were
examined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 neutrophils were resuspended in HBSS (Life
Technologies, Gaithersburg, MD) containing 10 mM HEPES (pH 7.5) and 1
mM calcium. To modify cellular MAP kinase activity, compound SB20358
(32, 33), a specific inhibitor for p38 MAP kinase
(IC50 = 0.6 µM 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
IC50 = 4 µM in vitro (34, 35), was purchased from New
England Biolabs (Beverly, MA). In addition, compound H7 (Seikagaku
Corp., Tokyo, Japan), a protein kinase C inhibitor with
Ki = 6 µM (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- (Sigma Chemical Co., St. Louis, MO),
10-8 M FMLP (Sigma Chemical Co.), 1 ng/ml GM-CSF (R&D
Systems, Minneapolis, MN), 20 ng/ml PMA (Sigma Chemical Co.), 1 µM
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 activated by distinct upstream kinases through phosphorylation of both threonine 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 phosphorylation of MAP kinases, following each stimulation, 4 x 106 neutrophils were harvested and resuspended in 140 µl 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 µM p-nitrophenyl phosphate, 1 mM diisopropylfluorophosphate, 0.7 mg/ml pepstatin A, 10 mg/ml leupeptin, and 2 mg/ml aprotinin) and solubilized with the addition of 140 µl of 2x Laemmli solution (9% (w/v) SDS, 6% (v/v) ß-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-µl samples) were electrophoresed through 10% SDS-PAGE and then transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) using a semidry 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-phosphorylated 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 chemiluminescence (ECL from Amersham, Arlington Heights, IL) reagents, and the phosphorylated MAP kinases were detected by autoradiography for variable 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 ß-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 horseradish 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 x 107) were harvested in 200 µl
cold sample buffer and treated with mild sonication for 10 s.
After centrifugation at 3000 x g for 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 initiated by the addition of an equal volume (30 µl) of
freshly prepared phosphorylation reaction mix containing: 30 mM HEPES
(pH 7.3), 20 mM MgCl2, 2 mM EGTA, 10 µM sodium
orthovanadate, 5 µM okadaic acid, 4 mM DTT, 30 µM H-7, 0.4 mM
[
-32P]ATP (105 cpm/pmol), and 0.5 µg
hsp27, to 30 µl cellular supernatant. The in vitro phosphorylation
reaction was conducted at 30°C for 10 min and stopped by addition of
60 µl 2x 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 x
106 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-, FMLP, GM-CSF, ionomycin, PMA, or LPS, as
described above, and cultured at 37°C under 5% CO2
condition. Medium was harvested at the various times as indicated in
figures, separated from cells by centrifugation at 2000 x
g for 5 min, and analyzed for IL-8 production by an RIA, as
described previously, with slight modifications (40). Briefly, 100 µl
neutrophil-conditioned medium was incubated for 1 h at 25°C with
100 µl chicken anti-human IL-8 Ab (40) diluted 1/2000 in PBS
containing 1% BSA. Human 125I-labeled IL-8 (DuPont NEN,
Boston, MA) at 70,000 to 80,000 cpm/ml in PBS was added (100 µl), and
the reaction mix was incubated for another hour at 25°C. The reaction
was stopped by addition of 500 µl cold PBS containing 10 mg/ml BSA,
and the immune complexes were precipitated using saturated ammonium
sulfate at 50% final concentration. The samples were centrifuged at
5000 x g for 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 x 107 cells/ml) were resuspended in HBSS
containing 145 mM cytochrome c, 2 mM sodium azide, 2 mM
CaCl2, and 2.4 mM MgCl2, and equilibrated to
37°C for 5 min. Cells (100 µl) were dispensed into prewarmed
microtiter plates, and superoxide anion production was initiated by
stimulating neutrophils with 25 ng/ml TNF-, 5 x
10-8 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 c at
550 nm (OD550) using a THERMOmax kinetic microplate reader
(Molecular Devices, Menlo Park, CA). Data were analyzed using the
SOFTmax program.
Chemotactic assays
Neutrophil chemotaxis induced by FMLP was assayed by a modified Boyden 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 Modified Dulbecco’s medium with 1% BSA, and loaded into upper wells of the Boyden chamber, which was separated from the lower wells by 3-µm cellulose nitrate filters (Millipore). To induce chemotaxis, 10-8 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 humidified 5% CO2 incubator at 37°C for 60 min, and the filters were removed, fixed, stained, and air dried. Cell migration was quantitated by a microsectioning 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 x distance migrated through the filter).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To better understand the regulation of MAP kinases in neutrophil
activation, freshly isolated cells were exposed to different stimuli,
including cytokines (TNF- 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 response to stimulation of TNF-, GM-CSF, FMLP, PMA, or ionomycin
(Fig. 1A). Cellular p44/42 MAP kinase was also
activated by exposure of neutrophils to GM-CSF, FMLP, PMA, and
ionomycin, but not to TNF- (Fig. 1B). The findings
were confirmed by examining total cellular p38 MAP kinase, and Figure 1C indicates that the differences observed for the induced
protein phosphorylation of the cellular MAP kinases did not result from
differences in loading or from cellular protein
digestion.
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The finding that TNF- could specifically activate p38 MAP
kinase, but not p44/42 MAP kinase (Fig. 1
, A and
B), led us to use this cytokine to study the role of
p38 MAP kinase in human neutrophil activation. In addition, recent
discovery of compound SB20358, a specific kinase inhibitor for p38 MAP
kinase with an IC50 = 0.6 µM in vitro that has no
apparent effect on other protein kinases, including p44/42 MAP kinase
or the stress-activated protein 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 µM 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 phosphorylation of
small heat-shock proteins (hsp25/27) (39, 44, 45). Enzymatic
activity of cellular MAPKAP kinase 2 was examined 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- 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 neutrophils with SB20358 to down-regulate cellular p38
MAP kinase completely inhibited the TNF--stimulated MAPKAP kinase 2
activation (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 downstream signaling of the
kinase cascade.
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-stimulated neutrophil IL-8 production
To investigate the physiologic function of p38 MAP kinase,
neutrophils were preincubated with SB20358, as above, and then exposed
to 25 ng/ml TNF-. 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.
Unstimulated human neutrophils produced a very low, but detectable,
level of IL-8 (Fig. 3A,
(-)/(-)), and SB20358 pretreatment of cells had no effect on basal
IL-8 production (Fig. 3A, SB20358/(-)). Stimulation of
neutrophils with TNF- induced a 27-fold increase in IL-8 production
at 16 h (Fig. 3A, (-)/TNF-). The TNF--mediated
increase in IL-8 production was abolished almost completely by
pretreatment of neutrophils with the p38 MAP kinase inhibitor SB20358
(Fig. 3A, SB20358/TNF-). Furthermore, dose analysis
indicates that the inhibitory effect of SB20358 on the TNF--induced
IL-8 production was concentration dependent and reached a maximal
inhibition at 0.15 µM (Fig. 3B), a concentration
that also inhibited the TNF--induced cellular p38 MAP
kinase activation in human neutrophils (data not shown).
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C shows that
pretreatment of human neutrophils with 4 µM compound PD98059, which
completely inhibited the FMLP-stimulated phosphorylation and activation
of cellular p44/42 MAP kinase (Fig. 5), had no effect on the
TNF--induced IL-8 production. Similarly, 6 µM compound H7, which
induced about 75% inhibition of the PMA-stimulated IL-8 production
(our unpublished observation), could not inhibit the TNF--induced
IL-8 production in human neutrophils (Fig. 3C).
Moreover, 0.6 µM SB20358 had no inhibitory 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 concentration used in this study. Taken
together, these results demonstrate that p38 MAP kinase plays a key
role in mediating the TNF--induced IL-8 production in human
neutrophils.
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-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-regulate cellular
p38 MAP kinase. Cells were then activated by 25 ng/ml TNF- or 20
ng/ml PMA stimulation, and the induced superoxide generation was
measured kinetically. In human neutrophils, a relatively low level
(0.714 ± 0.096 nmol superoxide/10 min/107 cells)
of spontaneous superoxide generation was detectable (Fig. 4A), and stimulation of
cells with TNF- induced a 3.3-fold increase (2.381 ± 0.274
nmol superoxide/10 min/107 cells) in superoxide generation
(Fig. 4B). Down-regulation of cellular p38 MAP kinase
by SB20358 dramatically inhibited the TNF--induced superoxide
generation (0.476 ± 0.038 nmol superoxide/10 min/107
cells, Fig. 4B), and slightly decreased the basal
level (0.238 ± 0.031 nmol superoxide/10 min/107
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--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-, but not for that triggered
by PMA.
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Exposure of human neutrophils to FMLP activated p38 and p44/42 MAP
kinases (Fig. 1). Thus, it is interesting to know what is cellular
function of each kinase. To down-regulate cellular kinase activity,
neutrophils were pretreated with the protein kinase inhibitors SB20358
or PD98059, respectively, as described above. Effect of the protein
kinase inhibitors on the FMLP-stimulated activation 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 activity of cellular
MAPKAP kinase 2 with an in vitro protein phosphorylation 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 chemotaxis index, as described
under Materials and Methods. Chemotaxis 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 chemotaxis was suppressed dramatically by
pretreatment of neutrophils with the p38 MAP kinase inhibitor SB20358,
but not influenced by 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.
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To investigate the effect of each MAP kinase on neutrophil
respiratory 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 exposed to FMLP,
and the induced superoxide generation was measured 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 ± 1.538 nmol superoxide/3 min/107 cells,
(-)/FMLP). Down-regulation of cellular p38 MAP kinase by SB20358
completely inhibited the FMLP-induced superoxide generation (0.762
± 0.104 nmol superoxide/3 min/107 cells, SB20358/FMLP).
However, inhibition of cellular p44/42 MAP kinase with PD98059 had no
inhibitory effect on the FMLP-induced respiratory burst (11.904 ±
1.538 nmol superoxide/3 min/107 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.
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The study shown in Figure 3 reveals that the p38 MAP kinase
activation was required for the neutrophil IL-8 production induced by
TNF-, 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 neutrophils, 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 functions (6), strongly induced IL-8 production (3.414
ng/107 cells vs basal level 0.162 ng/107
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
(Fig. 5). PMA, a potential protein kinase C activator, and ionomycin, 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/107 cells, respectively (Fig. 8). However,
down-regulation of cellular p38 MAP kinase by pretreatment 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/107 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 function in response to distinct
stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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 human 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 superoxide generation
stimulated by FMLP. Moreover, down-regulation of p38 MAP kinase
inhibited the IL-8 production stimulated by TNF-, 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 stimulation, as p44/22 MAP kinase does
(49). Our previous study revealed 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- or FMLP
remains to be determined.
In this study, we first demonstrated that p38 MAP kinase-signaling
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- induced the activation
and nuclear translocation of transcription factor NF-
B (51). Gene
transcription studies have revealed that the NF-B-like binding sites
located in the 5'-flanking region of the IL-8 gene are essential for
the transcriptional activation response to stimulation of TNF- (52
and 53). Considering these findings, it seems likely that in human
neutrophils, TNF- stimulates the activation of cellular p38 MAP
kinase, which in turn activates NF-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 IB, a cytoplasmic
protein that controls the nuclear translocation and activation of
NF-B (54).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. You-Li Zu, Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030-3505. E-mail address:
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-B, nuclear factor-B.
Received for publication August 18, 1997. Accepted for publication November 3, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced phosphorylation and activation of cytosolic phospholipase A2 are abrogated by an inhibitor of the p38 MAP kinase cascade in human neutrophils. Biochem. J. 319:17.
B pathway by inflammatory stimuli in human neutrophils. Blood 89:3421.B and its inhibitor, IB-. Proc. Natl. Acad. Sci. USA 90:2532.This article has been cited by other articles:
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N. R. Jog, V. R. Jala, R. A. Ward, M. J. Rane, B. Haribabu, and K. R. McLeish Heat Shock Protein 27 Regulates Neutrophil Chemotaxis and Exocytosis through Two Independent Mechanisms J. Immunol., February 15, 2007; 178(4): 2421 - 2428. [Abstract] [Full Text] [PDF]
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A. Cloutier, T. Ear, E. Blais-Charron, C. M. Dubois, and P. P. McDonald Differential involvement of NF-{kappa}B and MAP kinase pathways in the generation of inflammatory cytokines by human neutrophils J. Leukoc. Biol., February 1, 2007; 81(2): 567 - 577. [Abstract] [Full Text] [PDF]
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L. M. Lavigne, J. E. Albina, and J. S. Reichner beta-Glucan Is a Fungal Determinant for Adhesion-Dependent Human Neutrophil Functions J. Immunol., December 15, 2006; 177(12): 8667 - 8675. [Abstract] [Full Text] [PDF]
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L. E. Kilpatrick, S. Sun, D. Mackie, F. Baik, H. Li, and H. M. Korchak Regulation of TNF mediated antiapoptotic signaling in human neutrophils: role of {delta}-PKC and ERK1/2 J. Leukoc. Biol., December 1, 2006; 80(6): 1512 - 1521. [Abstract] [Full Text] [PDF]
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A. Jinnouchi, Y. Aida, K. Nozoe, K. Maeda, and M. J. Pabst Local anesthetics inhibit priming of neutrophils by lipopolysaccharide for enhanced release of superoxide: suppression of cytochrome b558 expression by disparate mechanisms J. Leukoc. Biol., December 1, 2005; 78(6): 1356 - 1365. [Abstract] [Full Text] [PDF]
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L. C. Parker, M. K. B. Whyte, S. K. Dower, and I. Sabroe The expression and roles of Toll-like receptors in the biology of the human neutrophil J. Leukoc. Biol., June 1, 2005; 77(6): 886 - 892. [Abstract] [Full Text] [PDF]
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G. Lominadze, M. J. Rane, M. Merchant, J. Cai, R. A. Ward, and K. R. McLeish Myeloid-Related Protein-14 Is a p38 MAPK Substrate in Human Neutrophils J. Immunol., June 1, 2005; 174(11): 7257 - 7267. [Abstract] [Full Text] [PDF]
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Z. T. Kneass and R. B. Marchase Protein O-GlcNAc Modulates Motility-associated Signaling Intermediates in Neutrophils J. Biol. Chem., April 15, 2005; 280(15): 14579 - 14585. [Abstract] [Full Text] [PDF]
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C. S. Hii, D. S. Anson, M. Costabile, V. Mukaro, K. Dunning, and A. Ferrante Characterization of the MEK5-ERK5 Module in Human Neutrophils and Its Relationship to ERK1/ERK2 in the Chemotactic Response J. Biol. Chem., November 26, 2004; 279(48): 49825 - 49834. [Abstract] [Full Text] [PDF]
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Z. T. Kneass and R. B. Marchase Neutrophils Exhibit Rapid Agonist-induced Increases in Protein-associated O-GlcNAc J. Biol. Chem., October 29, 2004; 279(44): 45759 - 45765. [Abstract] [Full Text] [PDF]
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R. Janssen, A. van Wengen, M. A. Hoeve, M. ten Dam, M. van der Burg, J. van Dongen, E. van de Vosse, M. van Tol, R. Bredius, T. H. Ottenhoff, et al. The Same I{kappa}B{alpha} Mutation in Two Related Individuals Leads to Completely Different Clinical Syndromes J. Exp. Med., September 7, 2004; 200(5): 559 - 568. [Abstract] [Full Text] [PDF]
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M. Aleman, P. Schierloh, S. S. de la Barrera, R. M. Musella, M. A. Saab, M. Baldini, E. Abbate, and M. C. Sasiain Mycobacterium tuberculosis Triggers Apoptosis in Peripheral Neutrophils Involving Toll-Like Receptor 2 and p38 Mitogen Protein Kinase in Tuberculosis Patients Infect. Immun., September 1, 2004; 72(9): 5150 - 5158. [Abstract] [Full Text] [PDF]
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T. Ohira, G. Bannenberg, M. Arita, M. Takahashi, Q. Ge, T. E. Van Dyke, G. L. Stahl, C. N. Serhan, and J. A. Badwey A Stable Aspirin-Triggered Lipoxin A4 Analog Blocks Phosphorylation of Leukocyte-Specific Protein 1 in Human Neutrophils J. Immunol., August 1, 2004; 173(3): 2091 - 2098. [Abstract] [Full Text] [PDF]
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M. Bouaouina, E. Blouin, L. Halbwachs-Mecarelli, P. Lesavre, and P. Rieu TNF-Induced {beta}2 Integrin Activation Involves Src Kinases and a Redox-Regulated Activation of p38 MAPK J. Immunol., July 15, 2004; 173(2): 1313 - 1320. [Abstract] [Full Text] [PDF]
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G. E. Brown, M. Q. Stewart, S. A. Bissonnette, A. E. H. Elia, E. Wilker, and M. B. Yaffe Distinct Ligand-dependent Roles for p38 MAPK in Priming and Activation of the Neutrophil NADPH Oxidase J. Biol. Chem., June 25, 2004; 279(26): 27059 - 27068. [Abstract] [Full Text] [PDF]
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B. Kasper, E. Brandt, S. Bulfone-Paus, and F. Petersen Platelet factor 4 (PF-4)-induced neutrophil adhesion is controlled by src-kinases, whereas PF-4-mediated exocytosis requires the additional activation of p38 MAP kinase and phosphatidylinositol 3-kinase Blood, March 1, 2004; 103(5): 1602 - 1610. [Abstract] [Full Text] [PDF]
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S. Singh, D. W. Powell, M. J. Rane, T. H. Millard, J. O. Trent, W. M. Pierce, J. B. Klein, L. M. Machesky, and K. R. McLeish Identification of the p16-Arc Subunit of the Arp 2/3 Complex as a Substrate of MAPK-activated Protein Kinase 2 by Proteomic Analysis J. Biol. Chem., September 19, 2003; 278(38): 36410 - 36417. [Abstract] [Full Text] [PDF]
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R. El Bekay, M. Alvarez, J. Monteseirin, G. Alba, P. Chacon, A. Vega, J. Martin-Nieto, J. Jimenez, E. Pintado, F. J. Bedoya, et al. Oxidative stress is a critical mediator of the angiotensin II signal in human neutrophils: involvement of mitogen-activated protein kinase, calcineurin, and the transcription factor NF-{kappa}B Blood, July 15, 2003; 102(2): 662 - 671. [Abstract] [Full Text] [PDF]
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A. A. Rarok, P. C. Limburg, and C. G. M. Kallenberg Neutrophil-activating potential of antineutrophil cytoplasm autoantibodies J. Leukoc. Biol., July 1, 2003; 74(1): 3 - 15. [Abstract] [Full Text] [PDF]
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Q. Chen, D. W. Powell, M. J. Rane, S. Singh, W. Butt, J. B. Klein, and K. R. McLeish Akt Phosphorylates p47phox and Mediates Respiratory Burst Activity in Human Neutrophils J. Immunol., May 15, 2003; 170(10): 5302 - 5308. [Abstract] [Full Text] [PDF]
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A. Holub, J. Byrnes, S. Anderson, L. Dzaidzio, N. Hogg, and A. Huttenlocher Ligand density modulates eosinophil signaling and migration J. Leukoc. Biol., May 1, 2003; 73(5): 657 - 664. [Abstract] [Full Text] [PDF]
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K. C. Malcolm and G. S. Worthen Lipopolysaccharide Stimulates p38-dependent Induction of Antiviral Genes in Neutrophils Independently of Paracrine Factors J. Biol. Chem., April 25, 2003; 278(18): 15693 - 15701. [Abstract] [Full Text] [PDF]
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 J. S. Park, J. Arcaroli, H.-K. Yum, H. Yang, H. Wang, K.-Y. Yang, K.-H. Choe, D. Strassheim, T. M. Pitts, K. J. Tracey, et al. Activation of gene expression in human neutrophils by high mobility group box 1 protein Am J Physiol Cell Physiol, April 1, 2003; 284(4): C870 - C879. [Abstract] [Full Text] [PDF]
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C. Stambe, R. C. Atkins, G. H. Tesch, A. M. Kapoun, P. A. Hill, G. F. Schreiner, and D. J. Nikolic-Paterson Blockade of p38{alpha} MAPK Ameliorates Acute Inflammatory Renal Injury in Rat Anti-GBM Glomerulonephritis J. Am. Soc. Nephrol., February 1, 2003; 14(2): 338 - 351. [Abstract] [Full Text] [PDF]
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J. A. Nick, S. K. Young, P. G. Arndt, J. G. Lieber, B. T. Suratt, K. R. Poch, N. J. Avdi, K. C. Malcolm, C. Taube, P. M. Henson, et al. Selective Suppression of Neutrophil Accumulation in Ongoing Pulmonary Inflammation by Systemic Inhibition of p38 Mitogen-Activated Protein Kinase J. Immunol., November 1, 2002; 169(9): 5260 - 5269. [Abstract] [Full Text] [PDF]
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J. Li, S. Kartha, S. Iasvovskaia, A. Tan, R. K. Bhat, J. M. Manaligod, K. Page, A. R. Brasier, and M. B. Hershenson Regulation of human airway epithelial cell IL-8 expression by MAP kinases Am J Physiol Lung Cell Mol Physiol, October 1, 2002; 283(4): L690 - L699. [Abstract] [Full Text] [PDF]
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M. Junghae and J. G. Raynes Activation of p38 Mitogen-Activated Protein Kinase Attenuates Leishmania donovani Infection in Macrophages Infect. Immun., September 1, 2002; 70(9): 5026 - 5035. [Abstract] [Full Text] [PDF]
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S. R. Yan, W. Al-Hertani, D. Byers, and R. Bortolussi Lipopolysaccharide-Binding Protein- and CD14-Dependent Activation of Mitogen-Activated Protein Kinase p38 by Lipopolysaccharide in Human Neutrophils Is Associated with Priming of Respiratory Burst Infect. Immun., August 1, 2002; 70(8): 4068 - 4074. [Abstract] [Full Text] [PDF]
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 M. Gouwy, S. Struyf, F. Mahieu, W. Put, P. Proost, and J. Van Damme The Unique Property of the CC Chemokine Regakine-1 to Synergize with Other Plasma-Derived Inflammatory Mediators in Neutrophil Chemotaxis Does Not Reside in Its NH2-Terminal Structure Mol. Pharmacol., July 1, 2002; 62(1): 173 - 180. [Abstract] [Full Text] [PDF]
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C. Wang, H. Hayashi, R. Harrison, B. Chiu, J. R. Chan, H. L. Ostergaard, R. D. Inman, J. Jongstra, M. I. Cybulsky, and J. Jongstra-Bilen Modulation of Mac-1 (CD11b/CD18)-Mediated Adhesion by the Leukocyte-Specific Protein 1 Is Key to Its Role in Neutrophil Polarization and Chemotaxis J. Immunol., July 1, 2002; 169(1): 415 - 423. [Abstract] [Full Text] [PDF]
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S. K. Roach and J. S. Schorey Differential Regulation of the Mitogen-Activated Protein Kinases by Pathogenic and Nonpathogenic Mycobacteria Infect. Immun., June 1, 2002; 70(6): 3040 - 3052. [Abstract] [Full Text] [PDF]
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M. Berger, S. Budhu, E. Lu, Y. Li, D. Loike, S. C. Silverstein, and J. D. Loike Different Gi-coupled chemoattractant receptors signal qualitatively different functions in human neutrophils J. Leukoc. Biol., May 1, 2002; 71(5): 798 - 806. [Abstract] [Full Text] [PDF]
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C. Rubel, G. C. Fernandez, F. A. Rosa, S. Gomez, M. B. Bompadre, O. A. Coso, M. A. Isturiz, and M. S. Palermo Soluble Fibrinogen Modulates Neutrophil Functionality Through the Activation of an Extracellular Signal-Regulated Kinase-Dependent Pathway J. Immunol., April 1, 2002; 168(7): 3527 - 3535. [Abstract] [Full Text] [PDF]
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B. Weinberger, D. L. Laskin, T. M. Mariano, V. R. Sunil, C. J. DeCoste, D. E. Heck, C. R. Gardner, and J. D. Laskin Mechanisms underlying reduced responsiveness of neonatal neutrophils to distinct chemoattractants J. Leukoc. Biol., December 1, 2001; 70(6): 969 - 976. [Abstract] [Full Text] [PDF]
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D. C. Cara, J. Kaur, M. Forster, D.-M. McCafferty, and P. Kubes Role of p38 Mitogen-Activated Protein Kinase in Chemokine-Induced Emigration and Chemotaxis In Vivo J. Immunol., December 1, 2001; 167(11): 6552 - 6558. [Abstract] [Full Text] [PDF]
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K. Suzuki, M. Hino, H. Kutsuna, F. Hato, C. Sakamoto, T. Takahashi, N. Tatsumi, and S. Kitagawa Selective Activation of p38 Mitogen-Activated Protein Kinase Cascade in Human Neutrophils Stimulated by IL-1{beta} J. Immunol., November 15, 2001; 167(10): 5940 - 5947. [Abstract] [Full Text] [PDF]
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H. Zarkesh-Esfahani, G. Pockley, R. A. Metcalfe, M. Bidlingmaier, Z. Wu, A. Ajami, A. P. Weetman, C. J. Strasburger, and R. J. M. Ross High-Dose Leptin Activates Human Leukocytes Via Receptor Expression on Monocytes J. Immunol., October 15, 2001; 167(8): 4593 - 4599. [Abstract] [Full Text] [PDF]
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M. O. Hannigan, L. Zhan, Y. Ai, A. Kotlyarov, M. Gaestel, and C.-K. Huang Abnormal Migration Phenotype of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2-/- Neutrophils in Zigmond Chambers Containing Formyl-Methionyl-Leucyl-Phenylalanine Gradients J. Immunol., October 1, 2001; 167(7): 3953 - 3961. [Abstract] [Full Text] [PDF]
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 M. J. Alcorn, J. L. Booth, K. M. Coggeshall, and J. P. Metcalf Adenovirus Type 7 Induces Interleukin-8 Production via Activation of Extracellular Regulated Kinase 1/2 J. Virol., July 15, 2001; 75(14): 6450 - 6459. [Abstract] [Full Text]
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 T. Nagata, M. Kansha, K. Irita, and S. Takahashi Propofol inhibits FMLP-stimulated phosphorylation of p42 mitogen-activated protein kinase and chemotaxis in human neutrophils Br. J. Anaesth., June 1, 2001; 86(6): 853 - 858. [Abstract] [Full Text] [PDF]
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E. Mollapour, D. C. Linch, and P. J. Roberts Activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate oxidase and phospholipase A2 are dissociated by inhibitors of the kinases p42ERK2 and p38SAPK and by methyl arachidonyl fluorophosphonate, the dual inhibitor of cytosolic and calcium-independent phospholipase A2 Blood, April 15, 2001; 97(8): 2469 - 2477. [Abstract] [Full Text] [PDF]
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 B. Y. Chin, A. Mohsenin, S. X. Li, A. M. K. Choi, and M. E. Choi Stimulation of pro-{alpha}1(I) collagen by TGF-{beta}1 in mesangial cells: role of the p38 MAPK pathway Am J Physiol Renal Physiol, March 1, 2001; 280(3): F495 - F504. [Abstract] [Full Text] [PDF]
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C. Kim and M. C. Dinauer Rac2 Is an Essential Regulator of Neutrophil Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activation in Response to Specific Signaling Pathways J. Immunol., January 15, 2001; 166(2): 1223 - 1232. [Abstract] [Full Text] [PDF]
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 S. Takahashi, Y. Keto, T. Fujita, T. Uchiyama, and A. Yamamoto FR167653, a p38 Mitogen-Activated Protein Kinase Inhibitor, Prevents Helicobacter pylori-Induced Gastritis in Mongolian Gerbils J. Pharmacol. Exp. Ther., January 1, 2001; 296(1): 48 - 56. [Abstract] [Full Text]
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R. KETTRITZ, A. SCHREIBER, F. C. LUFT, and H. HALLER Role of Mitogen-Activated Protein Kinases in Activation of Human Neutrophils by Antineutrophil Cytoplasmic Antibodies J. Am. Soc. Nephrol., January 1, 2001; 12(1): 37 - 46. [Abstract] [Full Text]
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J. A. Lehman, C. C. Paul, M. A. Baumann, and J. Gomez-Cambronero MAP kinase upregulation after hematopoietic differentiation: role of chemotaxis Am J Physiol Cell Physiol, January 1, 2001; 280(1): C183 - C191. [Abstract] [Full Text] [PDF]
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C. Dewas, M. Fay, M.-A. Gougerot-Pocidalo, and J. El-Benna The Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 1/2 Pathway Is Involved in formyl-Methionyl-Leucyl-Phenylalanine-Induced p47phox Phosphorylation in Human Neutrophils J. Immunol., November 1, 2000; 165(9): 5238 - 5244. [Abstract] [Full Text] [PDF]
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J. Jongstra-Bilen, V. L. Misener, C. Wang, H. Ginzberg, A. Auerbach, A. L. Joyner, G. P. Downey, and J. Jongstra LSP1 modulates leukocyte populations in resting and inflamed peritoneum Blood, September 1, 2000; 96(5): 1827 - 1835. [Abstract] [Full Text] [PDF]
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T. Adachi, B. K. Choudhury, S. Stafford, S. Sur, and R. Alam The Differential Role of Extracellular Signal-Regulated Kinases and p38 Mitogen-Activated Protein Kinase in Eosinophil Functions J. Immunol., August 15, 2000; 165(4): 2198 - 2204. [Abstract] [Full Text] [PDF]
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 Q. Jing, S.-M. Xin, W.-B. Zhang, P. Wang, Y.-W. Qin, and G. Pei Lysophosphatidylcholine Activates p38 and p42/44 Mitogen-Activated Protein Kinases in Monocytic THP-1 Cells, but Only p38 Activation Is Involved in Its Stimulated Chemotaxis Circ. Res., July 7, 2000; 87(1): 52 - 59. [Abstract] [Full Text] [PDF]
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P. Y. Coxon, M. J. Rane, D. W. Powell, J. B. Klein, and K. R. McLeish Differential Mitogen-Activated Protein Kinase Stimulation by Fc{gamma} Receptor IIa and Fc{gamma} Receptor IIIb Determines the Activation Phenotype of Human Neutrophils J. Immunol., June 15, 2000; 164(12): 6530 - 6537. [Abstract] [Full Text] [PDF]
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A. Mocsai, Z. Jakus, T. Vantus, G. Berton, C. A. Lowell, and E. Ligeti Kinase Pathways in Chemoattractant-Induced Degranulation of Neutrophils: The Role of p38 Mitogen-Activated Protein Kinase Activated by Src Family Kinases J. Immunol., April 15, 2000; 164(8): 4321 - 4331. [Abstract] [Full Text] [PDF]
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S. I. Simon, Y. Hu, D. Vestweber, and C. W. Smith Neutrophil Tethering on E-Selectin Activates {beta}2 Integrin Binding to ICAM-1 Through a Mitogen-Activated Protein Kinase Signal Transduction Pathway J. Immunol., April 15, 2000; 164(8): 4348 - 4358. [Abstract] [Full Text] [PDF]
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E. A. Albanyan, J. G. Vallejo, C. W. Smith, and M. S. Edwards Nonopsonic Binding of Type III Group B Streptococci to Human Neutrophils Induces Interleukin-8 Release Mediated by the p38 Mitogen-Activated Protein Kinase Pathway Infect. Immun., April 1, 2000; 68(4): 2053 - 2060. [Abstract] [Full Text] [PDF]
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J. A. Nick, S. K. Young, K. K. Brown, N. J. Avdi, P. G. Arndt, B. T. Suratt, M. S. Janes, P. M. Henson, and G. S. Worthen Role of p38 Mitogen-Activated Protein Kinase in a Murine Model of Pulmonary Inflammation J. Immunol., February 15, 2000; 164(4): 2151 - 2159. [Abstract] [Full Text] [PDF]
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N. Perskvist, L. Zheng, and O. Stendahl Activation of Human Neutrophils by Mycobacterium tuberculosis H37Ra Involves Phospholipase C{gamma}2, Shc Adapter Protein, and p38 Mitogen-Activated Protein Kinase J. Immunol., January 15, 2000; 164(2): 959 - 965. [Abstract] [Full Text] [PDF]
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S. A. Wadsworth, D. E. Cavender, S. A. Beers, P. Lalan, P. H. Schafer, E. A. Malloy, W. Wu, B. Fahmy, G. C. Olini, J. E. Davis, et al. RWJ 67657, a Potent, Orally Active Inhibitor of p38 Mitogen-Activated Protein Kinase J. Pharmacol. Exp. Ther., November 1, 1999; 291(2): 680 - 687. [Abstract] [Full Text]
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J. P. Lian, R. Huang, D. Robinson, and J. A. Badwey Activation of p90RSK and cAMP Response Element Binding Protein in Stimulated Neutrophils: Novel Effects of the Pyridinyl Imidazole SB 203580 on Activation of the Extracellular Signal-Regulated Kinase Cascade J. Immunol., October 15, 1999; 163(8): 4527 - 4536. [Abstract] [Full Text] [PDF]
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J. L. Johnson, E. E. Moore, D. A. Patrick, D. Y. Tamura, G. Zallen, D. J. Elzi, and C. C. Silliman Extracellular Signal-Related Kinase 1/2 and p38 Mitogen-Activated Protein Kinase Pathways Serve Opposite Roles in Neutrophil Cytotoxicity Arch Surg, October 1, 1999; 134(10): 1074 - 1078. [Abstract] [Full Text] [PDF]
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N. Geijsen, S. van Delft, J. A.M. Raaijmakers, J.-W. J. Lammers, J. G. Collard, L. Koenderman, and P. J. Coffer Regulation of p21rac Activation in Human Neutrophils Blood, August 1, 1999; 94(3): 1121 - 1130. [Abstract] [Full Text] [PDF]
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S. B. Rizoli, O. D. Rotstein, and A. Kapus Cell Volume-dependent Regulation of L-selectin Shedding in Neutrophils. A ROLE FOR p38 MITOGEN-ACTIVATED PROTEIN KINASE J. Biol. Chem., July 30, 1999; 274(31): 22072 - 22080. [Abstract] [Full Text] [PDF]
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R. M. Heuertz, S. M. Tricomi, U. R. Ezekiel, and R. O. Webster C-reactive Protein Inhibits Chemotactic Peptide-induced p38 Mitogen-activated Protein Kinase Activity and Human Neutrophil Movement J. Biol. Chem., June 18, 1999; 274(25): 17968 - 17974. [Abstract] [Full Text] [PDF]
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S. Arbabi, I. Garcia, G. J. Bauer, and R. V. Maier Alcohol (Ethanol) Inhibits IL-8 and TNF: Role of the p38 Pathway J. Immunol., June 15, 1999; 162(12): 7441 - 7445. [Abstract] [Full Text] [PDF]
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I. Feoktistov, A. E. Goldstein, and I. Biaggioni Role of p38 Mitogen-Activated Protein Kinase and Extracellular Signal-Regulated Protein Kinase Kinase in Adenosine A2B Receptor-Mediated Interleukin-8 Production in Human Mast Cells Mol. Pharmacol., April 1, 1999; 55(4): 726 - 734. [Abstract] [Full Text]
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K. K. Hale, D. Trollinger, M. Rihanek, and C. L. Manthey Differential Expression and Activation of p38 Mitogen-Activated Protein Kinase {alpha}, {beta}, {gamma}, and {delta} in Inflammatory Cell Lineages J. Immunol., April 1, 1999; 162(7): 4246 - 4252. [Abstract] [Full Text] [PDF]
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D. D. Browning, N. D. Windes, and R. D. Ye Activation of p38 Mitogen-activated Protein Kinase by Lipopolysaccharide in Human Neutrophils Requires Nitric Oxide-dependent cGMP Accumulation J. Biol. Chem., January 1, 1999; 274(1): 537 - 542. [Abstract] [Full Text] [PDF]
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 R. Huang, J. P. Lian, D. Robinson, and J. A. Badwey Neutrophils Stimulated with a Variety of Chemoattractants Exhibit Rapid Activation of p21-Activated Kinases (Paks): Separate Signals Are Required for Activation and Inactivation of Paks Mol. Cell. Biol., December 1, 1998; 18(12): 7130 - 7138. [Abstract] [Full Text]
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 J. Chen, Y. Sun, and G. J. Nabel Regulation of the Proinflammatory Effects of Fas Ligand (CD95L) Science, November 27, 1998; 282(5394): 1714 - 1717. [Abstract] [Full Text]
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J. P. Lian, R. Huang, D. Robinson, and J. A. Badwey Products of Sphingolipid Catabolism Block Activation of the p21-Activated Protein Kinases in Neutrophils J. Immunol., October 15, 1998; 161(8): 4375 - 4381. [Abstract] [Full Text] [PDF]
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P. A. Detmers, D. Zhou, E. Polizzi, R. Thieringer, W. A. Hanlon, S. Vaidya, and V. Bansal Role of Stress-Activated Mitogen-Activated Protein Kinase (p38) in {beta}2-Integrin-Dependent Neutrophil Adhesion and the Adhesion-Dependent Oxidative Burst J. Immunol., August 15, 1998; 161(4): 1921 - 1929. [Abstract] [Full Text] [PDF]
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R. A. Ward, M. Nakamura, and K. R. McLeish Priming of the Neutrophil Respiratory Burst Involves p38 Mitogen-activated Protein Kinase-dependent Exocytosis of Flavocytochrome b558-containing Granules J. Biol. Chem., November 17, 2000; 275(47): 36713 - 36719. [Abstract] [Full Text] [PDF]
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S. Bechoua and L. W. Daniel Phospholipase D Is Required in the Signaling Pathway Leading to p38 MAPK Activation in Neutrophil-like HL-60 Cells, Stimulated by N-Formyl-methionyl-leucyl-phenylalanine J. Biol. Chem., August 17, 2001; 276(34): 31752 - 31759. [Abstract] [Full Text] [PDF]
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J. E. Smolen, T. K. Petersen, C. Koch, S. J. O'Keefe, W. A. Hanlon, S. Seo, D. Pearson, M. C. Fossett, and S. I. Simon L-Selectin Signaling of Neutrophil Adhesion and Degranulation Involves p38 Mitogen-activated Protein Kinase J. Biol. Chem., May 19, 2000; 275(21): 15876 - 15884. [Abstract] [Full Text] [PDF]
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 O. Werz, J. Klemm, B. Samuelsson, and O. Radmark 5-Lipoxygenase is phosphorylated by p38 kinase-dependent MAPKAP kinases PNAS, May 9, 2000; 97(10): 5261 - 5266. [Abstract] [Full Text] [PDF]
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