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Originally published online as doi:10.1189/jlb.0104048 on August 24, 2004

Published online before print August 24, 2004
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(Journal of Leukocyte Biology. 2004;76:1047-1056.)
© 2004 by Society for Leukocyte Biology

Short-term delay of Fas-stimulated apoptosis by GM-CSF as a result of temporary suppression of FADD recruitment in neutrophils: evidence implicating phosphatidylinositol 3-kinase and MEK1-ERK1/2 pathways downstream of classical protein kinase C

Yasuko Kotone-Miyahara*, Kouhei Yamashita*, Kyung-Kwon Lee{dagger}, Shin Yonehara{dagger}, Takashi Uchiyama*, Masataka Sasada{ddagger} and Atsushi Takahashi*,1

* Department of Hematology and Oncology, Graduate School of Medicine,
{dagger} Graduate School of Biostudies, and
{ddagger} College of Medical Technology, Kyoto University, Japan

1 Correspondence: Division of Hematology and Oncology, Department of Internal Medicine, Kyoto University Hospital, 54 Shogoin Kawara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: atakahashi{at}ca.wakwak.com


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ABSTRACT
 
Granulocyte/macrophage colony-stimulating factor (GM-CSF) inhibits Fas-induced apoptosis of neutrophils. However, the exact step in the apoptotic pathway blocked by GM-CSF remained unclear. Here, we found that pretreatment of neutrophils with GM-CSF inhibits the recruitment of Fas-associated protein with death domain (FADD) to Fas, abolishing the formation of the death-inducing signaling complex required for Fas-induced apoptosis. Two-dimensional electrophoresis revealed that GM-CSF modifies the ratio of FADD subspecies. These GM-CSF-triggered changes were abrogated, and Fas-induced apoptosis was restored by an inhibitor of classical protein kinase C (PKC), Gö6976, and by the combination of a phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002, and an inhibitor of mitogen-activated protein kinase kinase (MEK)1, PD98059. Gö6976 blocked GM-CSF-elicited phosphorylation of Akt/PKB and extracellular signal-regulated kinase (ERK)1/2. These results indicated that GM-CSF suppresses Fas-induced neutrophil apoptosis by inhibiting FADD binding to Fas, through redundant actions of PI-3K and MEK1-ERK1/2 pathways downstream of classical PKC.

Key Words: death-inducing signaling complex • Gö6976 • LY294002 • PD98059 • caspase-8


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INTRODUCTION
 
Elimination of neutrophils by apoptotic cell death plays a crucial role in the resolution of inflammatory responses [1 ]. Various proapoptotic and antiapoptotic signals at the site of inflammation can interact on neutrophils and regulate their survival [2 ]. Ligation of death receptors, Fas/APO-1/CD95, and tumor necrosis factor receptor 1 (TNFR1) triggers neutrophil apoptosis, and proinflammatory cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF) antagonize their proapoptotic effects [3 4 5 ]. Macrophages release soluble Fas ligand (sFasL) together with undetermined soluble factor(s) and trigger Fas-dependent neutrophil apoptosis [6 ]. Dysregulation of the balance between proapoptotic and antiapoptotic signals can lead to prolonged inflammation and tissue damage. Neutrophils from patients with systemic inflammatory response syndrome show a blunted response to Fas ligation [7 ], suggesting that the predominance of antiapoptotic signals leads to such a serious pathophysiological state.

Activated death receptors such as Fas, TNF-related apoptosis-inducing ligand (TRAIL) receptor 1/death receptor 4 (DR4), and TRAIL receptor 2/DR5 form a complex called death-inducing signaling complex (DISC), containing death receptors, adapters such as Fas-associated death domain protein (FADD)/mediator of receptor-induced toxicity-1 (MORT1), and procaspase-8 [8 9 10 11 12 13 ], which when recruited to the DISC, is activated by dimerization and autoprocessing [14 ] and released to the cytoplasm. In type I cells [8 ], the activated "initiator" caspase-8 triggers a caspase cascade that activates downstream "effector" caspases such as caspase-3 and -6, which mediate apoptotic biochemical and morphological changes [15 , 16 ]. In type II cells, caspase-8 cleaves Bid, which induces mitochondrial release of cytochrome c [17 ], and Smac/DIABLO [18 ], resulting in the activation of another initiator caspase-9, which also triggers a caspase cascade.

Although a previous report proposed that GM-CSF inhibits Fas-stimulated neutrophil apoptosis at a step prior to caspase-8 activation [4 ], the exact process inhibited by GM-CSF remained to be clarified. Moreover, signaling from the GM-CSF receptor, which mediates suppression of Fas-induced apoptosis, was totally unknown. In the present study, we found that GM-CSF inhibits Fas-induced DISC formation by interfering with the recruitment of FADD to Fas. Two-dimensional gel electrophoresis revealed that FADD is post-translationally modified in GM-CSF-treated neutrophils. Inhibitor studies suggested that these GM-CSF-induced effects are mediated by redundant phosphatidylinositol 3-kinase (PI-3K) and mitogen-activated protein kinase kinase 1 (MEK1)-extracellular signal-regulated kinase (ERK)-1/2 pathways, acting downstream of classical protein kinase C (PKC). These results provide novel insights into the molecular mechanisms through which proapoptotic and antiapoptotic inflammatory signals interact in neutrophils.


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MATERIALS AND METHODS
 
Reagents
Gö6976, LY294002, and PD98059 (Calbiochem, San Diego, CA) were dissolved in dimethyl sulfoxide at 1, 10, and 100 mM, respectively, and stored at –20°C. GM-CSF was kindly provided by Schering-Plough Research Institute (Kenilworth, NJ), dissolved in Hanks’ balanced saline solution at 100 µg/ml, and stored at –20°C. Agonistic anti-human Fas immunoglobulin M (IgM) monoclonal antibody (mAb; CH-11) was purchased from Kamiya Biomedical Co. (Seattle, WA). Sources and stock solutions of other reagents were described previously [5 , 19 , 20 ].

Isolation and analysis of neutrophils
Human neutrophils were isolated from peripheral blood (PB) of healthy adult volunteers by sedimentation through two-step Percoll (Amersham Biosciences Co., Piscataway, NJ) gradients as described previously [21 ]. Freshly isolated neutrophils were resuspended in RPMI 1640, supplemented with 10% fetal bovine serum, and treated with the reagents indicated at 37°C in a humidified atmosphere containing 5% CO2. Flow cytometric analyses of neutrophil apoptosis were performed using propidium iodide staining of ethanol-permeabilized cells for DNA fragmentation as described previously [19 ].

Flow cytometric analysis of the surface expression of Fas
Neutrophils (2x106) were preincubated with pooled, heat-inactivated human AB group serum for 15 min on ice to inhibit nonspecific binding of antibodies. The cells were then washed with phosphate-buffered saline (PBS) and preincubated with a fluorescein isothiocyanate (FITC)-conjugated anti-CD95 (Fas) antibody (BD PharMingen, San Diego, CA) for 30 min on ice with protection from light. Cells were washed twice with PBS containing 0.1% bovine serum albumin and 0.1% sodium azide and were subjected to flow cytometry. FITC-conjugated mouse IgG1 was used as controls.

Immunoblotting
Whole cell extracts were obtained by boiling neutrophils fixed with 10% trichloroacetic acid in sodium dodecyl sulfate (SDS) sample buffer for 4 min. Proteins were resolved on SDS-polyacrylamide gels, transferred to Immobilon-P membranes (Millipore, Bedford, MA), stained, and visualized by enhanced chemiluminescence as described previously [19 ]. Primary antibodies used for these studies included: rabbit polyclonal antibody against the cytoplasmic region of human Fas (HF-1; kindly provided by Dr. Shigekazu Nagata, Osaka University Medical School, Japan) [22 ], ERK1 (Santa Cruz Biotechnology, Santa Cruz, CA), Akt1/PKB{alpha} phosphorylated at Ser473, and ERK1/2 phosphorylated at Thr202/Tyr204 (New England Biolabs, Beverly, MA); mouse mAb against FADD (Transduction Laboratories, Lexington, KY), caspase-8 (MBL, Nagoya, Japan), and actin (Roche Molecular Biochemicals, Indianapolis, IN); goat polyclonal antibody against Akt1 and ERK2 (Santa Cruz Biotechnology); and phosphoserine detection kit and phosphothreonine detection kit (nanoTools, Tenningen, Germany).

Immunoprecipitation of DISC
Cells suspended at a density of 2 x 107 cells/ml were stimulated with 1 µg/ml anti-Fas mAb (CH-11) plus 10 µg/ml cycloheximide (CHX) and incubated for the time indicated at 37°C. Then, cells were washed with ice-cold PBS, resuspended in 1 ml lysis buffer (30 mM Tris HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 10 mM EDTA, 10 mM EGTA, 100 mM NaF, 2 mM Na3VO4, 2 mM phenylmethylsulfonyl fluoride, 100 µM di-isopropylfluorophosphate, 1 µM calpeptin, 100 µg/ml aprotinin, and 10 µg/ml each chymostatin, leupeptin, antipain, and pepstatin), incubated at 4°C for 60 min with gentle inversion, and centrifuged for 10 min at 14,000 g at 4°C. The supernatants were obtained and incubated with 20 µl protein A-Sepharose beads (Amersham Biosciences) plus 2 µg/ml humanized anti-Fas mAb (HFE7A) [23 , 24 ] overnight at 4°C. Beads were washed five times with 0.5 ml ice-cold lysis buffer. For immunoblotting, proteins were eluted by boiling the beads in 10 µl 1x SDS sample buffer containing ß-mercaptoethanol.

Two-dimensional gel analysis of FADD
Neutrophils (1x108) were left untreated or treated with various reagents for 30 min at 37°C. Then, cells were lysed as described above for DISC immunoprecipitation. FADD was immunoprecipitated by incubation of the cell lysate with 20 µl protein A-Sepharose beads and 4 µg/ml anti-FADD mAb (BD PharMingen) overnight at 4°C. FADD was eluted by incubating the beads with 125 µl sample buffer {8 M urea, 2% 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate, 2% immobilized pH gradient buffer (pH 4–7; Amersham Biosciences), 18 mM dithiothreitol for 1 h at room temperature}. The first dimension isoelectric focusing was carried out using a Immobiline DryStrip (pH 4–7, 7 cm; Amersham Biosciences) and Multiphor II electrophoresis system (Amersham Biosciences) according to the manufacturer’s instructions. The second dimension, SDS-polyacrylamide gel electrophoresis (PAGE), was performed by placing the focused gel strip on the top of a 15% minigel. FADD was transferred to an Immobilon-P membrane by semidry transfer, stained, and visualized as described above.


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RESULTS
 
GM-CSF inhibition of Fas-induced procaspase-8 processing is mediated by classical PKCs
GM-CSF inhibited neutrophil apoptosis triggered by CH-11 anti-Fas IgM mAb (Fig. 1A ) as previously reported [3 , 4 ]. To clearly dissect the mechanism through which GM-CSF inhibits Fas-mediated apoptosis, we added CHX to facilitate Fas-induced death signaling. The Fas/CHX-induced apoptosis was also inhibited by GM-CSF (Fig. 1A) . The prominent suppression of Fas/CHX-induced neutrophil apoptosis by GM-CSF and the small degree of spontaneous apoptosis up to 3 h of incubation allowed us to perform inhibitor studies. We noticed that staurosporine (STS), a protein kinase inhibitor with a relatively broad spectrum [25 ] at 100 nM prevented the GM-CSF inhibition of Fas/CHX-induced apoptosis (Fig. 1A) . At this concentration, STS did not affect spontaneous or Fas/CHX-induced neutrophil apoptosis in the absence of GM-CSF (data not shown). It has been reported that Fas-induced activation of protease activity cleaving Ile-Glu-Thr-Asp-7-amino-4-trifluoromethylcoumarin (IETD-AFC), a relatively specific substrate for caspase-8, is suppressed in GM-CSF-treated neutrophils [4 ]. Based on this observation, the report proposes that GM-CSF inhibits Fas-stimulated apoptosis at a step prior to caspase-8 activation. As IETD-AFC can also be cleaved by other caspases [26 ], and it is possible that GM-CSF inhibits protease activity of processed caspase-8 rather than processing of procaspase-8, we examined the effect of GM-CSF on Fas-induced procaspase-8 processing. As shown in Figure 1B , GM-CSF suppressed Fas/CHX-induced processing of procaspase-8 (lane 3). The impaired procaspase-8 processing was reversed by STS (Fig. 1B , lane 4). Next, to test the possibility that GM-CSF down-regulates Fas as reported for TNFR1 [27 ], surface expression of Fas was analyzed by flow cytometry. As shown in Figure 2 , the level of Fas on neutrophils was not affected by GM-CSF or STS. These results suggest that GM-CSF inhibits Fas/CHX-induced neutrophil apoptosis at a step between Fas and procaspase-8 processing in a kinase-dependent manner.



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  		Figure 1. GM-CSF inhibits Fas/CHX-induced apoptosis upstream of procaspase-8 processing. Neutrophils (4x106/ml) were pretreated for 30 min with or without GM-CSF (100 ng/ml) in the absence or presence of staurosporine (STS; 100 nM). Then, cells were left untreated or stimulated with CH-11 anti-Fas mAb (100 ng/ml; Fas) in the absence or presence of CHX (10 µg/ml) for 3 h at 37°C. (A) Apoptotic neutrophils with hypodiploid DNA content were measured as described in Materials and Methods (mean±SEM of three independent experiments; *, P<0.001; **, P<0.01). (B) Immunoblotting was performed with antiprocaspase-8 antibody (1:1000 dilution).



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  		Figure 2. GM-CSF and STS do not affect Fas expression. Neutrophils (1x107/ml) were left untreated or treated with GM-CSF (100 ng/ml) and/or STS (100 nM) for 30 min at 37°C. Surface expression of Fas is indicated by shaded lines. Control cells stained with FITC-conjugated mouse IgG1 are shown as open lines.

As we tested various inhibitors of protein kinases, we found that Gö6976, an inhibitor of classical PKCs, highly selective for PKC{alpha} and PKCß isoforms [28 ], reverses the GM-CSF inhibition of Fas/CHX-induced apoptosis in a dose-dependent manner (Fig. 3A and 3B ). It is interesting that in parallel experiments, GM-CSF suppression of spontaneous apoptosis was not reversed by Gö6976 (Fig. 3A) , and GM-CSF inhibition of CHX-induced apoptosis was reversed (Fig. 3C) . In the absence of GM-CSF, Gö6976 did not significantly affect Fas/CHX-induced apoptosis (Fig. 3B) . Gö6976 alleviated the GM-CSF inhibition of procaspase-8 processing (Fig. 3D) . No reversal of GM-CSF inhibition of Fas/CHX-induced apoptosis was observed with AG490 (Janus kinase 2 inhibitor), wortmannin, LY294002 (PI-3K inhibitors), and PD98059 (a MEK1 inhibitor; data not shown), which potentially interfere with signaling pathways activated by GM-CSF. This inhibitor profile is distinct from those of GM-CSF suppression of spontaneous [29 30 31 ] and CHX-induced [32 ] apoptosis. The results suggest that GM-CSF inhibits Fas/CHX-induced neutrophil apoptosis predominantly through classical PKCs.



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  		Figure 3. GM-CSF inhibits FADD recruitment to Fas in a classical PKC-dependent manner. Neutrophils were pretreated with or without GM-CSF in the absence or presence of Gö6976 at the concentrations indicated (A) or at 5 µM (B; C, D, and F, lane 4) for 30 min before stimulation with CHX alone (10 µg/ml; C) or with CH-11 (100 ng/ml) plus CHX (10 µg/ml; Fas/CHX; A, B, and D–F) for the times indicated (E), for 3 h (A–C; D, lanes 2–4), or for 30 min (F). Apoptotic cells with hypodiploid DNA content were measured (A; B and C, mean±SEM of three independent experiments; *, P<0.03), and immunoblotting for procaspase-8 was performed (D) as in Figure 1 . The asterisks in D represent nonspecific, cross-reactive proteins. (E and F) DISC was immunoprecipitated (IP) from neutrophils (2x107 cells/lane) as described in Materials and Methods, subjected to immunoblotting, and the blots were cut to be probed for FADD (1:500 dilution of the antibody), procaspase-8 (1:3000 dilution of the antibody), and Fas (1:1000 dilution of the HF-1 antibody). Data shown in A and D–F represent three independent experiments with essentially identical results.

GM-CSF suppresses FADD recruitment and DISC formation through classical PKCs
The data indicating that GM-CSF interferes with a step between Fas and procaspase-8 processing prompted us to analyze the Fas-induced DISC formation that triggers autoprocessing of procaspase-8 [14 , 33 ]. Enhancement of Fas death signals with CHX and various technical improvements described in Materials and Methods enabled us to immunoprecipitate DISC from neutrophils. No significant recruitment of FADD to Fas was detected when neutrophils were treated with anti-Fas antibody alone or with CHX alone (data not shown). Recruitment of FADD and procaspase-8 to Fas peaked at 30 min after Fas/CHX stimulation (Fig. 3E , lane 3). Although DISC formation has been analyzed in only a few types of primary cells, the DISC formation in neutrophils seems to be relatively slow compared with other cell types such as T lymphocytes [34 ]. It is interesting that GM-CSF abolished the FADD recruitment to Fas (Fig. 3F , lane 3), indicating that this initial step in the DISC formation is the target for GM-CSF inhibition of Fas/CHX-induced apoptosis. The GM-CSF inhibition of FADD recruitment was reversed by Gö6976 (Fig. 3F , lane 4), suggesting that classical PKCs mediate the inhibitory effect of GM-CSF.

Modifications of FADD by GM-CSF
To gain insights into the mechanism through which GM-CSF blocks the FADD recruitment, we analyzed FADD protein by two-dimensional gel electrophoresis. In unstimulated neutrophils, five spots of FADD proteins were discerned (Fig. 4A , left upper panel). The spot 1 was the most predominant species, followed by the spots 3, 2, 4, and 5. As previous studies have demonstrated serine/threonine phosphorylation of FADD [35 , 36 ], we examined whether the FADD subspecies in neutrophils represent differentially phosphorylated forms of FADD protein. Immunoblotting analyses using mixtures of antiserine or antithreonine antibodies detected serine/threonine phosphorylation of the spot 2 alone (Fig. 4B) . GM-CSF caused a decrease in the spot 1 and an increase in the spot 3 (Fig. 4A , right upper panel). Consequently, the spot 3 became the most predominant species in GM-CSF-treated neutrophils. The spot 2 was not affected by GM-CSF. The amounts of the spots 4 and 5 were too small to consistently evaluate their changes. These modifications of FADD by GM-CSF were alleviated by Gö6976 (Fig. 4A , left lower panel), restoring the spot 1:spot 3 ratio above 1. Activation of PKCs with PMA caused FADD alterations essentially identical to GM-CSF (Fig. 4A , right lower panel). These results suggest that FADD is post-translationally modified by GM-CSF and that the modification is mediated by classical PKCs.



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  		Figure 4. FADD modifications by GM-CSF revealed by two-dimensional gel electrophoresis. (A) Neutrophils (1x108) were left untreated, treated with GM-CSF (100 ng/ml) in the absence or presence of Gö6976 (5 µM), or treated with phorbol myristate acetate (PMA; 10 ng/ml) for 30 min. FADD was immunoprecipitated (IP), resolved by two-dimensional gel electrophoresis, and detected by immunoblotting (IB; 1:500 dilution of the antibody). The small inset shows an overexposed film for better resolution of the spot 5. The data shown represent five independent experiments with essentially identical results. (B) FADD was immunoprecipitated from neutrophils left untreated for 30 min. Two-dimensional immunoblots were probed for proteins, phosphorylated at serines or at threonines.

We postulated that the GM-CSF inhibition of FADD recruitment is a result of a decrease in FADD species that can be recruited to DISC. To test this assumption, we examined which of the FADD subspecies participates in DISC formation. DISC was immunoprecipitated from Fas/CHX-stimulated neutrophils, and FADD in the DISC was analyzed by two-dimensional gel electrophoresis. We found that the spots 1, 3, 4, and 5 participate in the DISC formation (Fig. 5 ). The spot 2, which was not modified by GM-CSF (Fig. 4 , right upper panel), was not detected in the DISC. Contradictory to our expectation, the spot 3, which became predominant in the presence of GM-CSF (Fig. 4 , right upper panel), was more efficiently recruited to the DISC than the spot 1 (Fig. 5) . This observation suggests that the impaired FADD recruitment to Fas in the presence of GM-CSF is mediated by more complex mechanisms. Consistent with the ability of GM-CSF to abolish the DISC formation (Fig. 3F , lane 3), no FADD spot was coprecipitated with Fas when neutrophils were Fas/CHX-stimulated in the presence of GM-CSF (data not shown).



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  		Figure 5. Selected FADD subspecies are present in the DISC. Fas/CHX stimulation of neutrophils (1x108) for 30 min, immunoprecipitation (IP) of DISC, two-dimensional gel electrophoresis, and immunoblot (IB) detection of FADD were performed as described in Materials and Methods. Visualized spots are indicated by arrowheads as in Figure 4 . Spot 5 is visible in overexposed films (data not shown). The data shown represent three independent experiments with essentially identical results. IEF, Isoelectric focusing.

PI-3K and MEK1-ERK1/2 pathways are activated downstream of PKCs in GM-CSF-treated neutrophils and mediate FADD modifications and impaired FADD recruitment to Fas
Despite previous reports in other cell types that GM-CSF has antiapoptotic actions through PKC{alpha} [37 ], mediator(s) downstream of this classical PKC remained to be determined. PKCs are a family of serine/threonine protein kinases composed of at least 12 isozymes. They are classified into three subgroups with distinct tissue distribution, activators, and substrates: classical or conventional PKCs including {alpha}, ßI, ßII, and {gamma} isotypes; novel PKCs including {delta}, {epsilon}, {theta}, {eta}, and µ isotypes; and atypical PKCs including {zeta}, {iota}, and {lambda} isotypes [38 ]. Among classical PKCs, PKC{alpha}, PKCßI, and PKCßII are expressed in neutrophils [39 ]. Our observations below suggest that the GM-CSF signaling downstream of the classical PKCs responsible for suppression of Fas-induced apoptosis is mediated by PI-3K and MEK1-ERK1/2 pathways.

During the efforts to pursue agents that alleviate the GM-CSF inhibition of apoptosis, we noticed that the combination of LY294002 and PD98059 attenuates the GM-CSF inhibition of Fas/CHX-induced apoptosis (Fig. 6A ) and FADD recruitment (Fig. 6B) in neutrophils. In the absence of GM-CSF, LY294002 plus PD98059 did not affect Fas/CHX-induced apoptosis (Fig. 6A) .



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  		Figure 6. Redundant PI-3K and MEK1-ERK1/2 pathways act downstream of classical PKCs in GM-CSF blockade of Fas/CHX-induced apoptosis. (A and B) Neutrophils were pretreated with or without GM-CSF in the absence or presence of LY294002 (10 µM) plus PD98059 (50 µM) for 30 min before Fas/CHX stimulation for 3.5 h (A) or 30 min (B). (A) Apoptotic cells with hypodiploid DNA content were measured as in Figure 1A . Data shown are mean ± SEM of four independent experiments (*, P<0.04). (B) DISC was immunoprecipitated (IP) and immunoblotted for FADD and Fas. (C and D) Neutrophils (4x106) were left untreated or treated with GM-CSF (100 ng/ml) or with PMA (10 ng/ml) for 30 min in the absence or presence of Gö6976 (5 µM), LY294002 (10 µM), or PD98059 (50 µM). Whole cell extracts were immunoblotted and stained for phosphorylated Akt (1:1000 dilution; C, upper panels), total Akt (1 µg/ml; C, lower panels), phosphorylated ERK1/2 (1:1000 dilution; D, top panel), ERK2 (1 µg/ml; D, middle panel), and ERK1 (1 µg/ml; D, bottom panel).

Although GM-CSF is known to activate PKCs [40 ], and PKCs can activate the PI-3K-Akt and MEK1-ERK1/2 pathways in neutrophils [41 ], it remained to be clarified whether PKCs mediate GM-CSF-induced activation of PI-3K and ERK1/2. GM-CSF triggered LY294002-inhibitable phosphorylation of Akt/PKB (Fig. 6C , lanes 2 and 4), which indicates activation of PI-3K. Inhibition of classical PKCs with Gö6976 abolished the GM-CSF-induced Akt phosphorylation (Fig. 6C , lane 3), suggesting that classical PKCs mediate the GM-CSF-triggered activation of PI-3K in neutrophils. Likewise, Gö6976 potently inhibited GM-CSF-induced, PD98059-inhibitable phosphorylation of ERK1/2 (Fig. 6D , lane 3), suggesting that the MEK1-ERK1/2 pathway is activated by GM-CSF through classical PKCs. The suppression of PI-3K and MEK1-ERK1/2 pathways explains the ability of Gö6976 to reverse the GM-CSF inhibition of CHX-induced apoptosis (Fig. 3C) , which requires both pathways [32 ]. GM-CSF-induced Akt phosphorylation was not affected by PD98059 (Fig. 6C , lane 5) nor did LY294002 affect the GM-CSF-induced ERK1/2 phosphorylation (Fig. 6D , lane 4), indicating that PI-3K and MEK1-ERK1/2 are present on independent, parallel pathways. PMA induced phosphorylation of Akt, which was inhibited by LY294002 in human PB neutrophils (Fig. 6C , lanes 7 and 8), as previously reported for mouse bone marrow neutrophils [41 ]. PMA also triggered ERK1/2 phosphorylation, which was inhibited by PD98059 (Fig. 6D , lanes 7 and 9), suggesting that the PKC-cRaf-1-MEK1-ERK1/2 pathway [42 43 44 ] can be activated by PKCs in human PB neutrophils. Taken together, the results support the notion that classical PKCs mediate the GM-CSF activation of parallel PI-3K and MEK1-ERK1/2 pathways, which inhibit Fas/CHX-induced neutrophil apoptosis in a redundant way.

We next assessed whether the GM-CSF-triggered FADD modifications, which are also mediated by classical PKCs (Fig. 4A) , are dependent on or independent of the parallel PI-3K and MEK1-ERK1/2 pathways. As shown in Figure 7 , the GM-CSF-elicited FADD modifications were alleviated by the combination of LY294002 and PD98059, which alone, failed to affect the GM-CSF-induced FADD modifications (data not shown). These suggest that the parallel PI-3K and MEK1-ERK1/2 pathways converge on the FADD modifications. Although the inability to transfect mature neutrophils hindered us from performing the types of experiments such as exogenous expression of active MEK1, our result supports the notion that the PI-3K or MEK1-ERK1/2 pathway is sufficient for the FADD alterations, suppression of Fas/CHX-induced FADD recruitment, and blockade of Fas/CHX-induced apoptosis in GM-CSF-treated neutrophils.



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  		Figure 7. FADD modifications by GM-CSF are alleviated by combinatory blockade of PI-3K and MEK1-ERK1/2 pathways. Neutrophils were left untreated or treated with GM-CSF in the absence or presence of LY294002 (10 µM) plus PD98059 (50 µM). Two-dimensional gel analyses of FADD were performed as in Figure 4A . The data shown represent five independent experiments with essentially identical results. IP, Immunoprecipitation; IB, immunoblotting.

The effects of GM-CSF on FADD are reversible
We noticed that the inhibitory effect of GM-CSF was diminished markedly after 3 h of Fas/CHX stimulation (Fig. 8A ). This is consistent with a previous report that activated Fas blocks GM-CSF-induced antiapoptotic signaling in neutrophils, most likely through Src homology-2-containing tyrosine phosphatase 1 (SHP-1), which binds and inactivates Lyn tyrosine kinase [45 ]. The result prompted us to investigate whether the GM-CSF-induced changes in the Fas death pathway are canceled on subsequent Fas/CHX stimulation. As shown in Figure 8B , Fas/CHX stimulation of GM-CSF-treated neutrophils caused delayed recruitment of FADD and procaspase-8 to Fas after 150 min (lane 3). This delayed DISC formation is consistent with the onset of apoptosis in GM-CSF-treated neutrophils after 3 h of Fas/CHX stimulation (Fig. 8A) . We further examined whether the GM-CSF-induced FADD modifications are reversed over the same time course. Although the spot 3 was the most predominant FADD species after GM-CSF stimulation, the spot 1 became the most predominant, as in GM-CSF-untreated cells, after 150 min of Fas/CHX stimulation (Fig. 8C) . These results indicate that GM-CSF-induced effects on Fas death signaling are not persistent. This reversible nature of GM-CSF signals accounts for the observation in the present study that antiapoptotic GM-CSF effects were eventually overcome by irreversible, Fas-induced apoptotic processes involving proteolytic events.



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  		Figure 8. Reversal of FADD modifications and delayed DISC formation after Fas/CHX stimulation of GM-CSF-treated neutrophils. (A) Neutrophils pretreated for 30 min with (dashed lines) or without (solid lines) GM-CSF were left untreated (open symbols) or Fas/CHX-stimulated for the times indicated. Apoptotic neutrophils with hypodiploid DNA content were measured. (B and C) Neutrophils pretreated with GM-CSF for 30 min were Fas/CHX-stimulated for the time indicated. (B) DISC was immunoprecipitated (IP) and immunoblotted (IB) for FADD, procaspase-8, and Fas. (C) Two-dimensional gel analyses of FADD were performed as in Figure 4A .


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DISCUSSION
 
Although GM-CSF activates PKCs [40 ], and PKCs can inhibit Fas-induced apoptosis by inhibiting FADD recruitment to Fas [46 , 47 ] in cell lines, it remained to be determined whether GM-CSF uses this PKC-mediated mechanism when it inhibits Fas-induced apoptosis in normal cells such as neutrophils. In the present study, we demonstrate that GM-CSF inhibits Fas-induced neutrophil apoptosis by interfering with the FADD recruitment via classical PKCs. We then go on to provide evidence that parallel PI-3K and MEK1-ERK1/2 pathways are activated downstream of classical PKCs and mediate the GM-CSF suppression of FADD recruitment. Also, two-dimensional gel electrophoretic analyses display that GM-CSF causes characteristic, post-translational modifications of FADD through the same pathways.

It should be noticed that compelling evidence remains to be provided on the issue of whether GM-CSF activates PKCs in neutrophils. Although GM-CSF has been shown to activate PKCs in U937 cells [40 ], neither the production of diacylglycerol [48 ] nor the translocation of PKCs [49 ] has been detected in GM-CSF-treated neutrophils. Our observations might be compatible with the notion that GM-CSF does not increase PKC activity, and instead, the baseline PKC activity has a permissive role in the activation of PI-3K and MEK1-ERK1/2 pathways and in the inhibition of FADD recruitment to Fas by GM-CSF. Furthermore, Gö6976 action on a target distinct from classical PKCs [50 ] might provide a plausible explanation for the results in the present study. Nevertheless, the observation that GM-CSF causes Gö6976-inhibitable FADD modifications essentially identical to PMA supports the view that the GM-CSF effects on neutrophils are mediated by activation of classical PKCs.

GM-CSF inhibition of Fas/CHX-induced neutrophil apoptosis displayed inhibitor profiles, which are distinct from that of spontaneous apoptosis. In our experiments, the GM-CSF suppression of spontaneous neutrophil apoptosis was not attenuated by blockade of classical PKCs with Gö6976. It has been reported that LY294002 or PD98059 alone attenuates GM-CSF delay of spontaneous apoptosis, and the combination of these inhibitors produces no additive effect [29 ], although it remains controversial whether PD98059 significantly suppresses the GM-CSF survival effect [31 ]. AG490 partially inhibits the GM-CSF delay of spontaneous apoptosis [30 ]. In contrast, LY294002, PD98059, or AG490 alone failed to reverse the GM-CSF inhibition of Fas/CHX-induced apoptosis. Only the combination of LY294002 plus PD98059 could reverse the GM-CSF inhibition. It is likely that the basic mechanisms are different between spontaneous and Fas-induced neutrophil apoptosis [51 ], and both types of apoptosis are regulated by GM-CSF in distinct manners.

The data presented imply that the PI-3K or MEK1-ERK1/2 pathway is sufficient to suppress FADD recruitment to Fas. Further work should be directed at determining the underlying molecular mechanisms. Recruitment of procaspase-8, rather than FADD, to the DISC is impaired in the Akt1 transgenic T cells [52 ], suggesting that a downstream effector(s) of the PI-3K distinct from Akt1 mediates the GM-CSF blockade of FADD recruitment. An attractive possibility is that the PI-3K pathway interferes with the DISC formation by remodeling the actin cytoskeleton of neutrophils [53 , 54 ]. The actin filaments are required for the recruitment of FADD to Fas [55 ] and for efficient induction of apoptosis by anti-Fas mAb [56 ] and sFasL [55 ] in various cell types. It is difficult to conceive the possible mechanism through which the MEK1-ERK1/2 pathway suppresses the FADD recruitment. It also remains unsettled whether the MEK1-ERK1/2 and the PI-3K pathways work through the same or different mechanisms. We noticed that ERK2 coimmunoprecipitates with FADD in the absence or presence of GM-CSF (Y. Kotone-Miyahara and A. Takahashi, unpublished observations); however, its significance remains to be clarified. GM-CSF has been shown to increase the stability of antiapoptotic Mcl-1 protein in CHX-treated neutrophils through MEK1-ERK1/2 and PI-3K pathways [32 ]. However, PD98059 or LY294002 alone completely abrogates the GM-CSF-mediated increase in Mcl-1 stability [32 ], in contrast to the GM-CSF inhibition of Fas/CHX-induced apoptosis. Moreover, direct evidence is yet to be provided whether Mcl-1 mediates the ability of GM-CSF to inhibit neutrophil apoptosis.

It is interesting that as many as five FADD subspecies were detected in neutrophils. Among the subspecies resolved on two-dimensional gels, only the spot 2 was stained with antiphosphoserine and antiphosphothreonine antibodies. Elucidation of the nature of post-translational modifications awaits more precise characterization. The ability of GM-CSF to modify the ratio of FADD subspecies raises the possibility that these FADD alterations are responsible for the impaired FADD recruitment to Fas. It cannot be excluded that the impaired FADD recruitment is ascribed to alterations of Fas [57 ] and/or modifications of FADD distinct from those revealed by our two-dimensional gel analyses. Nonetheless, it should be noted that the GM-CSF-induced FADD modifications and the GM-CSF blockade of FADD recruitment have been located at the same point in the complex GM-CSF signaling pathways. Moreover, delayed recruitment of FADD to Fas was observed in GM-CSF-pretreated, Fas/CHX-stimulated neutrophils in parallel with the reversal of FADD modifications. Although our findings are in conflict with a simple hypothesis that GM-CSF down-regulates the FADD subspecies effectively recruited to the DISC, this does not exclude the possibility that the FADD modifications have an adverse effect on FADD recruitment by a more complex mechanism. It has been reported that a post-translational modification of the FADD protein disrupts its function in cell-cycle progression of T lymphocytes [58 ]. Integrated efforts are required to elucidate the effects of the GM-CSF-induced FADD alterations on various FADD-mediated processes [59 ].

The present results suggest that reversible GM-CSF antiapoptotic effects can be quickly overcome by strong Fas death signals. Future work should be directed at determining many parameters and variables in vivo that can affect the relative strength of GM-CSF and Fas signals, including local concentrations of GM-CSF and sFasL, kinetics of binding to cells expressing membrane-bound Fas ligand, and simultaneous exposure to other proapoptotic and apoptotic cytokines. Complemented by such approaches, this study should shed considerable light on interwound cross-talks between various survival and death signals underlying physiological and pathological events in vivo.

FADD functions as a proapoptotic adaptor that links procaspase-8 directly or indirectly to death receptors [11 , 12 , 60 61 62 63 ]. FADD has also been implicated in apoptosis induced by a variety of exogenous and endogenous signals including lipopolysaccharide [64 ], CHX [65 ], nonsteroidal, anti-inflammatory drugs [66 ], dsRNA-dependent protein kinase [67 ], Ras [68 ], and phosphatase and tensin homologue (PTEN) [69 ]. It also has nonapoptotic functions, such as embryonic development and T cell proliferation [59 60 61 ]. The present study adds further support to the idea that FADD acts as a versatile integrator of multiple intracellular and extracellular signals.


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ACKNOWLEDGEMENTS
 
We thank Dr. Shigekazu Nagata for rabbit polyclonal antibody against Fas, Susumu Kobayashi and Tomoharu Takeoka for technical and intellectual support, and Junko Hirai, Yukari Asano-Ashida, and Kaori Miyata for secretarial assistance.

Received January 28, 2004; revised June 30, 2004; accepted July 23, 2004.


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