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1
Departments of
*
Physiology and
Clinical Hematology, Osaka City University Medical School, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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.
IL-1 induced phosphorylation and activation of p38 MAPK and
phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38
MAPK was obtained by stimulation of cells with 300 U/ml IL-1 for 10
min. Extracellular signal-regulated kinase (ERK) was faintly
phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated
by IL-1. IL-1 primed neutrophils for enhanced release of
superoxide (O2-) stimulated by FMLP in
parallel with increased phosphorylation of p38 MAPK. IL-1 also
induced O2- release and up-regulation of CD11b
and CD15, and both responses were inhibited by SB203580 (p38 MAPK
inhibitor), suggesting that p38 MAPK activation mediates
IL-1-induced O2- release and up-regulation
of CD11b and CD15. Combined stimulation of neutrophils with IL-1 and
G-CSF, a selective activator of the ERK cascade, resulted in the
additive effects when the priming effect and phosphorylation of p38
MAPK and ERK were assessed. IL-1 induced phosphorylation of ERK and
JNK as well as p38 MAPK in human endothelial cells. These findings
suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is
selectively activated by IL-1 and activation of this cascade
mediates IL-1-induced O2- release and
up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and
the G-CSF receptor-ERK pathway work independently for activation of
neutrophils. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1, 2, 3, 4). Activation of
mature neutrophil functions by these cytokines may contribute not only
to augmenting the host-defense against invading microorganisms but also
to increasing the tissue damage at the inflammatory sites. The
mechanisms by which these cytokines activate mature neutrophils remain
to be determined. We have recently demonstrated that G-CSF, GM-CSF, and
TNF activate the overlapping but distinct mitogen-activated protein
kinase (MAPK)3 subtype
cascades in human neutrophils (5). The MAPK cascade is a major signaling system that is shared by various types of cells (6, 7). In mammalian cells, there are at least three MAPK subtypes; i.e., extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK). The ERK cascade appears to mediate signals promoting cell proliferation, differentiation, or survival, whereas the p38 MAPK and JNK cascades appear to be involved in the cell responses to stresses. Each MAPK subtype is activated by phosphorylation on threonine and tyrosine residues by upstream dual-specificity kinases, such as MAPK/ERK kinase (MEK), MAPK kinase (MKK) 3 or 6 (MKK3/6), and MKK4/7. Activation of the distinct MAPK subtype cascade is dependent on the types of cells and the stimuli used, and the functional role of each MAPK subtype may be different according to the types of cells. Our recent studies with human neutrophils show that G-CSF exclusively activates the MEK-ERK cascade; GM-CSF activates the MEK-ERK cascade strongly and the MKK3/6-p38 MAPK cascade weakly; TNF activates the MKK3/6-p38 MAPK cascade strongly and the MEK-ERK cascade weakly; and the JNK cascade is not activated by any cytokine (5). The differential activation of the MAPK subtype cascades may partly explain the differences of the effects of these cytokines on human neutrophil functions.
IL-1 plays a central role in the inflammatory responses and
activates various types of cells (8). IL-1 is known to
activate mature neutrophils and enhance superoxide
(O2-) release, chemotaxis, and
degranulation (4, 9, 10, 11, 12). IL-1 also promotes neutrophil
spreading (11) and prolongs neutrophil survival
(13). IL-1 may exert these effects through IL-1R type I
on neutrophils, although neutrophils predominantly express IL-1R type
II, which binds IL-1 but does not transduce a signal
(14). The mechanisms by which IL-1 activates mature
neutrophils through IL-1R type I are largely unknown. In regard to
activation of the MAPK subtype cascades, it has been reported that
IL-1 activates ERK, p38 MAPK, or JNK according to the types of
cells. For example, ERK, p38 MAPK, and JNK are activated in human
vascular endothelial cells (15, 16), human fibroblasts
(16), and human hepatoma cells (17); p38 MAPK
and JNK are activated in human epidermal carcinoma cells
(18); and JNK is selectively activated in rabbit
hepatocytes (19). These findings and our recent studies
(5) raise the possibility that the distinct MAPK subtype
cascades may be activated in human neutrophils stimulated by
IL-1.
In this paper, we studied activation of the MAPK subtype cascade in
human neutrophils stimulated by IL-1. The results show that in human
neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by
IL-1, and that this cascade is involved in IL-1-induced
O2- release and up-regulation
of CD11b and CD15. The results also suggest that the IL-1R-MKK3/6-p38
MAPK pathway and the G-CSFR-MEK-ERK pathway work independently for
activation of human neutrophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Highly purified recombinant human IL-1, G-CSF, GM-CSF, and
TNF produced by Escherichia coli were provided by Otsuka
Pharmaceutical (Tokushima, Japan), Kirin Brewery (Tokyo, Japan),
Schering-Plough (Osaka, Japan), and Dainippon Pharmaceutical (Osaka,
Japan), respectively. The specific activities of IL-1 and TNF were
2 x 107 U/mg protein and 3 x
106 U/mg protein, respectively. Endotoxin
contamination of each preparation was <100 pg/mg protein. Cytochrome
c type III, FMLP, and superoxide dismutase were purchased
from Sigma (St. Louis, MO); Conray was purchased from Mallinckrodt (St.
Louis, MO); Ficoll was purchased from Pharmacia Fine Chemicals
(Piscataway, NJ); and endothelial cell growth supplement was purchased
from BD Labware (Bedford, MA). PD98059 (MEK inhibitor) and rabbit
polyclonal Abs against Ser189/207-phosphorylated
MKK3/MKK6, ERK1/ERK2,
Thr202/Tyr204-phosphorylated
ERK1/ERK2, p38 MAPK,
Thr180/Tyr182-phosphorylated
p38 MAPK, JNK/stress-activated protein kinase, and
Thr183/Tyr185-phosphorylated
JNK/stress-activated protein kinase were purchased from New England
Biolabs (Beverley, MA). FITC-conjugated anti-human CD11b mAb
(BEAR1, mouse IgG1), anti-human CD15 mAb (80H5, mouse IgM),
anti-human MHC class I mAb (B9.12.1, mouse IgG2a), and
isotype-matched irrelevant Abs were purchased from
Immunotech (Marseille, France). The ECL Western blotting system was
purchased from Amersham (Arlington Heights, IL). SB203580 (p38 MAPK
inhibitor) was provided by SmithKline Beecham Pharmaceuticals (King of
Prussia, PA).
Preparation of cells
Human neutrophils were prepared from healthy adult donors as described previously (5) using dextran sedimentation, centrifugation with Conray-Ficoll, and hypotonic lysis of contaminated erythrocytes. Neutrophil fractions were suspended in HBSS containing 10 mM HEPES (pH 7.4), and contained >98% neutrophils. Primary human endothelial cells were harvested from human umbilical cord veins treated with 1250 U/ml dispase (20) and grown on 2.5% gelatin-precoated 60-mm culture dishes (Nunclon, Roskilde, Denmark) in M-199 containing 20% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 15 mM HEPES, 200 µg/ml heparin, and 60 µg/ml endothelial cell growth supplement. Cells between passages 2 and 4 were used.
Western blotting
Human neutrophils suspended in HBSS were prewarmed for 10 min at 37°C and were then stimulated with cytokines for 1–120 min at 37°C. The reactions were terminated by rapid centrifugation, and the pellets were frozen in liquid nitrogen after aspiration of the supernatant. The cell pellets were resuspended in ice-cold extraction buffer containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 100 µg/ml aprotinin, and 10 µg/ml leupeptin and were lysed for 60 min at 4°C. After rapid centrifugation, the supernatant was mixed 1:1 with 2x sample buffer (4% SDS, 20% glycerol, 10% 2-ME, and a trace amount of bromphenol blue dye in 125 mM Tris-HCl, pH 6.8), heated at 100°C for 5 min, and then frozen at -80°C until use. Samples were subjected to 10% SDS gel electrophoresis. After electrophoresis, proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane in a buffer containing 25 mM Tris, 192 mM glycine and 20% methanol at 2 mA/cm2 for 1.5 h at 25°C. Residual binding sites on the membrane was blocked by incubating the membrane in TBS (pH 7.6) containing 0.1% Tween 20 and 5% nonfat dry milk for 2 h at 25°C. The blots were washed in TBS containing 0.1% Tween 20 and then incubated with appropriate Ab overnight at 4°C. After washing, the membrane was incubated with anti-rabbit IgG Ab conjugated with HRP, and the Ab complexes were visualized by the ECL detection system as directed by the manufacturer. Endothelial cells were stimulated with cytokines for 10 min at 37°C. Cells were harvested using cell scraper and were resuspended in ice-cold extraction buffer. Endothelial cell extract was processed in the same manner described for neutrophils. Immunoreactive bands were quantified by a NIH Image program on a Macintosh computer.
Kinase assay of p38 MAPK
The kinase activity of p38 MAPK was determined by using a nonradioactive immunoprecipitation kinase assay kit (New England Biolabs) according to the manufacturer’s instructions. Phosphorylated p38 MAPK was immunoprecipitated by using immobilized mAb against Thr180/Tyr182-phosphorylated p38 MAPK. The resulting immunoprecipitates were incubated with GST-ATF-2 fusion protein in the presence of ATP. Phosphorylation of ATF-2 was determined by Western blotting using Ab against Thr71-phosphorylated ATF-2.
Determination of O2-release
O2- was assayed by superoxide dismutase-inhibitable reduction of ferricytochrome c, and the continuous assay was performed in a Hitachi 557 spectrophotometer (a double wavelength spectrophotometer; Hitachi, Tokyo, Japan), equipped with a thermostatted cuvette holder (37°C) as described (1). The final concentration of cytochrome c was 100 µM and the final cell concentration was 1 x 106 cells/ml. The amount of O2- release was calculated from cytochrome c reduced for 5 min after the addition of FMLP. In some experiments, O2- release was also determined by the end point assay (5). For the end point assay, the cell suspension in HBSS was added to a polypropylene tube (Falcon 2063; BD Labware) containing 100 µM ferricytochrome c with or without superoxide dismutase (200 U/ml) to obtain a final volume of 1 ml. Final cell concentration was 1 x 106 cells/ml. After incubation with appropriate stimuli for indicated periods at 37°C, the reduction of ferricytochrome c was measured at 550 nm with a reference wavelength at 540 nm.
Determination of expression of surface Ags
The effects of cytokines on surface expression of CD11b, CD15, and MHC class I were analyzed by flow cytometry. The whole blood was used for this assay to minimize the increased expression of surface Ags by incubation alone at 37°C (1, 21). After treatment of cells with appropriate stimuli for indicated periods, the cells were mixed with FITC-conjugated anti-human CD11b, anti-human CD15, or anti-human MHC class I mAb and were incubated for 30 min at 4°C. After the lysis of erythrocytes with FACS lysing solution (BD Biosciences), cells were washed with PBS and were suspended in PBS containing 2% paraformaldehyde. The binding of mAb was analyzed by flow cytometry with FACSCalibur (BD Biosciences, Mountain View, CA). Isotype-matched irrelevant Abs directly conjugated with FITC were used as controls. Granulocytes were gated on the basis of forward and side scatter. By this method, almost all other cells including lymphocytes, monocytes, and eosinophils could be excluded, and the cells analyzed were almost exclusively neutrophils. In each sample, >1 x 104 granulocytes were analyzed using CellQuest software (BD Biosciences). The changes in expression of surface Ags were analyzed using the values in the mean fluorescence intensity without subtracting the background.
Statistical analysis
Student’s t test was used to determine statistical significance. For the analysis of the changes in expression of surface Ags, paired Student’s t test was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human neutrophils in suspension were stimulated with G-CSF (50
ng/ml), GM-CSF (5 ng/ml), TNF (100 U/ml), or IL-1 (25 or 300 U/ml)
for 10 min at 37°C, and phosphorylation of p38 MAPK, ERK1/ERK2, and
JNK was analyzed by immunoblotting using Abs against phosphorylated
forms of each protein. As shown in Fig. 1
A, p38 MAPK was strongly
phosphorylated by TNF and GM-CSF, but not by G-CSF (5).
When the exposure time was prolonged, weak phosphorylation of p38 MAPK
was detected at 5–10 min after stimulation with G-CSF (see Fig. 6).
Under the same conditions, p38 MAPK was significantly phosphorylated by
300 U/ml IL-1 and marginally phosphorylated by 25 U/ml IL-1 (Fig. 1, A, B, and E). When neutrophils were
stimulated with IL-1 for 10 min, a significant increase in
phosphorylation of p38 MAPK was consistently observed at 100 U/ml and
maximal stimulation was obtained at 300 U/ml (Fig. 1, B and
E). IL-1-induced phosphorylation of p38 MAPK was rapid
and already detected at 1 min after stimulation with 300 U/ml IL-1,
and the maximal level was observed at 10 min followed by gradual
decrease of the level. The comparative study showed that the potency of
these cytokines to induce phosphorylation of p38 MAPK was TNF >
GM-CSF > IL-1 > G-CSF (Fig. 1, A and
B). The kinase activity of p38 MAPK was also increased by
stimulation with TNF, GM-CSF, or IL-1 when the kinase activity was
determined using ATF-2 as a substrate (Fig. 1C). Consistent
with the effect on phosphorylation of p38 MAPK, the potency of these
cytokines to increase p38 MAPK kinase activity was TNF >
GM-CSF > IL-1.
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, stimulation of
neutrophils with G-CSF (50 ng/ml) or TNF (100 U/ml) for 10 min resulted
in increased phosphorylation of ERK1 and ERK2 with predominant
phosphorylation of ERK2 (5). Under the same conditions, no
significant increase in phosphorylation of ERK1 and ERK2 was detected
even when the cells were stimulated with 300 U/ml IL-1 (Fig. 2, left panel). When the exposure time was prolonged, faint
phosphorylation of ERK1 and ERK2 was detected at 300 U/ml IL-1 with
predominant phosphorylation of ERK2 (Fig. 2, right panel).
In TNF- or IL-1-stimulated neutrophils, an additional band was
always detected just below the ERK2 band by the Ab against
phosphorylated ERK1/ERK2. The intensity of total ERK1/ERK2 band
detected by the Ab against ERK1/ERK2 was not significantly decreased
(<7%) by stimulation with TNF or IL-1, and an additional band was
not detected by the Ab against ERK1/ERK2 (Fig. 2, lower
panel). These findings suggest that the additional band is
unlikely to be a degradation product of ERK1/ERK2, but rather suggest
that this band is a definite protein different from ERK1/ERK2. It is
unknown whether this band is related to the ERK family or not.
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(300 U/ml) even when the
incubation time with IL-1 was prolonged up to 120 min (data not
shown). These findings are consistent with our recent observations
(5) that JNK is not phosphorylated in human neutrophils
stimulated by various agonists, including G-CSF, GM-CSF, and TNF,
despite the existence of JNK proteins.
Phosphorylation of MKK3/MKK6 in neutrophils stimulated by IL-1
In human neutrophils stimulated by GM-CSF or TNF, p38 MAPK and
MKK3/MKK6 were phosphorylated in parallel (5) (Fig. 1
D), supporting the idea that MKK3/MKK6 is an upstream
kinase for p38 MAPK (6, 7). As shown in Fig. 1D, stimulation of neutrophils with IL-1 (300 U/ml) for
10 min at 37°C resulted in increased phosphorylation of
MKK3/MKK6. Consistent with the effecton phosphorylation of p38 MAPK,
the potency of IL-1 to induce phosphorylation of MKK3/MKK6 was
less than that of TNF (Fig. 1). These findings suggest that
IL-1-induced activation of p38 MAPK may be, at least in part,
mediated through activation of MKK3/MKK6 in human neutrophils. When
cells were pretreated with PD98059 (50 µM), a MEK inhibitor
(22), for 20 min at 37°C, IL-1-induced
phosphorylation of ERK1/ERK2 was inhibited (data not shown)
(5), suggesting that the MEK-ERK cascade is also weakly
activated by IL-1.
Involvement of p38 MAPK in IL-1-induced
O2- release
As shown in Fig. 3, IL-1 primed
human neutrophils and enhanced FMLP-induced
O2- release in dose- and
time-dependent manners (4). The optimal priming was
obtained by pretreatment of cells with 300 U/ml IL-1 for 10 min at
37°C. The priming effect of IL-1 was parallel to phosphorylation
of p38 MAPK induced by this cytokine (Figs. 1E and 3). These
findings raise the possibility that p38 MAPK may be involved in
IL-1-induced priming. However, this possibility could not be
determined in the present experiments, because
O2- release induced by FMLP
itself was strongly inhibited by SB203580, a p38 MAPK inhibitor (Table I) (23, 24, 25). By contrast,
FMLP-induced O2- release in
untreated or IL-1-primed cells was not affected by PD98059 (Table I). These findings are consistent with the fact that activation of ERK
in IL-1-stimulated neutrophils was negligible and suggest that ERK
is not involved in IL-1-mediated priming. The experiments were also
performed using G-CSF-primed cells, and the results were essentially
similar to those observed in IL-1-primed cells (Table I). Thus, it
appears that ERK is unlikely to be involved in the priming induced by
G-CSF as well as IL-1, and possible involvement of p38 MAPK in the
priming remains to be determined.
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also induced O2-
release in neutrophils (4). The potency of IL-1 to
induce O2- release was less
than that of TNF (Fig. 4). Our previous
studies show that activation of p38 MAPK may mediate TNF-induced
O2- release in human
neutrophils, as evidenced by the finding that TNF-induced
O2- release was significantly
inhibited by pretreatment of cells with SB203580 (Fig. 4) (5, 23). Under the same condition, IL-1-induced
O2- release was almost
completely inhibited by SB203580 (Fig. 4), implying that p38 MAPK may
be also involved in IL-1-induced
O2- release.
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- and TNF-induced up-regulation
of CD11b and CD15
Certain cell surface Ags such as CD11b are known to be
up-regulated by stimulation of neutrophils with cytokines
(1). As shown in Fig. 5, stimulation of neutrophils with TNF resulted in a significant increase
in surface expression of CD11b and CD15, but not MHC class I. The
values in the mean fluorescence intensity of CD11b and CD15 in
TNF-stimulated cells were 5.11 ± 1.02 (p
< 0.01, n = 7) and 1.70 ± 0.25
(p < 0.01, n = 6) times
greater than those in unstimulated control cells, respectively. Under
the same condition, IL-1, like TNF, was found to induce significant
up-regulation of CD11b and CD15, but not MHC class I (Fig. 5). The
potency of IL-1 to induce up-regulation of CD11b and CD15 was much
less than that of TNF. The values in the mean fluorescence intensity of
CD11b and CD15 in IL-1-stimulated cells were 1.52 ± 0.15
(p < 0.01, n = 7) and
1.12 ± 0.03 (p < 0.05, n
= 4) times greater than those in unstimulated control cells,
respectively. The finding that surface expression of CD15 is
up-regulated by stimulation with IL-1 or TNF is consistent with the
previous report that a part of CD15 Ag is localized in specific
granules of neutrophils and surface expression of CD15 is increased by
stimulation with FMLP plus cytochalasin B (26). The
fluorescence intensity of CD11b and CD15 in unstimulated control cells
incubated at 37°C was greater than that in cells kept on ice
(p < 0.01 for CD11b (n = 10)
and p < 0.05 for CD15 (n = 5),
respectively), indicating that surface expression of CD11b and CD15 was
increased by incubation alone at 37°C (Fig. 5) (1).
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-induced
up-regulation of CD11b by 
80% (p < 0.01,
n = 7) and TNF-induced up-regulation of CD11b by
40% (p < 0.01, n = 7),
respectively (Fig. 5, A and B). The less
inhibitory effect of SB203580 on TNF-induced up-regulation of CD11b may
be ascribed to stronger activation of p38 MAPK and/or activation of
other pathways observed in TNF-stimulated cells (5).
Increased expression of CD11b by incubation alone was also inhibited by
SB203580 (p < 0.05, n = 6)
(Fig. 5). This effect of SB203580, however, did not affect the
statistical significance for the inhibitory effect of this inhibitor on
IL-1- and TNF-induced up-regulation of CD11b. The similar results
were obtained for the effect of SB203580 on IL-1- and TNF-induced
up-regulation of CD15. Pretreatment of cells with SB203580 inhibited
up-regulation of CD15 induced by IL-1 (p <
0.05, n = 5) or TNF (p < 0.02,
n = 5) (Fig. 5C; data not shown for
IL-1). By contrast, surface expression of MHC class I was neither
affected by incubation alone at 37°C nor altered by stimulation of
cells with IL-1 or TNF regardless of the presence or absence of
SB203580 (Fig. 5D; data not shown for SB203580). It has been
demonstrated that CD11b and CD15, but not MHC class I, are localized,
besides on the cell surface, in specific or tertiary granules
(26, 27). Thus, it is conceivable that increased
expression of CD11b and CD15 by incubation alone may result from
mobilization from internal stores during incubation, which may be
mediated by slight activation of p38 MAPK by incubation alone (Fig. 6). Additional control experiments were
performed in each condition using an isotype-matched irrelevant Ab
directly conjugated with FITC. The results showed that the
autofluorescence and the nonspecific binding of Ab were not affected by
treatment of cells with the inhibitor and/or the cytokines (data not
shown). Thus, the results indicate that CD11b and CD15, but not MHC
class I, are selectively up-regulated by stimulation of neutrophils
with IL-1 or TNF, and activation of p38 MAPK may be involved in
IL-1- and TNF-induced up-regulation of CD11b and CD15.
Independent activation of neutrophils by IL-1 and G-CSF
The data shown in Figs. 1 and 2 indicate that stimulation of human
neutrophils with IL-1 results in predominant activation of the p38
MAPK cascade and weak or negligible activation of the ERK cascade.
These findings contrast well with the effects of G-CSF on human
neutrophils, in which the ERK cascade is predominantly activated
(5). Weak phosphorylation of p38 MAPK was also detected in
human neutrophils stimulated by G-CSF (50 ng/ml) when the exposure time
was prolonged (Fig. 6). Then, using IL-1 and G-CSF in combination,
the possible cross-talk between the p38 MAPK cascade and the ERK
cascade could be explored. As shown in Fig. 6, phosphorylation of
ERK1/ERK2 induced by G-CSF (50 ng/ml) or IL-1 (300 U/ml) was
transient; i.e., the maximal level was observed at 5–10 min after
stimulation with G-CSF or IL-1, and the phosphorylated bands
disappeared within 60 min. On the other hand, the increased level of
phosphorylation of p38 MAPK was still detected at 60 min after
stimulation with IL-1 or G-CSF (Fig. 6). Thus, the kinetics of
phosphorylation of p38 MAPK and ERK1/ERK2 induced by IL-1 were
similar to those induced by G-CSF, whereas the kinetics of
phosphorylation of p38 MAPK were different from those of ERK1/ERK2.
Increased phosphorylation of p38 MAPK in unstimulated control cells was
sometimes detected when cells were incubated for >10 min (Fig. 6). It
is likely that the p38 MAPK cascade in control cells may be slightly
activated by stresses such as cell preparation procedures and
incubation of cells in the buffer solution. Stimulation of neutrophils
with IL-1 and G-CSF in combination resulted in the additive effects
when the increased levels and the kinetics of phosphorylation of p38
MAPK and ERK1/ERK2 were assessed (Fig. 6). Consistent with these
findings, pretreatment of neutrophils with IL-1 (300 U/ml) and G-CSF
(50 ng/ml) in combination resulted in the additive priming effect on
FMLP-induced O2- release (Table II). The potency of IL-1 to prime the
cells for enhanced release of
O2- was almost identical to
that of G-CSF. In addition, IL-1-induced
O2- release (triggering effect)
was not affected by the combined addition of G-CSF, which by itself is
unable to induce O2- release
(4) (data not shown).
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or TNF
In human neutrophils, p38 MAPK was predominantly phosphorylated by
IL-1 and relatively higher concentrations (300 U/ml) of IL-1 were
required for maximal stimulation. On the other hand, maximal
stimulation of endothelial cells is obtained at much lower
concentrations of IL-1 (10–25 U/ml) (20, 28), the
concentration which showed negligible or marginal effects on human
neutrophils (
Figs. 1–3). These differences between the cells may
be attributed to the differences of IL-1 receptors, intracellular
signaling pathways, or both. Then, we studied phosphorylation of MAPK
subtypes in endothelial cells stimulated by IL-1 or TNF. As shown in
Fig. 7, stimulation of endothelial cells
with IL-1 (25 U/ml) or TNF (100 U/ml) for 10 min at 37°C resulted
in increased phosphorylation of ERK1/ERK2, p38 MAPK, and JNK1/JNK2
(15, 16).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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results in predominant activation of p38
MAPK, negligible activation of ERK1/ERK2, and no activation of JNK.
Selective activation of p38 MAPK by IL-1 appears to be specific to
human neutrophils, since previous studies show that JNK is consistently
activated by IL-1 in various types of cells, and additional
activation of ERK or p38 MAPK is observed depending on the types of
cells (15, 16, 17, 18, 19). In fact, we found that stimulation of
human endothelial cells with IL-1 resulted in activation of JNK,
ERK, and p38 MAPK (15, 16). We also found that IL-1
induced activation of JNK, ERK, and p38 MAPK in human monocytes (data
not shown). These findings indicate that IL-1-induced activation of
MAPK subtype cascades is strictly regulated according to the types of
cells. Parallel phosphorylation of MKK3/MKK6 and p38 MAPK suggest that
IL-1, like TNF and GM-CSF (5), activates the
MKK3/MKK6-p38 MAPK cascade in human neutrophils. MKK3 may be an
upstream kinase of p38 MAPK in human neutrophils, since it has been
recently demonstrated that human neutrophils possess MKK3, but not MKK6
(29).
The present experiments show that p38 MAPK activation is functionally
correlated with IL-1- and TNF-induced activation of
O2- release and up-regulation
of CD11b (2 integrin) and CD15 (a ligand for P-selectin)
(30). The potency of cytokines (TNF, GM-CSF, and IL-1)
to induce activation of p38 MAPK was TNF > GM-CSF > IL-1
and was identical to that to induce activation of
O2- release and up-regulation
of CD11b and CD15 (data not shown for GM-CSF) (1, 2, 3, 4, 5). In
addition, O2- release and
up-regulation of CD11b and CD15 induced by these cytokines were
inhibited by SB203580 (data not shown for GM-CSF-induced up-regulation
of CD11b) (5). These findings and our recent studies
(5, 31) taken together suggest that p38 MAPK activation is
critically important for O2-
release and up-regulation of CD11b and CD15 induced by cytokines,
including TNF, GM-CSF, and IL-1. It has been reported that p38 MAPK
can phosphorylate serine residues of
p47phox, a component of NADPH oxidase
(32). Thus, it is possible that phosphorylation of
p47phox by p38 MAPK may, at least in part,
mediate activation of O2-
release in neutrophils stimulated by these cytokines. Recent studies
suggest that p38 MAPK is involved in actin reorganization in
endothelial cells stimulated by vascular endothelial growth factor
(33) or platelet-derived growth factor (34)
and macrophages stimulated by TNF (35). Since
up-regulation of CD11b and CD15 on neutrophils may reflect
degranulation, a function closely associated with actin dynamics, it is
possible that p38 MAPK activation by IL-1 or TNF may cause
up-regulation of CD11b and CD15 through actin reorganization.
Stimulation of neutrophils with IL-1 and G-CSF in combination
resulted in the additive effects when the increased levels and the
kinetics of phosphorylation of p38 MAPK and ERK1/ERK2 were assessed.
These findings indicate that neither augmentation nor interference
occurs in activation of p38 MAPK and ERK in neutrophils stimulated with
IL-1 and G-CSF in combination, and suggest that the IL-1R-MKK3-p38
MAPK pathway and the G-CSFR-MEK-ERK pathway work independently for
activation of human neutrophils. Consistent with this is the finding
that pretreatment of neutrophils with IL-1 and G-CSF in combination
resulted in the additive priming effect on FMLP-induced
O2- release. It is unlikely
that cell populations activated by IL-1 and G-CSF are different from
each other, since all neutrophils express G-CSF receptors
(36) and show increased expression of CD11b in response to
stimulation with G-CSF (1) as well as IL-1 (Fig. 5).
It should be noted that relatively higher concentrations (300 U/ml) of
IL-1 were required for optimal activation of human neutrophils,
whereas lower concentrations (10–25 U/ml) of IL-1 were sufficient
for optimal activation of human endothelial cells (20, 28). This difference may be explained by predominant expression
of IL-1R type II (a decoy receptor) on human neutrophils
(37) and exclusive expression of the signal tranducing
type I receptor on human endothelial cells (38). IL-1
at lower concentrations can induce expression of adhesion molecules
such as ICAM-1 on endothelial cells (39), which helps
neutrophils accumulate at the inflammatory sites. Thus, it is
conceivable that neutrophils recruited to the inflammatory sites may
bind IL-1 via type II receptor with minimal activation of
neutrophils and decrease the concentration of IL-1 in the milieu at
the inflammatory sites, which may contribute to preventing the
excessive inflammatory reactions. On the other hand, neutrophils
activated by relatively higher concentrations of IL-1 may contribute
to augmenting the host defense.
The present experiments show that in human neutrophils the MKK3-p38
MAPK cascade is selectively activated by IL-1 and that activation of
this cascade mediates IL--induced
O2- release and up-regulation
of CD11b and CD15. The results also suggest that the IL-1R-MKK3-p38
MAPK pathway and the G-CSFR-MEK-ERK pathway work independently for
activation of human neutrophils.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Seiichi Kitagawa, Department of Physiology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail address: kitagawas{at}med.osaka-cu.ac.jp
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MEK, MAPK/ERK kinase; MKK, MAPK kinase.
Received for publication July 27, 2000. Accepted for publication September 7, 2001.
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References |
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