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Department of Medicine, Chest Institute, Tokyo Women’s Medical College, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intriguing clues to the mechanism underlying expression of neutrophil apoptosis have begun to emerge from recent observations. For example, transient elevation of cytosolic-free Ca2+ induced by low doses of calcium ionophores inhibits apoptosis in neutrophils (3). Conversely, intracellular ROS3 induce apoptosis in neutrophils as well as other cell types (4, 5, 6). Furthermore, the autocrine interaction of neutrophil Fas with soluble or cell-associated Fas ligand may cause spontaneous apoptosis that occurs in normal neutrophils (7, 8). However, the signal-transduction pathway mediating neutrophil apoptosis remains unclear.
Recent studies indicate the involvement of tyrosine phosphorylation events in signaling pathways that results in neutrophil apoptosis. For example, treatment of neutrophils with inhibitors of tyrosine kinases and phosphatases alters the rate of neutrophil apoptosis (9). Furthermore, GM-CSF induces a rapid activation of Lyn, a Src family tyrosine kinase, whereas Lyn antisense treatment of neutrophils reverses the survival-promoting effect of GM-CSF (10). These studies suggest that neutrophils utilize a specific signaling pathway dependent on tyrosine phosphorylation and dephosphorylation to trigger their apoptotic protocol.
p38-MAPK, a MAPK family member, is a serine/threonine kinase activated
by phosphorylation of tyrosine and threonine residues (11). This kinase
is phosphorylated and activated by many cellular stresses and
inflammatory stimuli. To date, its physiologic roles have not been
defined, but it seems to be involved in the regulation of important
cellular responses such as apoptosis and inflammatory reactions. In
neutrophils, p38-MAPK has been shown to be activated by agonistic
stimulation with LPS, TNF-
, GM-CSF, platelet-activating factor,
FMLP, and IL-8 (12, 13, 14, 15, 16). However, the functional significance of
p38-MAPK in neutrophils is unknown.
In this study, we present evidence that p38-MAPK plays an important role in spontaneous apoptosis in neutrophils. We found that p38-MAPK is continuously phosphorylated and activated during the program of spontaneous apoptosis. Inhibition of p38-MAPK by SB203580 and specific antisense RNA delayed spontaneous apoptosis. These findings suggest that p38-MAPK activity is involved in spontaneous apoptosis in neutrophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All reagents for cell culture were obtained from Life Technologies (Gaithersburg, MD). MTT, bisBenzimide (Hoechst33258), thymus DNA, genistein, N-acetylcysteine, catalase, leupeptin, aprotinin, PMSF, ß-glycerophosphate, sodium vanadate, DTT, and polymyxin B sulfate were purchased from Sigma (St. Louis, MO). Recombinant human GM-CSF, anti-Fas Ab (CH-11), protein A plus G agarose, and PD98059 were purchased from Pharma Biotechnologie (Hannover, Germany), Medical and Biological Laboratories (Nagoya, Japan), Oncogene Research Product (Cambridge, MA), and Biomol Research Laboratories (Plymouth Meeting, PA), respectively. SB203580 was a generous gift of SmithKline Beecham Pharmaceuticals (King of Prussia, PA). The phosphorylated forms of the proteins p38-MAPK, ERK, and JNK were obtained from New England Biolabs (Beverly, MA). MAPKAP kinase-2-GST was purchased from Upstate Biotechnology (Lake Placid, NY).
Neutrophil preparation and cell culture
Human neutrophils were isolated from EDTA-anticoagulated venous blood samples by dextran sedimentation and centrifugation on a Histopaque gradient (without endotoxin; Sigma), as previously described (17). Contaminating erythrocytes were removed by hypotonic water lysis. The isolated neutrophils were washed twice with PBS and resuspended in RPMI 1640 containing 5% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. To avoid accidental neutrophil activation and clumping, neutrophil preparations were carefully washed by centrifugation at 200 x g and aspiration of the supernatant, followed by gentle resuspension with a pipet, and sudden changes in temperature were avoided (10). Endotoxin was not detected (<5 pg/ml) in Histopaque, culture medium, or FCS by a Limulus lysate test (Endospec-ST; Seikagaku, Tokyo, Japan). The purity of neutrophil populations was >95% on May-Grünwald-Giemsa stain, and neutrophil viability was >98%, as determined by trypan blue dye exclusion. Less than 5% of neutrophil preparations showed a polarized shape, a sensitive marker for neutrophil activation (18). Unless otherwise stated, to assay for apoptosis, cell survival, and ROS generation, 105 cells were incubated in 200 µl medium in 96-well round-bottom plates (Becton Dickinson, Lincoln Park, NJ) at 37°C in a humidified incubator containing 5% CO2. For p38-MAPK assay and immunoblotting, 5 x 106 cells were suspended in 10 ml medium in a polypropylene tube (Becton Dickinson) and cultured as above.
Normal human fetal lung fibroblasts (IMR-90; Clonetics, San Diego, CA) and human bronchial epithelial cells (16HBE) were cultured in DMEM or 1:1 Ham’s F-12/DMEM, respectively, supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Antisense oligonucleotide treatment of cells
Phosphorothiorate-modified antisense oligonucleotides specific for p38-MAPK (5'-GTCTTGTTCAGCTCCTGC-3') (19) as well as sense (5'-GCAGGAGCTGAACAAGAC-3') and scrambled (5'-TGCTTAGTTCTCGTCCGC-3') oligonucleotides were synthesized. Neutrophils were incubated with 1 µM of each oligonucleotide in serum-free RPMI 1640 for 6 h before addition of 5% heat-inactivated FCS. After 24 h of culture, the cells were prepared for apoptosis assay and immunoblot analysis, as described below.
Apoptosis assay
Aliquots of neutrophils were cytospun on glass slides and dried. Slides were stained with May-Grünwald-Giemsa. Apoptosis was assessed based on nuclear pyknosis or chromatin condensation together with cytoplasmic vacuolation on oil immersion microscopy (2). Three hundred cells were scored in each experiment to determine the percentage of apoptotic cells.
Apoptosis was also evaluated by detection of cytosolic histone-bound DNA fragments. Briefly, 2 x 104 cells were incubated in a 96-well round-bottom plate for 12 h. The plate was centrifuged and cell pellets were analyzed for histone-bound DNA fragments using the cell detection ELISA kit (Cell Death Detection ELISAPLUS; Boeringer Mannheim, Indianapolis, IN), according to the manufacturer’s instruction. The principle of this test is based on the detection of mono- and oligonucleosomes in the cytoplasmic fractions of cell lysates by using biotinylated antihistone- and peroxidase-coupled anti-DNA Abs. The enrichment of mono- and oligonucleosomes released into the cytoplasm is calculated as absorbance of incubated cells/absorbance of freshly isolated cells.
Cell survival assay
Cell survival was evaluated by both a colorimetric MTT assay and a fluorescence cellular DNA assay. For MTT assay, 0.5 mg/ml MTT was added to culture plates for the last 4 h of incubation. Then the plates were centrifuged and cell pellets were lysed in 200 µl DMSO and 25 µl Sorenson’s buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The OD was measured at 570 nm on a microplate immunoreader (Bio-Rad, Hercules, CA). For DNA assay, culture plates were centrifuged and cell pellets were lysed in 100 µl distilled water, followed by a cycle of freezing and thawing. Then, cell lysates were solubilized in 100 µl of TNE buffer (10 mM Tris, 1 mM EDTA, 2 M NaCl, pH 7.4) containing 10 µg/ml Hoechst33258. The fluorescence intensities were read at respective excitation and emission wavelengths of 350 and 460 nm on a Cytofluor II multiplate fluorometer (Perseptive Biosystems, Framigham, MA). DNA content was determined in comparison with reference curves generated with known amounts of thymus DNA.
Immunoblot analysis for protein phosphorylation
Cell lysates were solubilized in RIPA buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.4, 0.5% Nonidet P-40, and 0.1% SDS) containing 10 µg/ml leupeptin, 1 mM PMSF, 10 µg/ml aprotinin, and 1 mM sodium orthovanadate, and centrifuged at 10,000 x g for 30 min at 4°C. The supernatant containing equivalent amounts of protein (50 µg) was fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with Abs, all of which were used at a dilution of 1/1000. Primary Ab was detected by horseradish peroxidase-conjugated Ab (1:2500), which in turn was visualized using enhanced chemoluminescence (SuperSignal; Pierce, Rockford, IL). OD of positive bands was measured with the National Institute of Health Image software.
p38-MAPK assay
Cell pellets (5 x 106 cells) were lysed in 500
µl of lysis buffer (150 mM NaCl, 20 mM Tris-Cl, pH 7.4, 1 mM EDTA, 1
mM EGTA, 1% Triton-X, 2.5 mM sodium pyrophosphate, 1 mM
ß-glycerophosphate, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, 1
mM PMSF) and centrifuged at 10,000 x g for 30 min at
4°C. The supernatant containing equivalent amounts of protein (250
µg) was precleared with protein A plus G agarose and
immunoprecipitated with 4 µl of anti-p38-MAPK Ab for 16 h at
4°C. The immunocomplex was captured by protein A plus G agarose for
4 h. Bead pellets were washed twice in lysis buffer and twice in
kinase buffer (25 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM
ß-glycerophosphate, 2 mM DTT, 100 mM sodium orthovanadate). The beads
were incubated in 30 µl of kinase buffer containing 12.5 µg/ml
MAPKAP kinase-2-GST, 30 µM ATP, and 10 µCi
[
-32P]ATP for 20 min at 30°C. The reaction was
terminated by the addition of 10 µl 5x SDS sample buffer and heating
to 95°C for 5 min. Samples were resolved on a 12% acrylamide
SDS-PAGE gel and subjected to autoradiography. The immunoprecipitated
samples were also analyzed for p38-MAPK by immunoblotting to determine
whether the same amount of p38-MAPK was immunoprecipitated. The OD of
each band was determined, and the ratio of phosphorylated MAPKAP
kinase-2-GST/immunoprecipitated p38-MAPK was plotted.
Immunocytochemistry for phosphorylation of p38-MAPK
Venous blood was drawn and immediately smeared on a glass slide. Blood cells were fixed in 3% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with phosphospecific Ab to p38-MAPK (1:50) or control rabbit IgG. Primary Ab was detected by FITC-conjugated Ab. Cells were counterstained with propidium iodide and observed under fluorescence microscopy.
Statistics
Results are presented as mean ± SEM. Comparisons were made by Student’s t test or ANOVA with Scheffe’s correction where appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When cultured in DMEM containing 5% FCS, neutrophils underwent
apoptosis constitutively, with 25.1% of cells apoptotic at 16 h
and 94.3% apoptotic by 72 h, as assessed by light microscopy
(Fig. 1
). The constitutive rate of
apoptosis in neutrophils shown in this work is within the range
reported previously (2, 3, 4, 5). Incubation of neutrophils with 50 µM of
SB203580, a selective p38-MAPK inhibitor that has no inhibitory action
on ERK and JNK (20), significantly inhibited apoptosis at each time
point, with delayed onset of apoptosis by approximately 24 h (Fig. 1A). Genistein, a broad spectrum tyrosine kinase inhibitor,
modestly inhibited apoptosis. In contrast, PD98059, an inhibitor of ERK
kinase, had no effect on the rate of neutrophil apoptosis. Inhibition
of apoptosis by SB203580 was dose dependent and was observed at a
concentration as low as 5 µM, which is comparable with effective
concentrations (1–25 µM) reported previously (12, 20, 21, 22, 23, 24, 25) (Fig. 1B). SB203580 also inhibited nucleosomal DNA fragmentation,
as assessed by a cell death ELISA assay (Fig. 2), which detects cytosolic histone-bound
DNA fragments formed in cells undergoing apoptosis. The inhibition of
neutrophil apoptosis by SB203580 was supported by the fact that this
compound prolonged survival of neutrophils, as assessed by an MTT assay
(Fig. 3A), and by measurement
of residual DNA in culture wells (Fig. 3B). In contrast to
SB203580, the ERK inhibitor PD98059 did not affect neutrophil survival,
consistent with the apoptosis data. These results suggest that the
pharmacologic inhibition of p38-MAPK, but not ERK, delays spontaneous
apoptosis with resultant extension of survival in neutrophils.
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A). When cells were
treated with the antisense oligonucleotides, neutrophils exhibited a
decreased level of apoptosis in comparison with cells treated with
sense or scrambled oligonucleotides (Fig. 4B). These
experiments suggest that p38-MAPK activity may play a role in
spontaneous apoptosis in neutrophils.
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To assess p38-MAPK activity in neutrophils, we evaluated the
phosphorylation and activation of p38-MAPK. Phosphorylation of
p38-MAPK, ERK, and JNK was assessed by SDS-PAGE fractionation of
equivalent amounts (50 µg) of protein from each sample, followed by
immunoblotting with phosphospecific Abs. In freshly isolated
neutrophils (time 0), a significant level of p38-MAPK phosphorylation
was detected (Fig. 5A). This
basal phosphorylation level was sustained during a 24-h period, while
neutrophils in parallel cultures increasingly underwent apoptosis with
time (data not shown). In contrast to p38-MAPK, ERK and JNK showed no
significant phosphorylation during the same period of culture (Fig. 5A). This was not due to the absence of ERK and JNK proteins
because these proteins were detectable by immunoblotting with Abs to
ERK and JNK. To determine whether phosphorylation of p38-MAPK is also
seen in quiescent cultures of other cell types, we evaluated
fibroblasts (IMR-90) and bronchial epithelial cells (16HBE). In
contrast to neutrophils, fibroblasts and bronchial epithelial cells
cultured in quiescent conditions showed no apparent phosphorylation of
p38-MAPK (Fig. 5B).
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C).
Stimulation of neutrophils with PMA modestly potentiated
phosphorylation of p38-MAPK (Fig. 5C). Fourth, endotoxin was
not detected (<5 pg/ml) in cell preparation reagents or culture medium
by a Limulus lysate test. Fifth, inclusion of the endotoxin
inhibitor polymyxin B (100 U/ml) in culture medium did not affect the
phosphorylation of p38-MAPK (Fig. 5D). Sixth, to avoid
endotoxin contamination and accidental cell activation during cell
preparation, fresh blood smears made on glass slides were immunostained
with a phosphospecific p38-MAPK Ab. A low but significant level of
p38-MAPK phosphorylation was detectable in neutrophils in blood smears
(Fig. 6).
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). These
experiments indicate that p38-MAPK in neutrophils is continuously
phosphorylated and activated during the program of spontaneous
apoptosis. Furthermore, a lack of phosphorylation in ERK and JNK
confirms that p38-MAPK is selectively phosphorylated during
spontaneous apoptosis.
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We examined whether p38-MAPK activity was affected by Fas ligand,
GM-CSF, and intracellular ROS, which are all known to affect the rate
of neutrophil apoptosis (4, 5, 6, 7, 8, 29). The agonistic Fas Ab CH-11 (100
ng/ml) shortened neutrophil survival and promoted apoptosis (Fig. 8). In contrast, GM-CSF (100 ng/ml) and
the antioxidants catalase (500 U/ml) and N-acetylcysteine
(500 µg/ml) extended survival and inhibited apoptosis (Fig. 8).
However, none of these reagents or H2O2
significantly affected p38-MAPK phosphorylation and activation in
neutrophils (Fig. 9, A and
B). These results indicate that p38-MAPK function in
neutrophils is independent of signaling pathways triggered by Fas
ligand, GM-CSF, and ROS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies suggest the importance of tyrosine phosphorylation and dephosphorylation events in signaling pathways that result in neutrophil apoptosis. For example, a study showed that increased tyrosine-phosphorylated proteins were detected in GM-CSF-treated neutrophils that showed increased survival, while genistein, a broad tyrosine kinase inhibitor, abrogated the survival-promoting effect of GM-CSF (9). Another study indicated that the tyrosine kinase lyn is specifically involved in GM-CSF-mediated signaling that promotes neutrophil survival (10). These studies indicate that tyrosine phosphorylation of certain proteins is necessary for the inhibition of apoptosis by GM-CSF. On the other hand, the present study indicates that tyrosine phosphorylation events are also important for the induction of apoptosis that occurs spontaneously. Our data suggest that continuous activation of p38-MAPK is involved in spontaneous apoptosis in neutrophils.
Endotoxin contamination during and after cell preparation is a constant problem that may prime neutrophils and confuse results. Although endotoxin has been shown to activate p38-MAPK in neutrophils (13, 14), we could not obtain any evidence favoring the possibility that p38-MAPK phosphorylation and activation seen in freshly isolated neutrophils were due to endotoxin contamination or accidental neutrophil activation. However, although a relationship between apoptosis and p38-MAPK is clearly demonstrated, our results may neither exclude contamination by undetectable levels of endotoxin, nor rule out the necessity of a certain degree of neutrophil activation for constitutive activation of p38-MAPK. It should also be emphasized that because cell preparation procedures induce some degree of neutrophil activation (18), extrapolation of in vitro data to in vivo situations requires caution.
Nevertheless, additional evidence for a close relationship between
neutrophil apoptosis and p38-MAPK is provided by a recent paper
published after our original submission of this manuscript (30). Frasch
et al. have reported that SK & F 86002, a specific p38-MAPK inhibitor,
suppressed neutrophil apoptosis induced by stress stimuli such as UV,
hyperosmolarity, and sphingosine (30). In contrast to us, they observed
no inhibition of spontaneous apoptosis by the p38-MAPK inhibitor.
However, there are several methodologic differences between their study
and our own. First, they used a different p38-MAPK inhibitor (SK & F
86002) than we used (SB203580). Second, they assessed the effect of the
p38-MAPK inhibitor on spontaneous apoptosis only at one time point (24
h). Third, they assessed apoptosis by fluorometric measurement of DNA
content in propidium iodide-stained cells by flow cytometry. It should
be noted that in our experiments, when compared with morphologic (Fig. 1B) and MTT-based assays (Fig. 3A), the
fluorometric measurement of DNA content (Fig. 3B) detected a
lower degree of inhibition of apoptosis by the same dose (10 µM) of
p38-MAPK inhibitor. It is likely that the different methods of
assessing apoptosis detect various stages of apoptosis. Fourth,
neutrophils isolated in their study may not have exhibited basal
phosphorylation and activation of p38-MAPK. However, in accordance with
our own, their figures show low but significant levels of basal
phosphorylation and activation of p38-MAPK at time 0 and 90 min of
incubation.
Recent studies suggest that spontaneous neutrophil apoptosis is elicited by the interaction between the constitutively expressed Fas and Fas ligand molecules in neutrophils (7, 8, 31). In the present study, we found that an agonistic Fas Ab, which promoted neutrophil apoptosis, did not affect the phosphorylation and activation of p38-MAPK. This finding is consistent with a recent report that the incubation of neutrophils with anti-Fas Ab did not affect the tyrosine phosphorylation or tyrosine phosphatase activity of the cells (31) nor the phosphorylation and activation of p38-MAPK (30). In Jurkat T lymphocytes, however, Fas has been shown to activate p38-MAPK (32, 33). Although we cannot explain these differences, our data suggest that p38-MAPK works independent of or in parallel with Fas-Fas ligand systems in neutrophils.
Our finding that p38-MAPK phosphorylation and activation were not affected by stimulation of neutrophils with the Fas agonist or GM-CSF raises the question of how p38-MAPK in neutrophils is continuously activated without any need for apparent external stimuli. We initially hypothesized that ROS generated intracellularly may contribute to continuous phosphorylation and activation of p38-MAPK. This hypothesis was based on the following evidence. First, previous investigators showed that ERK, another MAPK family member, is activated following treatment of neutrophils with the oxidizing agent diamide and H2O2 (34). Second, we have found recently that ROS act as an activator of p38-MAPK in fibroblasts depleted of thiols.4 Third, like other aerobic cells, neutrophils constitutively generate ROS within cells and undergo apoptosis in response to ROS accumulation (4, 5, 35). In support of this, we observed that peroxides, as detected by CDCFH (6-carboxy-2'7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester)) fluorescence, are accumulated within neutrophils cultured in quiescent conditions (unpublished data). Fourth, we have found in the present study that the antioxidants catalase and N-acetylcysteine inhibited spontaneous apoptosis and extended survival of neutrophils. Taken together, these lines of evidence confirm that ROS play an important role in spontaneous apoptosis in neutrophils. However, the lack of effect of these antioxidants and extragenous H2O2 on P38-MAPK phosphorylation and activation we observed suggests that ROS generation does not lie upstream of p38-MAPK activation.
In other mammalian cell types such as COS-1, COS-7, and HeLa cells, the small GTP-binding proteins Rac and Cdc42 (36, 37) and different MAPK kinases (MKK-3, MKK-4, MKK-6) (38, 39) have been implicated as upstream regulators of p38-MAPK. In neutrophils, however, the upstream regulators of p38-MAPK signaling remain ill defined. In this respect, a recent report suggested that activation of p38-MAPK by LPS in neutrophils uses MKK-3, but not Raf, mitogen-activated protein/ERK-1, or mitogen-activated protein/ERK-2 (14). Other reports showed that activation of p38-MAPK by chemoattractants is mediated through a pathway involving phosphatidylinositol 3-kinase, protein kinase C, and Ca2+ (15, 16). In turn, changes in Ca2+ have been shown to affect the constitutive rate of neutrophil apoptosis (3). To date, intracellular events preceding p38-MAPK phosphorylation and activation in neutrophils have not been defined. Further studies will be needed to determine what mediates the continuous phosphorylation and activation of p38-MAPK that lead to apoptosis in neutrophils.
Activation of p38-MAPK has been implicated in extracellular stress-induced apoptosis in other cell types such as neuronal cells and lymphocytes (40, 41). However, the mechanisms by which p38-MAPK activation participates in apoptosis are unknown. In our findings in neutrophils, inhibition of p38-MAPK activity by SB203580 and by antisense oligonucleotides delayed apoptosis, but could not abolish its expression. In addition, the phosphorylation and activation of p38-MAPK were detected even in freshly isolated neutrophils that had not yet expressed the morphologic features of apoptosis (i.e., at time 0). These findings suggest that p38-MAPK activation in neutrophils is not required for the execution of apoptosis, but rather is required for the constitutive activation of other necessary components of a cell death pathway. It should also be emphasized that SB203580 effectively inhibited p38-MAPK activity, yet only inhibited apoptosis by 50%. This suggests a partial role of p38-MAPK in the program of spontaneous apoptosis in neutrophils. Nevertheless, the continuous phosphorylation and activation of p38-MAPK presented in this work may reflect the constitutively expressed apoptotic program in neutrophils, which rapidly die via apoptosis in vitro and in vivo (1, 2).
The functional role of p38-MAPK in neutrophils has not previously been
demonstrated. Several reports documented that p38-MAPK is activated
following stimulation of neutrophils with various agonists such as LPS,
TNF-, GM-CSF, platelet-activating factor, FMLP, IL-8, and
protein kinase C (12, 13, 14, 15, 16). However, many of these agonists activate ERK
as well (15, 26, 42), indicating multiple MAPK activation by
inflammatory stimuli. Our findings suggest that the activation of
p38-MAPK may serve specifically to limit the longevity of neutrophil
survival following activation.
Indeed, it seems clear that the constitutive rate of apoptosis in neutrophils is the major determinant of their survival and functional longevity at inflamed sites (1). Although elevation of intracellular Ca2+ (3) or cAMP (43) by agonistic stimuli has been shown to inhibit neutrophil apoptosis, the effect of inflammatory mediators such as FMLP, C5a, IL-1, and IL-8 on neutrophil apoptosis remains controversial (34, 44, 45). Based on our findings, activation of p38-MAPK by inflammatory mediators may activate an apoptotic program to counteract the antiapoptotic effects of inflammatory signals. This may be important to limit the toxic potential of neutrophils. The validity of this hypothesis remains to be determined.
In summary, our data suggest that p38-MAPK plays a role in driving spontaneous apoptosis of neutrophils. Clarification of this role perhaps awaits further investigations of its regulators and targets. Definition of the p38-MAPK-mediated pathway will provide clues to the complex mechanism underlying neutrophil apoptosis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Atsushi Nagai, Department of Medicine, Chest Institute, Tokyo Women’s Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
3 Abbreviations used in this paper: ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; GM-CSF, granulocyte-macrophage colony-stimulating factor; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAP, mitogen-activated protein kinase-activated protein; MKK, mitogen-activated protein kinase kinase; MTT, 3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide.
4 K. Aoshiba, S. Yasui, K. Nishimura, and A. Nagai. Thiol depletion induces apoptosis through an ordered cell death pathway in cultured lung fibroblasts. Submitted for publication.
Received for publication February 19, 1998. Accepted for publication October 16, 1998.
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 M.-J. Hsu, S.-S. Lee, and W.-W. Lin Polysaccharide purified from Ganoderma lucidum inhibits spontaneous and Fas-mediated apoptosis in human neutrophils through activation of the phosphatidylinositol 3 kinase/Akt signaling pathway J. Leukoc. Biol., July 1, 2002; 72(1): 207 - 216. [Abstract] [Full Text] [PDF]
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 G. S. Kerby, V. Cottin, F. J. Accurso, F. Hoffmann, E. D. Chan, V. A. Fadok, and D. W. H. Riches Impairment of macrophage survival by NaCl: implications for early pulmonary inflammation in cystic fibrosis Am J Physiol Lung Cell Mol Physiol, July 1, 2002; 283(1): L188 - L197. [Abstract] [Full Text] [PDF]
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P. M. De Souza, H. Kankaanranta, A. Michael, P. J. Barnes, M. A. Giembycz, and M. A. Lindsay Caspase-catalyzed cleavage and activation of Mst1 correlates with eosinophil but not neutrophil apoptosis Blood, May 1, 2002; 99(9): 3432 - 3438. [Abstract] [Full Text] [PDF]
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 A. R. Khaled, A. N. Moor, A. Li, K. Kim, D. K. Ferris, K. Muegge, R. J. Fisher, L. Fliegel, and S. K. Durum Trophic Factor Withdrawal: p38 Mitogen-Activated Protein Kinase Activates NHE1, Which Induces Intracellular Alkalinization Mol. Cell. Biol., November 15, 2001; 21(22): 7545 - 7557. [Abstract] [Full Text] [PDF]
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H. Lundqvist-Gustafsson, S. Norrman, J. Nilsson, and A. Wilsson Involvement of p38-mitogen-activated protein kinase in staphylococcus aureus-induced neutrophil apoptosis J. Leukoc. Biol., October 1, 2001; 70(4): 642 - 648. [Abstract] [Full Text] [PDF]
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J. Varghese, S. Chattopadhaya, and A. Sarin Inhibition of p38 Kinase Reveals a TNF-{{alpha}}-Mediated, Caspase-Dependent, Apoptotic Death Pathway in a Human Myelomonocyte Cell Line J. Immunol., June 1, 2001; 166(11): 6570 - 6577. [Abstract] [Full Text] [PDF]
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R. G. Deschesnes, J. Huot, K. Valerie, and J. Landry Involvement of p38 in Apoptosis-associated Membrane Blebbing and Nuclear Condensation Mol. Biol. Cell, June 1, 2001; 12(6): 1569 - 1582. [Abstract] [Full Text] [PDF]
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M. Goebeler, R. Gillitzer, K. Kilian, K. Utzel, E.-B. Brocker, U. R. Rapp, and S. Ludwig Multiple signaling pathways regulate NF-{kappa}B-dependent transcription of the monocyte chemoattractant protein-1 gene in primary endothelial cells Blood, January 1, 2001; 97(1): 46 - 55. [Abstract] [Full Text] [PDF]
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A. Savoie, V. Lavastre, M. Pelletier, T. Hajto, K. Hostanska, and D. Girard Activation of human neutrophils by the plant lectin Viscum album agglutinin-I: modulation of de novo protein synthesis and evidence that caspases are involved in induction of apoptosis J. Leukoc. Biol., December 1, 2000; 68(6): 845 - 853. [Abstract] [Full Text]
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A. Villunger, L. A. O'Reilly, N. Holler, J. Adams, and A. Strasser Fas Ligand, Bcl-2, Granulocyte Colony-Stimulating Factor, and p38 Mitogen-activated Protein Kinase: Regulators of Distinct Cell Death and Survival Pathways in Granulocytes J. Exp. Med., August 28, 2000; 192(5): 647 - 658. [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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 J. Lotem, R. Kama, and L. Sachs Suppression or induction of apoptosis by opposing pathways downstream from calcium-activated calcineurin PNAS, October 12, 1999; 96(21): 12016 - 12020. [Abstract] [Full Text] [PDF]
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 H. Kankaanranta, P. M. De Souza, P. J. Barnes, M. Salmon, M. A. Giembycz, and M. A. Lindsay SB 203580, an Inhibitor of p38 Mitogen-Activated Protein Kinase, Enhances Constitutive Apoptosis of Cytokine-Deprived Human Eosinophils J. Pharmacol. Exp. Ther., August 1, 1999; 290(2): 621 - 628. [Abstract] [Full Text]
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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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