From the Department of Pediatrics, ¶ Department
of Medicine, and ** Program in Molecular Signal Transduction, National
Jewish Medical and Research Center and the
Department of
Medicine, University of Colorado School of Medicine,
Denver, Colorado 80206
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ABSTRACT |
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Human neutrophils undergo apoptosis spontaneously
when cultured in vitro; however, the signal transduction
pathways involved remain largely unknown. In some cell types, c-Jun
NH2-terminal kinase and p38 mitogen-activated protein
kinase (MAPK) have been implicated in the pathways leading to
stress-induced apoptosis. In this study, we begin to define two
pathways leading to apoptosis in the neutrophil induced either by
stress stimuli (UV, hyperosmolarity, sphingosine) or by anti-Fas
antibody or overnight culture in vitro (spontaneous
apoptosis). Apoptosis induced by stress stimuli activated p38 MAPK, and
apoptosis was inhibited by the specific p38 MAPK inhibitor,
6-(4-Fluorophenyl)-2.3-dihydro-5-(4-puridinyl)imidazo(2,1-)thiazole dihydrochloride. Furthermore, differentiation of HL-60 cells toward the
neutrophil phenotype resulted in a loss in c-Jun
NH2-terminal kinase activation with concomitant
acquisition of formylmethionylleucylphenylalanine-stimulatable and stress-inducible p38 MAPK activity as well as apoptosis blockade by
the p38 MAPK inhibitor. In contrast, anti-Fas-induced or spontaneous apoptosis occurred independent of p38 MAPK activation and was not
blocked by the inhibitor. Both pathways appear to utilize member(s) of
the caspase family, since pretreatment with either Val-Ala-Asp-fluoromethyl ketone or Asp-Glu-Val-Asp-fluoromethyl ketone
inhibited apoptosis induced by each of the stimuli. We propose the
presence of at least two pathways leading to apoptosis in human
neutrophils, a stress-activated pathway that is dependent on p38 MAPK
activation and an anti-FAS/spontaneous pathway that is p38
MAPK-independent.
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INTRODUCTION |
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Human neutrophils have a life span of only hours following release from bone marrow into systemic circulation. At sites of inflammation, neutrophils are short lived and, under normal circumstances, undergo apoptosis with subsequent recognition and removal by phagocytic cells such as macrophages (1-3). Cultured in vitro, neutrophils isolated from the blood undergo apoptosis spontaneously, with greater than 50% of the population becoming apoptotic within 16 h (1, 2, 4). It has been assumed that the neutrophil half-life in vitro is a reflection of their life span in vivo and that this cell possesses an "inbuilt" clock leading inexorably to apoptosis within a certain time range after maturation in, or release from, the bone marrow. In inflammatory lesions, the presence of neutrophil stimuli such as granulocyte-macrophage colony-stimulating factor or lipopolysaccharide could extend this life span by inhibiting apoptosis (5-8), while interleukin-10 might shorten it by enhancing the apoptotic process (9). Removal of apoptotic inflammatory cells by macrophages before they lyse and release their toxic contents may represent an important mechanism for limiting tissue injury and for resolution of inflammation (10-13). Thus, induction and control of neutrophil apoptosis appears to be central to resolution or persistence of an inflammatory state.
Recently, a number of gene products have been identified as important regulators involved in the pathways leading to apoptosis. However, the relative involvement of any one gene product may vary depending on the cell type. Although apoptosis appears to be a universal phenomenon observed in virtually all cells, the specific signal transduction pathways mediating the death program can be biochemically and functionally distinct in response to different stimuli as well as vary between cell types.
Neutrophil intracellular signal transduction in response to a wide variety of stimuli appears to utilize the MAP1 kinase cascades. Each MAP kinase cascade involves phosphorylation and activation of a MAP ERK kinase kinase, which in turn activates members of the MAP ERK kinases (MEK). MEKs then activate a specific member of the MAPK family by dual phosphorylation of a threonine and tyrosine residue. This system of parallel intracellular signaling pathways activated in response to a specific external stimulus and leading to a unique set of functional responses is well defined in yeast and is now recognized to exist in most mammalian cells including the human neutrophil. Three distinct MAPKs have been identified to date in mammalian cells: p42/p44 ERKs are activated by growth factors (14); JNK/stress-activated protein kinase is potently activated by irradiation and other environmental stresses such as hyperosmolarity (15, 16); and p38 MAPK is activated by proinflammatory cytokines, osmotic stress, and UV irradiation (16). Irradiation and other stress stimuli are known to induce apoptosis in a variety of cell types (17, 18). Accordingly, JNK and/or p38 have been implicated by some investigators in the process leading to apoptosis in response to these stimuli (17, 18). In Jurkat cells, JNK and p38 MAPK activation have been coupled to Fas-induced apoptosis, and it appears as though this coupling requires the activation of ICE-like proteases (caspases) (19).
Both Fas and the caspase superfamily have emerged as key players in the regulation of the apoptotic pathway. The Fas/FasL system has been extensively studied in T cells and has been shown to be intimately involved in the removal of autoreactive B cells (20) as well as in activation-induced T cell death (21-23). Recently, it has been reported that spontaneous apoptosis in neutrophils may be in part mediated by Fas/FasL (24, 25). However, the signal transduction pathway leading from Fas receptor cross-linking to apoptosis in the neutrophil is unknown.
The caspase superfamily contain a highly homologous group of proteins that can be subdivided into three categories including ICE-like (caspase 1), CPP32-like (caspase 3), and ICH-1-like (caspase 2) proteases. The involvement of caspases in apoptosis has been demonstrated in a number of ways including the use of tri- and tetrapeptide inhibitors that take advantage of the different substrate specificity of the enzymes or by the cowpox viral protein, CrmA, which is a natural inhibitor of caspase 1 (26, 27). Several lines of evidence have suggested an involvement of caspase 1 and caspase 3 in Fas-mediated apoptosis. For instance, treatment with either YVAD-cmk, an inhibitor of caspase 1, or DEVD-fmk, an inhibitor of caspase 3, prevents apoptosis induced by Fas cross-linking in Jurkat and U937 cells (28). Although there is detailed information regarding the involvement of caspases in apoptosis in other cell types, little is known about the role of these enzymes in neutrophil apoptosis.
In this report, we examine signal transduction pathways leading to apoptosis in human neutrophils induced by in vitro culture (spontaneous apoptosis), Fas cross-linking, or stress stimuli. At least two pathways leading to apoptosis have been identified, both requiring caspase involvement, a stress-activated pathway that is p38 MAPK-dependent and a spontaneous/Fas-mediated pathway that appears to be p38 MAPK-independent.
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EXPERIMENTAL PROCEDURES |
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Materials--
Endotoxin free reagents and plastics were used in
all experiments. Neutrophils were resuspended in Krebs-Ringer phosphate buffer, pH 7.2, with 0.2% dextrose. fMLP, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, bovine serum albumin fraction V,
propidium iodide, and tissue culture grade Me2SO were
purchased from Sigma. Protein A-Sepharose was purchased from Zymed
(South San Francisco, CA). DEVD-fmk and VAD-fmk were purchased from
Enzyme Systems (Dublin, CA). Anti-JNK1(C-17) rabbit and goat polyclonal antibody, anti-p38(C-20) polyclonal antibody, anti-ERK2(C-16) polyclonal antibody, c-JUN-(1-79), and ATF-2-(1-96) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-p38 phosphospecific polyclonal, horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody and PD98059 were purchased from Calbiochem. A MAP kinase assay kit and anti-Fas IgM (CH-11) were
purchased from Upstate Biotechnology Inc. (Lake Placid, NY). 6-(4-Fluorophenyl)-2.3-dihydro-5-(4-puridinyl)imidazo(2,1-)thiazole dihydrochloride (SK & F 86002-A2, or SK & F 86002) was kindly provided by SmithKline Beecham Pharmaceuticals (King of Prussia, PA).
Induction of Apoptosis-- Apoptosis was induced by exposure of neutrophils or HL-60 cells resuspended to either 5 × 106 cells/ml or 20 × 106 cells/ml in Krebs-Ringer phosphate buffer or RPMI 1640 plus 10% FBS and plated in a 12-well tissue culture plate at 1 ml/well, to UV irradiation (254 nm) on a UV transilluminator for 10 min, sphingosine, 450 mosM (0.25 M sucrose in Krebs-Ringer phosphate buffer), anti-Fas IgM, or 18-24 h culture in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% FBS (spontaneous apoptosis) followed by incubation at 37 °C for the times indicated.
p38 MAPK Immunoprecipitation and Kinase Assay--
p38 MAP
kinase activity measurements were performed essentially as described
(29, 30). Either HL-60 cells cultured in RPMI 1640 with 10% FBS or
neutrophils isolated by the plasma Percoll method (31) were resuspended
to 20 × 106/ml in RMPI 1640 supplemented with 10%
FBS. 20 × 106 cells (1 ml) were plated in each well
of a 12-well tissue culture plate. Cells were stimulated with UV
irradiation, 1 µM sphingosine, 400 ng/ml anti-Fas IgM,
450 mosM, incubated for the indicated times at 37 °C,
harvested, and lysed in RIPA lysis buffer. p38 MAP kinase was
immunoprecipitated with 1 µg/ml anti-p38 rabbit polyclonal antibody.
Protein A-Sepharose beads with bound antibody were resuspended in 50 µl of kinase reaction mix containing 20 mM Hepes, pH 7.6, 200 µM MgCl2, 20 µM ATP, 20 µCi of [-32P]ATP, 2 mM dithiothreitol,
100 µM sodium orthovanadate, 25 mM
-glycerolphosphate (pH 7.2), and 500 ng recombinant fragment of
activated transcription factor 2 (ATF-2-(1-96)). Reactions were
terminated with 4× Laemmli buffer (32), and proteins were separated by
10% SDS-PAGE and blotted to nitrocellulose membrane. Activity was
visualized as the phosphorylation of the ATF-2 fragment by
autoradiography.
JNK Immunoprecipitation and Kinase Assay-- JNK immunoprecipitation and kinase assay was carried out as described previously (29). HL-60 cells or neutrophils were resuspended and stimulated as described for p38 immunoprecipitation and kinase assay. Following stimulation, cells were harvested and lysed with anti-phosphotyrosine lysis buffer (29). JNK1 was immunoprecipitated with 1 µg/ml anti-JNK1 rabbit polyclonal antibody. Beads were resuspended in 40 µl of kinase reaction mixture (29) containing 500 ng of recombinant fragment of c-Jun-(1-79). Activity was visualized following SDS-PAGE as the phosphorylation of the c-Jun fragment by autoradiography.
ERK Immunoprecipitation and Kinase Assay-- Neutrophils were resuspended as described for p38 MAP kinase assay. Cells were pretreated with or without 100 nM fMLP for 10 min, or 30 µM PD98059 for 30 min either alone or prior to UV irradiation to induce apoptosis and lysed in RIPA lysis buffer. ERK2 was immunoprecipitated with anti-ERK2 rabbit polyclonal antibody. ERK2 kinase activity was determined by using a MAP kinase assay kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. For Western blotting, neutrophils were resuspended to 10 × 106 cells/ml in RPMI 1640 supplemented with 10% fetal calf serum, and 10 × 106 cells (1 ml) were plated into the appropriate number of wells in a 12-well tissue culture plate. Cells were stimulated for the times indicated, harvested by centrifugation at 14,000 rpm for 20 s, and resuspended in 500 µl of RIPA lysis buffer. ERK2 was immunoprecipitated with 1 µg/ml anti-ERK2 rabbit polyclonal antibody, as described above, washed three times with RIPA lysis buffer, and resuspended with 30 µl of RIPA lysis buffer. Samples were boiled for 5 min with 4× Laemmli sample buffer, resolved on 10% SDS-PAGE, and blotted to nitrocellulose membrane.
Western Immunoblotting--
Following p38 MAPK, JNK, or ERK
kinase assays, the membranes were blocked overnight in blocking buffer
(25 mM Tris-HCl, pH 7.8, 190 mM NaCl, 0.2%
Tween 20, 5% bovine serum albumin) at 4 °C with agitation and
probed with anti-p38 rabbit polyclonal, anti-JNK1 goat polyclonal, or
anti-ERK2 rabbit polyclonal antibody diluted 1:1000 in 1× wash buffer
(25 mM Tris-HCl, pH 7.8, 190 mM NaCl, 0.2%
Tween 20) plus 1% bovine serum albumin for 2 h at room
temperature with rocking. The membranes were washed twice for 30 min
each with 1× wash buffer and incubated with either horseradish
peroxidase-conjugated anti-rabbit antibody (for p38 and ERK) or
horseradish peroxidase conjugated anti-goat antibody (for JNK) for 45 min at room temperature with rocking. The membranes were washed two
times for 30 min each with 1× wash buffer, and proteins were
visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech)
according to the manufacturer's instructions. The membranes were
stripped in 62.5 mM Tris-HCl, pH 7.8, 100 mM
-mercaptoethanol, 2% SDS for 30 min at 50 °C; washed two times
for 10 min each with 1× wash at room temperature with rocking; blocked
overnight in blocking buffer at 4 °C; and reprobed with
anti-p38-phosphospecific, anti-ERK-phosphospecific, or horseradish
peroxidase-conjugated anti-phosphotyrosine monoclonal antibody.
HL-60 Differentiation-- HL-60 cells were maintained at no greater than 1 × 106 cells/ml in RPMI 1640 supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. For differentiation, HL-60 cells were harvested and resuspended at 0.2 × 106 cells/ml in RPMI 1640 supplemented with 10% FBS and 1.25% Me2SO and incubated at 37 °C for up to 5 days. Differentiation was determined by the ability of cells to reduce nitro blue tetrazolium (33).
Analysis of DNA Fragmentation by Flow Cytometry--
Neutrophils
were resuspended to 5 × 106 cells/ml in RPMI 1640 supplemented with 10% FBS and plated into each well of a 12-well tissue culture plate (5 × 106 cells/well). Following
stimulation, 1 × 106 cells were harvested and placed
directly into 80% ethanol in phosphate-buffered saline and fixed
overnight at 20 °C. Cells were harvested by centrifugation and
resuspended in 500 µl of phosphate-buffered saline. RNA was removed
by treatment with 0.25 mg of RNase A at 37 °C for 30 min. Propidium
iodide (50 µg/ml stock in phosphate-buffered saline) was added to a
final concentration of 500 ng/ml, and samples were stored at 4 °C in
the dark until analysis. DNA fragmentation analysis was carried out on
the Becton Dickinson FACScalibur. Apoptosis was scored by the
appearance of a sub-G0 peak.
Morphological Assessment of Apoptosis-- Following stimulation, 5 × 105 cells were resuspended in 1 ml of cytospin buffer (phosphate-buffered saline, 20 mM EDTA, 1% FBS). One hundred microliters of the cell suspension was cytocentrifuged onto a slide, fixed and stained with a modified Wright-Giemsa stain, and analyzed by light microscopy. Neutrophils counted in randomly selected fields were scored as apoptotic, normal, or necrotic based on their nuclear morphology.
Statistics-- S.D. values were calculated from at least three independent experiments with duplicate readings within each experiment.
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RESULTS |
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Selective Activation of p38 MAPK with Stimuli That Induce Apoptosis in Human Neutrophils-- While the susceptibility of neutrophils to spontaneous apoptosis (1, 2, 4) or to Fas cross-linking (24) is well described, the sensitivity of neutrophils to other apoptosis-inducing stimuli, such as UV irradiation or hyperosmolarity, is less well understood. Propidium iodide staining and flow cytometric analysis or stained cytospin preparations were used to determine DNA fragmentation and nuclear condensation (respectively) characteristic of apoptosis in human neutrophils in response to a variety of stimuli (Fig. 1). Neutrophils exposed to overnight culture in vitro (spontaneous apoptosis (Fig. 1b)), anti-Fas (Fig. 1c), and UV irradiation (Fig. 1d) all show reduced PI staining, evident by the appearance of a sub-G0 peak, characteristic of apoptosis. Likewise, these stimuli as well as treatment with sphingosine and culture in hyperosmolar conditions (Fig. 1e) showed an increase in nuclear condensation over time to levels comparable with the amount of DNA fragmentation analyzed by PI staining and flow cytometry. Morphological features of apoptosis, including nuclear condensation and membrane blebbing, were seen in neutrophils treated with UV irradiation followed by incubation for 4 h at 37 °C (Fig. 1f). Apoptosis was also confirmed by demonstrating DNA fragmentation with typical internucleosomal cleavage and laddering on agarose gels (data not shown). These data demonstrate that in addition to spontaneous and anti-Fas-induced apoptosis, neutrophils undergo apoptosis with stress stimuli in a rapid and relatively synchronous manner.
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p38 MAPK Involvement-- To determine more directly whether p38 MAPK activation was necessary for apoptosis to occur, a specific p38 MAP kinase inhibitor, SK & F 86002, was used (30, 39, 40). Neutrophils pretreated with the inhibitor were allowed to undergo apoptosis either spontaneously in overnight culture or by stimulation with UV irradiation, hyperosmolarity, sphingosine, or anti-Fas, and the degree of apoptosis was determined (Table I). Consistent with the activation of p38 MAPK by the different stimuli, treatment with SK & F 86002 (10 µM) protected the cells from UV irradiation, hyperosmolarity, and sphingosine-induced apoptosis but had no effect on anti-Fas or spontaneous apoptosis, suggesting that p38 MAPK activation is required for signal transduction leading to stress-induced apoptosis in human neutrophils. A dose-response of the SK & F 86002 on p38 activity shows that concentrations of 10 µM and lower were efficient at completely inhibiting p38 activity (data not shown and Ref. 30). Pretreatment of neutrophils with PD98059, an inhibitor of MEK1 kinase that is responsible for the phosphorylation and activation of p42/p44 ERKs (41) had no effect on UV-induced apoptosis, thus supporting the lack of p42/p44 ERK involvement (Fig. 4).
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Switch in MAP Kinase Usage during Differentiation-- In contrast to neutrophils, the HL-60 cell line, a cell that can be terminally differentiated to the neutrophil phenotype by treatment with Me2SO, demonstrates activation of both JNK and p38 MAPK in response to UV irradiation (Fig. 6). JNK has been implicated in ceramide-induced apoptosis in undifferentiated HL-60 cells (42). As both JNK and p38 MAPK were activated by UV irradiation in the undifferentiated HL-60 cell, SK & F 86002 was used to determine whether p38 MAPK or JNK was the dominant kinase involved in the pathway leading to apoptosis. HL-60 cells pretreated with and without SK & F 86002 prior to UV irradiation were incubated at 37 °C over a range of times. The p38 MAPK inhibitor failed to prevent UV-induced apoptosis in undifferentiated HL-60 cells (Fig. 5a). It has been suggested by Whitmarsh et al. (43) that some p38 MAPK inhibitors can inhibit JNK activation. A dose-response analysis of JNK activation with increasing concentrations of SK & F 86002 demonstrates that at 10 µM, the concentrations used in this study, there was no inhibition of JNK activity (Fig. 5b). These results suggest that, in HL-60 cells, UV-induced apoptosis does not require p38 MAPK and are consistent with the possibility that signaling occurs through the JNK MAP kinase cascade in this cell.
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Effects of fMLP on UV-induced Apoptosis-- Although p38 MAPK is activated under conditions that induce apoptosis, it is also activated by the chemoattractant fMLP and utilized in pathways leading to cell activation for superoxide anion release, actin assembly, adherence, calcium influx, and chemotaxis (44, 45). Therefore, we tested the effects of fMLP directly and also on UV-induced apoptosis. Neutrophils were pretreated with fMLP for 10 min prior to UV irradiation and allowed to incubate at 37 °C for 4 h. fMLP treatment did not directly induce apoptosis and, in fact, inhibited DNA fragmentation induced by UV irradiation although p38 MAPK was activated under these conditions (Fig. 7, a and b). Furthermore, fMLP did not have an effect under conditions where p38 MAPK was not activated, such as anti-Fas-induced or spontaneous apoptosis (Fig. 7, c and d).
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Involvement of CED3/ICE-like Proteases in Apoptosis-- As mentioned above, p38 MAPK seemed to be involved in stress-induced apoptosis and did not appear to participate in anti-Fas-induced or spontaneous apoptosis, suggesting an alternative pathway leading to DNA fragmentation. Another class of enzymes implicated in apoptosis are Ced-3/ICE-like proteases, or caspases. We sought to determine whether member(s) of the caspase family were involved in stress-induced, anti-FAS-induced, or spontaneous apoptosis. In these studies, two inhibitors were used; VAD-fmk is an irreversible caspase 1-like protease inhibitor, and DEVD-fmk inhibits the caspase 3-like proteases (50, 51). Both inhibited apoptosis induced by any of the stimuli (Fig. 9).
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DISCUSSION |
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The requirement for JNK and/or p38 MAPK in stress-induced or Fas-mediated apoptosis has been a matter of controversy. In some cell types, JNK activation has been uncoupled from apoptosis, although it is activated with stimuli that also induce apoptosis. For instance, in MCF7 cells, JNK activation has been dissociated from TNF receptor-mediated apoptosis (52). In contrast, in PC-12 cells JNK activation is required for apoptosis induced by nerve growth factor withdrawal (17). Likewise, there appears to be a requirement for JNK activation in ceramide-mediated apoptosis in U937 cells, suggesting that there is coordinated regulation of the apoptotic pathway by both the sphingomyelinase and the JNK pathways (18). UV irradiation has recently been reported to accelerate apoptosis in human neutrophils, although the mechanism by which apoptosis was mediated remains unclear (34). We have shown that in neutrophils, there is selective activation of the p38 MAPK, and not JNK, in response to stress stimuli that induce apoptosis providing a possible mechanism of stress-induced apoptosis in these cells. Several lines of evidence support this conclusion. In addition to activation of p38 MAPK, but not JNK or p42/p44 ERKs, with stress stimuli (Fig. 2), we have shown that inhibition of p38 MAPK with SK & F 86002 protects from apoptosis induced by UV irradiation, hyperosmolarity, and sphingosine (Table I).
Furthermore, as HL-60 cells differentiated toward the neutrophil
phenotype, there was a loss in JNK activation in response to UV
irradiation, with concomitant acquisition of fMLP-activable p38 MAPK
activity. The notion that p38 MAPK is involved in neutrophil apoptosis
is supported further by the observation that differentiated HL-60 cells
were protected from UV-induced apoptosis by the p38 inhibitor, whereas
undifferentiated HL-60 cells were unaffected by that treatment. Of
interest, no stimulus has been identified to date that leads to
activation of JNK in neutrophils using the current assay conditions,
although the JNK protein is present in the cell (Fig. 2). These results
strongly suggest that the requirement for either JNK or p38 MAPK in
stress-induced apoptosis varies depending on the cell type as well as
on the context in which the kinase is activated. For instance, in
addition to stimuli that induce apoptosis, such as -irradiation (37,
38, 53), UV irradiation (15), and anti-Fas antibody (19, 35), JNK activation has been associated with activation stimuli, including T
cell activation signals (37, 38, 54), CD40 ligation (55, 56), and
growth factors (57). Chen et al. (37, 38, 57) have shown
that although JNK is activated by these various stimuli, resulting in
different biological responses (i.e. proliferation versus apoptosis), the kinetics of activation determines the
fate of the cell, such that a rapid and transient activation is
associated with proliferation, while persistent and sustained JNK
activation results in apoptosis. A similar situation is observed in the
neutrophil where p38 MAPK, although it appears to be involved in the
pathway leading to stress-induced apoptosis, is also activated under
conditions that promote cell survival, such as treatment with
chemoattractants such as fMLP. The kinetics of p38 MAPK activation
induced by fMLP is rapid and transient (30), where peak activity is
observed within 2 min following fMLP stimulation and has returned to
background levels by 10 min. In contrast, in response to UV irradiation
and other stress stimuli, activation is delayed and sustained, reaching maximal activity after 30 min of stimulation and maintaining activity for 1-2 h (Fig. 3). It is possible that the differences in activation kinetics of p38 MAPK by fMLP and UV irradiation in the neutrophil could
contribute to the distinct biological responses elicited by each
stimulus. Even when the potentially inhibitory ERK activation was
blocked by treatment of neutrophils with PD98059 prior to fMLP
stimulation, the transient p38 MAPK activation was apparently not
enough to induce apoptosis (data not shown), supporting the hypothesis
that the duration of p38 MAPK activation contributes to whether the
cell will survive or undergo apoptosis.
There appear to be two distinct outcomes associated with p38 MAPK
activation in the neutrophil: cell activation and apoptosis. This
suggests that the microenvironment in which p38 MAPK is activated determines the ultimate biological response to be mediated by p38 MAPK.
We and others (30, 46) have shown that in neutrophils, fMLP induces
robust p42/p44 ERK activity. p42/p44 ERKs are activated by growth
factors and are hypothesized to be involved in cell survival (17, 41,
48). The relative balance between JNK and p42/p44 ERK activity in PC-12
cells has been reported to determine whether the cell will survive or
undergo apoptosis (17). Similarly, protection from TNF--induced
apoptosis by fibroblast growth factor-2 requires the activation of
p42/p44 ERK (48). Based on the results presented in this study, we
suggest a similar situation where, although p38 MAPK activation is
involved in the signal transduction pathway leading to stress-induced
apoptosis, the "death" signal mediated by p38 MAPK can be
overridden by the survival signal generated by activation of p42/p44
ERK. However, since fMLP stimulation evokes a variety of cellular
responses, it is possible that there may be additional, as of yet
unidentified mechanisms of cell survival. For instance, activation of
NF-
B is critical in the regulation of cytokine-induced gene
expression and has been associated with protection of TNF-induced
apoptosis in 3T3 and Jurkat cells (58, 59) as well as in
radiation-induced apoptosis in transfected HT1080 cells (60). In the
neutrophil, NF-
B activation occurs in response to TNF as well as
fMLP (61). It is possible, therefore, that protection of UV-induced
apoptosis by fMLP occurs at the level of NF-
B activation. The
observation that inhibition of UV-induced apoptosis by fMLP can be
reversed by the MEK1 kinase inhibitor PD98059 strongly suggests that
fMLP protection requires p42/p44 ERK activation. Preliminary evidence
(data not shown) suggests that NF-
B activation by fMLP was not
reduced in the presence of the MEK1 kinase inhibitor, PD98059,
suggesting that NF-
B was not involved in the protection of
UV-induced apoptosis by fMLP.
In addition to stress-induced apoptosis, neutrophils undergo apoptosis spontaneously when cultured in vitro as well as by Fas receptor cross-linking; however, it appears as though apoptosis by these pathways is independent of p38 MAPK, since treatment of neutrophils with anti-Fas antibody induces apoptosis in the absence of p38 MAPK activation. Similarly, the p38 MAPK inhibitor had no effect on Fas-mediated or spontaneous apoptosis, suggesting further an alternative signal transduction pathway leading to apoptosis. However, one cannot rule out the possibility of different isoforms of p38 MAPK involved in these pathways, the activities of which may not be detected by the available methods. Since Fas-induced and spontaneous apoptosis appear to follow the same pathway, it is possible that spontaneous apoptosis involves the Fas/FasL system. It has been shown previously (24) and confirmed by us (data not shown) that apoptosis induced by overnight culture in vitro could be partially inhibited (50%) by an antagonistic anti-Fas IgG antibody. This raises the question of whether neutrophils have an inbuilt clock that determines when the cell will die, or whether the life span is in part dependent on whether the neutrophil encounters FasL as it is filtered through the liver and spleen. It is also possible that the in vivo life span is a combination of both Fas/FasL expression on the neutrophil as well as the presence of an inbuilt clock such that as the neutrophil ages, Fas and/or FasL expression is up-regulated, therefore increasing the likelihood of Fas/FasL ligation either by another neutrophil or another cell expressing FasL, the interaction of which would subsequently induce apoptosis. Both Fas and FasL have been detected on human neutrophils (24); however, whether Fas and/or FasL expression increases as neutrophils are cultured in vitro remains to be determined.
The Ced-3/ICE protease superfamily has been shown to be required for multiple pathways leading to apoptosis; however, it appears as though different apoptosis-inducing stimuli may utilize different members of the protease family, and the relevant protease(s) for these different apoptotic pathways have not been defined. In an attempt to distinguish stress-induced from Fas-mediated apoptosis pathways, we used tetrapeptide inhibitors of the Ced-3/ICE protease superfamily. We observed that treatment with either VAD-fmk or DEVD-fmk virtually blocked apoptosis induced by any stimuli used, whether it was stress-stimuli, anti-Fas, or spontaneous. Furthermore, we have shown that p38 MAPK activation was independent of caspase activity, suggesting that either p38 MAPK and caspase activation are distinct from each other or that p38 MAPK activation is upstream of caspase activation. The implication of these results is that although both p38 MAPK and member(s) of the caspase family appear to be involved in the pathway leading to stress-induced apoptosis, it is possible that their activities may follow parallel pathways in response to the same stimuli. In addition, these results suggest further that the anti-Fas-induced and spontaneous apoptosis pathways, both of which are independent of p38 MAPK activation, may converge with the stress-induced pathway at the level of the caspase(s).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM48211 and HK50319 (to P. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Tel.: 303-398-1282; Fax: 303-398-1381; E-mail: fraschc{at}njc.org.
1
The abbreviations used are: MAP,
mitogen-activated protein; MAPK, mitogen-activated protein kinase; JNK,
c-Jun NH2-terminal kinase; FasL, Fas ligand; ATF-2,
activated transcription factor-2; MEK, MAP-ERK kinase; ERK,
extracellular signal-regulated kinase; ICE, interleukin-1-converting
enzyme; TNF, tumor necrosis factor; NF-
B, nuclear factor-kappa B;
PAGE, polyacrylamide gel electrophoresis; fMLP,
formylmethionylleucylphenylalanine; FBS, fetal bovine serum; RIPA,
radioimmune precipitation; SK & F 86002, 6-(4-Fluorophenyl)-2.3-dihydro-5-(4-puridinyl)imidazo(2,1-
)thiazole dihydrochloride; VAD-fmk, Val-Ala-Asp-fluoromethyl ketone;
DEVD-fmk, Asp-Glu-Val-Asp-fluoromethyl ketone.
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REFERENCES |
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