Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.1104690 on April 21, 2005

Published online before print April 21, 2005
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(Journal of Leukocyte Biology. 2005;78:301-309.)
© 2005 by Society for Leukocyte Biology

Type I and type II interferons delay human neutrophil apoptosis via activation of STAT3 and up-regulation of cellular inhibitor of apoptosis 2

Erina Sakamoto*, Fumihiko Hato*, Takayuki Kato*, Chikahiko Sakamoto*, Mika Akahori*, Masayuki Hino{dagger} and Seiichi Kitagawa*,1

* Departments of Physiology and
{dagger} Clinical Hematology, Osaka City University Graduate School of Medicine, Japan

1 Correspondence: Department of Physiology, Osaka City University Graduate School of Medicine, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail: kitagawas{at}med.osaka-cu.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have recently demonstrated that granulocyte-colony stimulating factor (G-CSF) delays human neutrophil apoptosis via up-regulation of cellular inhibitor of apoptosis 2 (cIAP2), which is dependent on activation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Here, we show that type I and type II interferons (IFNs), which bind to the distinct receptors, exert the antiapoptotic effect on human neutrophils through the similar mechanism. IFN-{alpha} (type I IFN) and IFN-{gamma} (type II IFN), like G-CSF, delayed human neutrophil apoptosis through the protein synthesis-dependent mechanism. Stimulation of neutrophils with IFN-{alpha} or IFN-{gamma} resulted in tyrosine phosphorylation of STAT1 and STAT3 but not phosphorylation of STAT5, Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase. IFN-{alpha} and IFN-{gamma} induced the expression of transcripts of cIAP2 and suppressor of cytokine signaling 1 and 3, but not cIAP1, Mcl-1, and A1. IFN-{alpha}- and IFN-{gamma}-induced up-regulation of cIAP2 mRNA and protein, phosphorylation of STAT3, and antiapoptotic effect were inhibited significantly by pretreatment of cells with AG490, a specific inhibitor of JAK2. These findings suggest that cIAP2 expression is up-regulated by IFN-{alpha} and IFN-{gamma} through, at least in part, activation of the JAK2-STAT3 pathway, and increased expression of the cIAP2 protein may contribute to an IFN-{alpha}- and IFN-{gamma}-mediated antiapoptotic effect on human neutrophils.

Key Words: signal transducer and activator of transcription 3 • AG490 • granulocyte-colony stimulating factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human neutrophils are short-lived cells and undergo spontaneous apoptosis during culture. Spontaneous neutrophil apoptosis is delayed or accelerated in the presence of various cytokines such as granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, interferon-{alpha}/ß (IFN-{alpha}/ß), IFN-{gamma}, and tumor necrosis factor {alpha} (TNF-{alpha}) [1 2 3 4 5 6 ]. Modulation of neutrophil apoptosis by inflammatory cytokines may be closely associated with the outcome of inflammation. However, neither the mechanisms regulating spontaneous neutrophil apoptosis nor the mechanisms by which cytokines exert apoptotic or antiapoptotic effect on neutrophils are understood fully. Possibilities may include up- or down-regulation of molecules associated with apoptosis, which may include the Bcl-2 family proteins such as Bcl-XL, Mcl-1, A1, and Bax and the inhibitor of apoptosis (IAP) family proteins [1 , 7 8 9 10 ]. For example, it has been reported that G-CSF up-regulates the expression of cellular IAP2 (cIAP2), survivin, and A1 [1 , 7 , 10 ] and down-regulates the expression of Bax [8 ]. GM-CSF up-regulates the expression of Mcl-1 and survivin [3 , 4 , 10 ] and down-regulates the expression of Bax-{alpha} [9 ], whereas TNF-{alpha} down-regulates the expression of Bcl-XL [9 ]. However, the results reported are contradictory. In regard to the signaling pathways, Lyn, extracellular signal-regulated kinase (ERK), phosphatidylinositol-3 kinase (PI-3K), and Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) have been proposed to be involved in a GM-CSF-mediated antiapoptotic effect on human neutrophils [4 , 9 10 11 12 ]. Our recent study shows that G-CSF induces selective up-regulation of cIAP2 through activation of the JAK2-STAT3 pathway, which may contribute to a G-CSF-mediated antiapoptotic effect on human neutrophils [1 ].

IFNs play an important role in the host defense against invading microorganisms and help integrate early innate immune responses with later events mediated by the adaptive immune system. Two types of IFN with distinct immunological properties are recognized. The type I IFN is comprised of multiple IFN-{alpha} subtypes, IFN-ß, IFN-{varepsilon}, and IFN-{tau}, all of which are structurally related and bind to a common receptor. IFN-{gamma} is the sole type II IFN, structurally unrelated to type I IFN, and binds to a different receptor [13 , 14 ]. STAT1 activation is primarily required for many IFN-dependent responses. In addition, recent studies have demonstrated that the STAT1-independent signaling pathway also plays an important role in some IFN-dependent responses [15 ]. IFN-{gamma} is known to enhance neutrophil phagocytosis and microbicidal activity, to induce the expression of Fc receptor for immunoglobulin G (IgG) I and gp91-phox transcripts in neutrophils, and to exert the antiapoptotic effect on human neutrophils [16 17 18 ]. IFN-{alpha}/ß is also known to exert the antiapoptotic effect on human neutrophils [5 ]. However, the signals provoked by the binding of IFNs to their receptors on human neutrophils as well as the mechanisms by which IFNs exert the antiapoptotic effect on human neutrophils are largely unknown. In this paper, we show that IFN-{alpha} (type I IFN) and IFN-{gamma} (type II IFN) delay human neutrophil apoptosis through the protein synthesis-dependent mechanism, and the expression of cIAP2 is selectively up-regulated by stimulation with IFN-{alpha} or IFN-{gamma} at the protein as well as mRNA level. The results also show that IFN-{alpha}- or IFN-{gamma}-induced up-regulation of cIAP2 is mediated through, at least in part, activation of the JAK2-STAT3 pathway, and the increased expression of cIAP2 may contribute to IFN-{alpha}- or IFN-{gamma}-mediated antiapoptotic effect on human neutrophils.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Recombinant human (rh)G-CSF, IFN-{gamma}, and TNF-{alpha} produced by Escherichia coli were provided by Kirin Brewery (Tokyo, Japan), Shionogi Pharmaceutical (Osaka, Japan), and Dainippon Pharmaceutical (Osaka, Japan), respectively. rhGM-CSF and IFN-{alpha} (IFN-{alpha}-2b), produced by E. coli, were provided by Schering-Plough (Osaka, Japan). Endotoxin contamination of each preparation was <100 pg/mg protein. Ficoll and the enhanced chemiluminescence (ECL) Western blotting system were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Conray was purchased from Mallinckrodt (St. Louis, MO). Cycloheximide was purchased from Sigma Chemical Co. (St. Louis, MO). AG490, a JAK2 inhibitor, was purchased from Calbiochem (San Diego, CA). Rabbit polyclonal antibodies against Tyr701-phosphorylated STAT1, Tyr705-phosphorylated STAT3, Tyr694-phosphorylated STAT5, Thr202/Tyr204-phosphorylated ERK1/2, p38 mitogen-activated protein kinase (MAPK), Thr180/Tyr182-phosphorylated p38 MAPK, and Ser473-phosphorylated Akt and goat anti-rabbit IgG antibody, conjugated with horseradish peroxidase (HRP), were purchased from Cell Signaling Technology (Beverley, MA). Rabbit polyclonal antibody against cIAP2 was purchased from Santa Cruz Biotechnology (CA). Rabbit polyclonal antibody against Bax was purchased from PharMingen (San Diego, CA). Rabbit polyclonal antibody against suppressor of cytokine signaling 3 (SOCS3) was purchased from American Research Products (Belmont, MA). Goat polyclonal antibody (AF245) against human IFN-{alpha}/ß receptor 1 (IFNAR1) and normal goat IgG were purchased from R&D Systems (Minneapolis, MN).

Preparation of cells
Human neutrophils and mononuclear cells were prepared from healthy adult donors as described previously [1 ] using dextran sedimentation, centrifugation with Conray-Ficoll, and hypotonic lysis of contaminating erythrocytes. Neutrophil fractions contained >98% neutrophils. Lymphocytes were further purified from mononuclear cells by centrifugal elutriation in a Hitachi SRR6Y elutriation rotor (Hitachi, Tokyo, Japan), as described previously [1 ]. Lymphocyte fractions contained >99% lymphocytes. Cells were suspended in Hanks’ balanced salt solution (HBSS) containing 10 mM HEPES (pH 7.4). For the experiments with cell cultivation, cells were suspended in RPMI 1640 supplemented with 10% fetal calf serum (FCS).

Cell culture and determination of apoptosis
Neutrophils (5x106/ml) suspended in RPMI 1640 were cultivated in 5% CO2/95% humidified air at 37°C. When required, IFN-{alpha} (1000 U/ml), IFN-{gamma} (1000 U/ml), G-CSF (50 ng/ml), or AG490 (50 µM) was added to the culture medium. We used 50 µM AG490 throughout the present experiments, as the effect of 50 µM AG490 on human neutrophils and monocytes was essentially identical to that of 100 µM AG490 [1 , 2 , 19 ]. HL-60 cells were grown in RPMI 1640 supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml). DNA content in the cells was determined by propidium iodide (PI) staining and flow cytometry with FACSCalibur (Becton Dickinson, Mountain View, CA), as described previously [1 ]. For determination of DNA content, cells (1x106) were placed in 70% ethanol in phosphate-buffered saline and stored at –20°C until use. Cells were treated with DNase-free RNase (50 µg/ml) and PI (50 µg/ml) for 15 min at room temperature. Samples were kept at 4°C in the dark until analysis. Cells with decreased DNA content (hypodiploid cells) were considered as cells undergoing apoptosis-associated DNA degradation [1 ]. Total viable cells were counted using the trypan blue exclusion test, and the data are expressed as the percentage of seeded cells.

Western blotting
Human neutrophils, lymphocytes, or HL-60 cells suspended in HBSS were prewarmed for 10 min at 37°C and were then stimulated with cytokines for 10–60 min at 37°C. When required, cells were pretreated with AG490 (50 µM) for 30 min at 37°C before stimulation with cytokines. The reactions were terminated by rapid centrifugation, and the pellets were frozen in liquid nitrogen after aspiration of the supernatant. The cell pellets were resuspended in ice-cold solution containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, and 10 µg/ml leupeptin and were lysed for 10 min at 4°C. After rapid centrifugation, the supernatant was mixed 1:1 with 2x sample buffer [4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% mercaptoethanol, and a trace amount of bromophenol blue dye in 125 mM Tris-HCl, pH 6.8], heated at 100°C for 5 min, and then frozen at –80°C until use. Samples were subjected to 10% SDS gel electrophoresis. After electrophoresis, proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol at 2 mA/cm2 for 1.5 h at 25°C. Residual binding sites on the membrane were blocked by incubating the membrane in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and 5% nonfat dry milk for 2 h at 25°C. The blots were washed in Tris-buffered saline containing 0.1% Tween 20 and then incubated with appropriate antibody overnight at 4°C. After washing, the membrane was incubated with anti-rabbit IgG antibody conjugated with HRP, and the antibody complexes were visualized by the ECL detection system, as directed by the manufacturer. Immunoreactive bands were quantified by National Institutes of Health Image program on a Macintosh computer.

In some experiments, the reactions were terminated by the addition of trichloroacetic acid (TCA). The final concentration of TCA was 10%. The cells were washed with acetone containing 10 mM dithiothreitol and were lysed with the 1x sample buffer. Samples were subjected to 10% SDS gel electrophoresis, and the immunoblotting was performed as mentioned above. We found that the method with TCA fixation could effectively prevent the proteolysis of STAT3 and Akt, both of which, but not ERK and p38 MAPK, were easily cleaved by granule-derived serine proteases during preparation of neutrophil lysates with the conventional lysis buffer [19 20 21 ].

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from neutrophils with the guanidine isothiocyanate method. To generate cDNA, 1000 ng RNA was used for each reaction. The reaction mixtures (20 µl) contained an RNA, random primer pd(N)6 (1 µM), RNase inhibitor (0.5 U/µl; Roche Molecular Biochemicals, Mannheim, Germany), deoxy-unspecified nucleoside 5'-triphosphate (dNTP) mixture (500 µM of each dNTP), and Omniscript reverse transcriptase (0.2 U/µl, Qiagen, Valencia, CA), and the reaction mixtures were incubated for 60 min at 37°C. Semiquantitative RT-PCR analysis was performed using a GeneAmp PCR system model 9700 (Perkin Elmer, Norwalk, CT). PCR reaction mixtures (25 µl) contained cDNA, dNTP mixture (200 µM of each dNTP), MgCl2 (2 mM), Taq DNA polymerase (0.025 U/µl, Fermentas AB, Vilnius, Lithuania), and the forward and reverse primers.

The following primer pairs were used. For each set, the forward and reverse primers and the accession number are listed: cIAP1, 5'-AGC TGT TGT CAA CTT CAG ATA CCA CT-3', 5'-TGT TTC ACC AGG TCT CTA TTA AAG CC-3', U45879; cIAP2, 5'-ACT TGA ACA GCT GCT ATC CAC ATC-3', 5'-GTT GCT AGG ATT TTT CTC TGA ACT GTC-3', U45878; Mcl-1, 5'-CTC TCA TTT CTT TTG GTG CCT-3', 5'-ATT CCT GAT GCC ACC TTC TA-3', AF198614; A1, 5'-GTT TGA AGA CGG CAT CAT T-3', 5'-ACA AAG CCA TTT TCC CAG-3', U27467; SOCS3, 5'-CTC GCC ACC TAC TGA ACC CTC-3', 5'-AAG CGG GGC ATC GTA CTG GT-3', AF159854; SOCS1, 5'-AGA CCC CTT CTC ACC TCT TG-3', 5'-CTG CAC AGC AGA AAA ATA AAG C-3', NM-003745; Bax, 5'-ATG CGT CCA CCA AGA AGC-3', 5'-AAA CTG GTG CTC AAG GCC-3', L22473.1; 18S rRNA, 5'-TCC GAT AAC GAA CGA GAC TC-3', 5'-CAG GGA CTT AAT CAA CGC AA-3', M10098; and ß-actin, 5'-CCA ACC GCG AGA AGA TGA C-3', 5'-GGA AGG AAG GCT GGA AGA GT-3', X00351.

The conditions for PCR amplification were as follows: denaturation for 30 s at 94°C, annealing for 30 s at 56°C, and elongation for 30 s at 72°C with 23 cycles for SOCS3, SOCS1, Bax, and ß-actin and 27 cycles for cIAP1, cIAP2, Mcl-1, A1, and 18S rRNA. The PCR products were analyzed by electrophoresis on a 2% agarose gel containing ethidium bromide. The PCR products were purified, and the nucleotide sequence was analyzed for confirmation.

Real-time PCR analysis
For quantitative determination of the transcripts, the real-time PCR analysis was performed with LightCycler (Roche Molecular Biochemicals), as directed by the manufacturer. The reaction mixtures (20 µl) contained MgCl2 (3 mM), the forward and reverse primers (0.5 µM), the fluorescein isothiocyanate (FITC)-conjugated probe (0.2 µM), LightCycler Red 640 (LC Red 640)-conjugated probe (0.4 µM), LightCycler-DNA master hybridization probe (0.4 µM), and the cDNA (1 µl) [1 ]. As an internal control, 18S rRNA was used. The primer sequences are described above, and the hybridization probe sets are as follows: cIAP2, 5'-GAT TGC ATC TTC TGA ATG GTC TTC TCC-(FITC)-3' and 5'-(LC Red 640)-GGT TCC AAA TGG ATA ATT GAT GAC TCT G-(phosphate)-3'; A1, 5'-AGA AAC TTC TAC GAC AGC AAA TTG CC-(FITC)-3' and 5'-(LC Red 640)-CGG ATG TGG ATA CCT ATA AGG AGA TTT C-(phosphate)-3'; and 18S rRNA, 5'-GGA CAT CTA AGG GCA TCA CAG ACC T-(FITC)-3' and 5'-(LC Red 640)-TTA TTG CTC AAT CTC GGG TGG CT-(phosphate)-3'.

Statistical analysis
An ANOVA followed by a multiple comparison test or Student’s t-test was used to determine statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IFN-{alpha}, IFN-{gamma}, and G-CSF delay human neutrophil apoptosis through the protein synthesis-dependent mechanism
Human neutrophils underwent spontaneous apoptosis during culture. Spontaneous neutrophil apoptosis was delayed in the presence of IFN-{alpha}, IFN-{gamma}, or G-CSF (Fig. 1 ), confirming the previous reports [1 , 2 , 5 , 18 ]. The potency of these cytokines to prolong neutrophil survival was G-CSF > IFN-{gamma} > IFN-{alpha} when assessed at 72 h after cultivation. Significant antiapoptotic effect of IFN-{alpha} was detected during 24 h after cultivation but undetected at 48 h (Fig. 1A) . Spontaneous neutrophil apoptosis was accelerated in the presence of cycloheximide, and the antiapoptotic effect of all these cytokines was almost completely abolished by cycloheximide. These findings were confirmed, not only by counting the total viable cells but also determining the DNA fragmentation (Fig. 1) . These findings indicate that like G-CSF [1 , 2 ], IFN-{alpha} and IFN-{gamma} exert the antiapoptotic effect on human neutrophils through the protein synthesis-dependent mechanism.



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  		Figure 1. IFN-{alpha}, IFN-{gamma}, and G-CSF delay human neutrophil apoptosis through the protein synthesis-dependent mechanism. Neutrophils (5x106/ml) were cultivated with IFN-{alpha} (1000 IU/ml), IFN-{gamma} (1000 IU/ml), or G-CSF (50 ng/ml) in the presence or absence of cycloheximide (CHX; 10 µg/ml). When required, cells were preincubated with cycloheximide for 20 min at 37°C before the addition of IFN-{alpha}, IFN-{gamma}, or G-CSF. (A) After cultivation for the indicated periods, total viable cells were counted. The data are expressed as means ± SD of four experiments. *, #, Significantly greater as compared with control cells cultivated in the absence of cycloheximide (*, P<0.01; #, P<0.05). +, Significantly greater as compared with cells cultivated with IFN-{alpha} or IFN-{gamma} in the absence of cycloheximide (+, P<0.05). (B) After cultivation for 18 h, DNA fragmentation was determined. The data are expressed as means ± SEM of five experiments. *, Significantly greater as compared with control cells (*, P<0.01); #, significantly greater as compared with IFN-{alpha}-treated cells (#, P<0.05).

 
IFN-{alpha} and IFN-{gamma} induce tyrosine phosphorylation of STAT1 and STAT3 in human neutrophils
The major signaling molecule activated in various types of cells stimulated by IFN-{alpha} or IFN-{gamma} is STAT1 [13 14 15 ]. As shown in Figure 2A and 2B , stimulation of human neutrophils with IFN-{alpha} or IFN-{gamma} for 10 min resulted in significant tyrosine phosphorylation of STAT1. Both IFNs also induced tyrosine phosphorylation of STAT3, but not STAT5. Under the same conditions, G-CSF and GM-CSF induced strong tyrosine phosphorylation of STAT3. STAT1 was phosphorylated by stimulation with GM-CSF, but not with G-CSF, whereas STAT5 was phosphorylated by stimulation with G-CSF or GM-CSF. Neither STAT1 nor STAT3 was phosphorylated by stimulation with TNF-{alpha} (Fig. 2A and 2B) . The potency of IFN-{alpha} and IFN-{gamma} to induce tyrosine phosphorylation of STAT3 was almost identical, whereas IFN-{gamma} was slightly more potent than IFN-{alpha} in inducing tyrosine phosphorylation of STAT1. In lymphocytes, STAT1 and STAT3 were strongly tyrosine-phosphorylated by stimulation with IFN-{alpha} and weakly phosphorylated by stimulation with IFN-{gamma} (Fig. 2A) .



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  		Figure 2. Phosphorylation of STAT1 and STAT3 in neutrophils stimulated by IFN-{alpha}, IFN-{gamma}, or G-CSF. (A) Neutrophils or lymphocytes were stimulated with IFN-{alpha} (1000 IU/ml), IFN-{gamma} (1000 IU/ml), G-CSF (50 ng/ml), or GM-CSF (5 ng/ml) for 10 min at 37°C. The immunoblotting was performed using antibodies against the phosphorylated forms of STAT1 (P-STAT1), STAT3, STAT5, ERK1/2, and p38. The cell lysates equivalent to 3.75 x 106 neutrophils or 2.5 x 106 lymphocytes were loaded onto each lane. The equal loading of proteins onto each lane was confirmed by immunoblotting using antibody against p38. The results shown are representative of five independent experiments. (B) Neutrophils were stimulated with GM-CSF (5 ng/ml) or TNF-{alpha} (100 U/ml) for 10 min or IFN-{alpha} (1000 IU/ml) or IFN-{gamma} (1000 IU/ml) for the indicated periods at 37°C. In this experiment, the reactions were terminated by the addition of TCA. The immunoblotting was performed using antibodies against the phosphorylated form of Akt, STAT1, and STAT3. The equal loading of proteins onto each lane was confirmed by immunoblotting using antibodies against p38, STAT1, and STAT3. The densitometric data for phosphorylation of STAT1 and STAT3 are also shown. The results shown are representative of three independent experiments. (C) Neutrophils were stimulated with IFN-{alpha} (1000 IU/ml), IFN-{gamma} (1000 IU/ml), or G-CSF (50 ng/ml) for the indicated periods at 37°C. The immunoblotting was performed using antibodies against the phosphorylated form of STAT1 and STAT3. The cell lysates equivalent to 3.75 x 106 neutrophils were loaded onto each lane. The cell lysates equivalent to 1 x 106 HL-60 cells stimulated with IFN-{gamma} (1000 IU/ml) for 10 min were used as a positive control for phosphorylation of STAT1. The results shown are representative of three independent experiments. (D) Neutrophils or lymphocytes were preincubated with neutralizing antibody (Ab; AF245; 5 µg/ml) against human IFNAR1 or control goat IgG antibody for 30 min and thereafter, stimulated with IFN-{alpha} (1000 IU/ml) for 10 min at 37°C. The immunoblotting was performed using antibody against the phosphorylated form of STAT3. The cell lysates equivalent to 3.4 x 106 neutrophils or 0.7 x 106 lymphocytes were loaded onto each lane. The results shown are representative of three independent experiments.

 
In contrast to G-CSF and GM-CSF, both of which induced phosphorylation of ERK1/2 and p38, neither IFN-{alpha} nor IFN-{gamma} induced phosphorylation of ERK1/2 and p38 (Fig. 2A) [20 ]. No significant phosphorylation of ERK1/2 and p38 was observed, even when the incubation time with IFN-{alpha} or IFN-{gamma} was prolonged for up to 60 min (data not shown). To determine the possible activation of PI-3K, phosphorylation of Akt was analyzed as a marker of PI-3K activation. As shown in Figure 2B , Akt was strongly phosphorylated by stimulation with GM-CSF or TNF-{alpha}. Conversely, no significant phosphorylation of Akt was detected in response to stimulation with IFN-{alpha} or IFN-{gamma}.

IFN-{alpha}-induced tyrosine phosphorylation of STAT1 and STAT3 was rapid and already detected at 3 min after stimulation with IFN-{alpha}, and the maximal level was observed at 5–10 min, followed by a gradual decrease of the level (Fig. 2C) . Similar kinetics were observed in tyrosine phosphorylation of STAT1 and STAT3 induced by IFN-{gamma} (Fig. 2C) . G-CSF-induced STAT3 phosphorylation was detected within 3 min after stimulation, and the maximal level was observed at 10 min, followed by a gradual decrease of the level. Tyrosine phosphorylation of STAT1 in G-CSF-stimulated neutrophils was undetected, even when the incubation time was prolonged for up to 60 min (Fig. 2C) .

IFN-{alpha}-induced STAT3 phosphorylation in human neutrophils and lymphocytes was abolished completely by neutralizing antibody (AF245) against human IFNAR1, a signal-transducing chain of the type I IFN receptor, indicating that IFN-{alpha}-induced STAT3 phosphorylation is mediated by the specific IFN-{alpha}/ß receptor expressed on neutrophils and lymphocytes (Fig. 2D) . The STAT3 isoform predominantly phosphorylated in neutrophils stimulated by IFN-{alpha} or IFN-{gamma} was STAT3{gamma} when neutrophil lysates were prepared using the conventional lysis buffer (Fig. 2A 2C and 2D) [1 , 21 ]. In this regard, we have demonstrated recently that STAT3{alpha}, but not STAT3{gamma}, is primarily expressed in human neutrophils, and STAT3{gamma} is rapidly generated from STAT3{alpha} by limited proteolysis with granule-derived serine proteases during preparation of neutrophil lysates with the conventional lysis buffer [21 ]. To prevent the proteolytic cleavage of proteins during preparation of cell lysates, the reactions were terminated by the addition of TCA. The method with TCA fixation could effectively prevent the proteolysis of STAT1 and STAT3 during preparation of cell lysates, and strong phosphorylation of STAT3{alpha} as well as STAT1 was consistently detected in neutrophils stimulated by IFN-{alpha}, IFN-{gamma}, or GM-CSF (Fig. 2B) .

IFN-{alpha}- or IFN-{gamma}-induced phosphorylation of STAT3 and antiapoptosis is inhibited by AG490
As shown in Figure 3A and 3B , G-CSF-induced phosphorylation of STAT3 and antiapoptosis was significantly inhibited by AG490, confirming our previous report [1 ]. Under the same conditions, IFN-{alpha}- or IFN-{gamma}-induced phosphorylation of STAT3, but not STAT1, was also inhibited significantly by AG490 (Fig. 3A) . There was little or no IFN-{alpha}-induced phosphorylation of STAT1 when neutrophils were preincubated for 30 min at 37°C before stimulation with IFN-{alpha}. IFN-{alpha}- or IFN-{gamma}-induced antiapoptotic effect on human neutrophils was also inhibited significantly by AG490 (Fig. 3B) . These findings suggest that IFN-{alpha} and IFN-{gamma}, like G-CSF, exert the antiapoptotic effect on human neutrophils, at least in part, via activation of STAT3. Selective inhibition of STAT3 phosphorylation by AG490 may be ascribed to inhibition of JAK2, as AG490 is a selective inhibitor of JAK, especially JAK2 [22 ]. In addition, IFN-{alpha} can activate tyrosine kinase 2, JAK1, and JAK2, and IFN-{gamma} can activate JAK1 and JAK2, although IFN-{alpha}- or IFN-{gamma}-mediated activation of these kinases may vary according to the cell types [13 , 23 ].



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  		Figure 3. Effect of AG490 on IFN-{alpha}-, IFN-{gamma}-, or G-CSF-induced phosphorylation of STAT1 and STAT3, antiapoptosis, and expression of SOCS1 and SOCS3. (A) Neutrophils were stimulated with IFN-{alpha} (1000 IU/ml), IFN-{gamma} (1000 IU/ml), or G-CSF (50 ng/ml) for 10 min at 37°C. When required, cells were pretreated with AG490 (50 µM) for 30 min at 37°C. The immunoblotting was performed using antibodies against the phosphorylated forms of STAT1 and STAT3. The cell lysates equivalent to 3.75 x 106 neutrophils were loaded onto each lane. The cell lysates equivalent to 1 x 106 HL-60 cells, stimulated with IFN-{gamma} (1000 IU/ml) for 10 min, were used as a positive control for phosphorylation of STAT1. The results shown are representative of three independent experiments (top and bottom panels). The ratio of phosphorylated STAT3 to p38 band intensity was calculated, and the value of cells stimulated with G-CSF in the absence of AG490 is expressed as 1. The densitometric data are expressed as means ± SD of three experiments (middle panel). *, Significantly inhibited by AG490 (*, P<0.01). (B) Neutrophils (5x106/ml) were preincubated with AG490 (50 µM) for 30 min and thereafter, cultivated with IFN-{alpha} (1000 IU/ml), IFN-{gamma} (1000 IU/ml), or G-CSF (50 ng/ml). After cultivation for 24 h, total viable cells were counted. The data are expressed as means ± SD of three experiments (upper panel). After cultivation for 18 h, DNA fragmentation was determined. The data are expressed as means ± SD of three experiments (lower panel). *, Significantly inhibited by AG490 (*, P<0.05). (C) Neutrophils were cultivated with IFN-{alpha} (1000 IU/ml) or IFN-{gamma} (1000 IU/ml) for 1 h, and thereafter, the expression of SOCS1 and SOCS3 mRNA was analyzed by semiquantitative RT-PCR. When required, cells were preincubated with AG490 (50 µM) for 30 min. As an internal control, ß-actin was used. The results shown are representative of three independent experiments (top panel). The densitometric data are expressed as means ± SD of three experiments (middle panel). *, Significantly inhibited by AG490 (*, P<0.01). Neutrophils were cultivated with IFN-{alpha} (1000 IU/ml) or IFN-{gamma} (1000 IU/ml) for 3 h, and thereafter, the expression of SOCS3 protein was analyzed by immunoblotting. When required, cells were preincubated with AG490 (50 µM) for 30 min. In this experiment, the reactions were terminated by the addition of TCA. The results shown are representative of three independent experiments (bottom panel).

 
As shown in Figure 3C , SOCS3 and SOCS1 mRNA expression was induced by stimulation of neutrophils with IFN-{alpha} or IFN-{gamma}, and IFN-{alpha}- or IFN-{gamma}-induced expression of SOCS3 but not SOCS1. mRNA was inhibited significantly by pretreatment of cells with AG490. These findings suggest that IFN-{alpha}- or IFN-{gamma}-mediated induction of SOCS3 mRNA in neutrophils is mediated partly by STAT3 [24 ]. Despite the induced expression of SOCS3 mRNA by stimulation with IFN-{alpha} or IFN-{gamma}, the level of SOCS3 protein was not increased, at least during 3 h after stimulation (Fig. 3C) .

Up-regulation of cIAP2 by IFN-{alpha} or IFN-{gamma}
The IFN-{alpha}- or IFN-{gamma}-mediated antiapoptotic effect on neutrophils was sensitive to cycloheximide, suggesting that the synthesis of certain antiapoptotic proteins is induced by stimulation with IFN-{alpha} or IFN-{gamma}. To determine the antiapoptotic molecules induced by IFN-{alpha} or IFN-{gamma}, neutrophils were stimulated with IFN-{alpha} or IFN-{gamma} for 1 h, and thereafter, the expression of apoptosis-related gene transcripts, including cIAP1, cIAP2, Mcl-1, A1, and Bax, was analyzed by semiquantitative RT-PCR. Among these transcripts, cIAP2 was up-regulated selectively by stimulation with IFN-{alpha} or IFN-{gamma} (Fig. 4 ). Up-regulation of cIAP2 mRNA by stimulation with IFN-{alpha} or IFN-{gamma} was confirmed by the real-time PCR analysis (Fig. 5A ), which also revealed that IFN-{alpha}- or IFN-{gamma}-induced up-regulation of cIAP2 mRNA was inhibited significantly by AG490. Under the same conditions, the expression of A1 mRNA was unaffected by stimulation with IFN-{alpha} or IFN-{gamma} nor affected by AG490 (Fig. 5A) . Consistent with increased expression of cIAP2 mRNA, stimulation of neutrophils with IFN-{alpha} or IFN-{gamma} resulted in an elevated level of cIAP2 protein. In addition, IFN-{alpha}- or IFN-{gamma}-induced up-regulation of cIAP2 protein was inhibited significantly by AG490. Under the same conditions, G-CSF-induced up-regulation of cIAP2 protein was also inhibited by AG490, confirming our previous report (Fig. 5B) [1 ]. The level of Bax protein in neutrophils was unaltered during 8 h cultivation with IFN-{alpha}, IFN-{gamma}, or G-CSF (data not shown).



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  		Figure 4. Effects of IFN-{alpha} and IFN-{gamma} on expression of apoptosis-related gene transcripts in neutrophils, which were cultivated with IFN-{alpha} (1000 IU/ml) or IFN-{gamma} (1000 IU/ml) for 1 h, and thereafter, the expression of cIAP1, cIAP2, Mcl-1, A1, and Bax mRNA was analyzed by semiquantitative RT-PCR. As an internal control, ß-actin and 18S rRNA were used. The results shown are representative of three independent experiments.

 


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  		Figure 5. Effect of AG490 on up-regulation of cIAP2 mRNA and protein in neutrophils stimulated by IFN-{alpha} or IFN-{gamma}. (A) Neutrophils (5x106/ml) were preincubated with AG490 (50 µM) for 30 min and thereafter, cultivated in the presence or absence of IFN-{alpha} (1000 IU/ml) or IFN-{gamma} (1000 IU/ml) for 1 h. The expression of cIAP2 and A1 mRNA was analyzed by the real-time PCR. As an internal control, 18S rRNA was used. The ratio of cIAP2 or A1 to 18S rRNA was calculated, and the ratio at 0 h is expressed as 1. The data are expressed as means ± SEM of four to six experiments. *, Significantly greater as compared with control cells (*, P<0.01). #, ##, Significantly inhibited by AG490 (#, P<0.05; ##, P<0.01). The expression of A1 mRNA was not increased significantly by stimulation with IFN-{alpha} or IFN-{gamma} (P=0.15 for IFN-{alpha}-treated cells vs. control cells; P=0.26 for IFN-{gamma}-treated cells vs. control cells). (B) Neutrophils were preincubated with AG490 (50 µM) for 30 min and thereafter, cultivated in the presence or absence of IFN-{alpha} (1000 IU/ml), IFN-{gamma} (1000 IU/ml), or G-CSF (50 ng/ml) for 3 h. The expression of cIAP2 protein was analyzed by immunoblotting. As a control, p38 was used. In this experiment, the reactions were terminated by the addition of TCA. The results shown are representative of five independent experiments (upper panel). The ratio of the cIAP2-to-p38 band intensity was calculated, and the value of cells stimulated with G-CSF in the absence of AG490 is expressed as 1. The densitometric data are expressed as means ± SD of three experiments (lower panel). The level of cIAP2 protein was increased significantly (P<0.01) by stimulation with IFN-{alpha}, IFN-{gamma}, or G-CSF. *, Significantly inhibited by AG490 (*, P<0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have recently reported that the IAP family members [cIAP1, cIAP2, and X-linked IAP (XIAP)] are expressed in human neutrophils, and G-CSF induces up-regulation of cIAP2 through activation of the JAK2-STAT3 pathway, which contributes to a G-CSF-mediated antiapoptotic effect on human neutrophils [1 ]. The present experiments show that IFN-{alpha} (type I IFN) and IFN-{gamma} (type II IFN), which bind to the distinct receptors, exert the antiapoptotic effect on human neutrophils through the similar mechanism. IFN-{alpha} and IFN-{gamma}, like G-CSF, delayed human neutrophil apoptosis through the protein synthesis-dependent mechanism and induced selective up-regulation of cIAP2. IFN-{alpha}- and IFN-{gamma}-induced up-regulation of cIAP2 mRNA and protein, phosphorylation of STAT3, and antiapoptotic effect were inhibited significantly by AG490. These findings support the concept that cIAP2 expression is up-regulated by IFN-{alpha} and IFN-{gamma} through, at least in part, activation of the JAK2-STAT3 pathway, and up-regulation of cIAP2 protein plays an important role in IFN-{alpha}- and IFN-{gamma}-induced antiapoptotic effect on human neutrophils.

It has been established that STAT1 plays a major role in mediating the actions of IFN-{alpha} and IFN-{gamma} in various cell types. STAT1 is implicated in innate immunity, inhibition of cell growth, and promotion of apoptosis [25 ]. The requirement of STAT1 for growth inhibition and apoptosis may be ascribed to its ability to up-regulate caspases and the cdk inhibitor p21 [25 , 26 ]. Conversely, considerable evidences indicate that the STAT1-independent signaling pathway also plays an important role in the actions of IFN-{alpha} and IFN-{gamma} [15 , 27 ]. The STAT1-independent signaling pathway may include STAT3, STAT5, nuclear factor (NF)-{kappa}B, and p38 [28 29 30 31 32 ]. The present experiments show that IFN-{alpha} and IFN-{gamma} induce activation of STAT1 and STAT3, but not STAT5, ERK, p38, and Akt in human neutrophils, and STAT3 may be involved in the antiapoptotic effect of IFN-{alpha} and IFN-{gamma} on human neutrophils. It has been reported that STAT3 activation could induce several antiapoptotic molecules such as Bcl-2, Bcl-XL, Mcl-1, survivin, and cIAP2 according to the cell types and the stimuli used [1 , 33 34 35 36 37 ]. The present experiments show that cIAP2 is up-regulated selectively by STAT3 activation in human neutrophils stimulated by IFN-{alpha} or IFN-{gamma}, in which both could induce the opposite effect on the cell fate (apoptosis or survival), depending on the cell types used [13 14 15 ]. It is possible that the cell fate in response to IFN-{alpha} or IFN-{gamma} may be determined partly by the balance of proapoptotic STAT1 and antiapoptotic STAT3 activation, which may vary according to the cell types. It is likely that predominant activation of STAT3 may support survival, whereas predominant activation of STAT1 may promote apoptosis. In fact, it has been shown that constitutively activated STAT3 antagonizes the proapoptotic effect of activated STAT1 in Me180 cells [38 ]. The present experiments show that stimulation of human neutrophils with IFN-{alpha} or IFN-{gamma} results in predominant activation of STAT3, but not STAT1. By contrast, we found that in human hematopoietic progenitor cells (CD34+ cells), STAT1 as well as STAT3 was strongly phosphorylated by stimulation with IFN-{alpha} or IFN-{gamma} (data not shown). These findings suggest that the signaling pathway from IFN-{alpha} or IFN-{gamma} receptors to STAT1 activation may be down-regulated during differentiation into mature neutrophils, as observed in that from TNF-{alpha} receptors to c-Jun amino-terminal kinase activation [20 ]. These findings also support the concept that a physiological role of IFN-{alpha} or IFN-{gamma} is to promote neutrophil survival.

It has been reported recently that IFN-ß delays human neutrophil apoptosis through activation of NF-{kappa}B, which may occur downstream of PI-3K and protein kinase C-{delta} [5 ]. In contrast to other cytokines such as GM-CSF and TNF-{alpha}, which induce phosphorylation of Akt, IFN-{alpha}/ß-mediated activation of PI-3K in human neutrophils appears to be unaccompanied with activation of Akt, although the reason for the lack of Akt phosphorylation as well as the PI-3K-dependent and Akt-independent pathway remains to be elucidated [5 , 30 , 39 ]. In this regard, it is of interest that phosphorylated STAT3 could function as an adaptor to couple PI-3K to IFNAR1, suggesting that STAT3 could activate NF-{kappa}B via PI-3K in certain cell types stimulated by IFN-{alpha}/ß [40 ]. These findings suggest that IFN-{alpha}-induced up-regulation of cIAP2 in human neutrophils might be partly mediated by the STAT3-PI-3K-NF-{kappa}B pathway, as cIAP2 is inducible by NF-{kappa}B activation [41 ]. However, it should be noted that cIAP2, but not cIAP1 and A1, is selectively up-regulated by IFN-{alpha}, IFN-{gamma}, or G-CSF, despite the fact that cIAP1, cIAP2, and A1 are inducible by NF-{kappa}B activation, and NF-{kappa}B is not activated in neutrophils stimulated by G-CSF [1 , 7 , 42 43 44 ]. These findings suggest that cytokine-induced up-regulation of cIAP2 is unlikely to be solely ascribed to NF-{kappa}B activation. In fact, the expression of the IAP family members may be regulated by different mechanisms, depending on the cell types and the stimuli used. For example, p38 is involved in CD40-induced cIAP2 expression in dendritic cells but not in B cells [45 ]. MAPK/ERK kinase or PI-3K is involved in GM-CSF-induced up-regulation of survivin in myeloid leukemia cells, and constitutive STAT3 activation results in survivin expression in primary effusion lymphoma cells [36 , 46 ]. In any case, the present experiments show that IFN-{alpha} and IFN-{gamma}, like G-CSF, exert the antiapoptotic effect on human neutrophils, at least in part via up-regulation of cIAP2 at the transcriptional level. It is possible that the expression of cIAP2 protein may also be regulated at the post-transcriptional level. Furthermore, the JAK2-independent mechanisms such as NF-{kappa}B also may be possibly involved in the IFN-{alpha}- and IFN-{gamma}-mediated antiapoptotic effect on human neutrophils, as the antiapoptotic effect of these cytokines was not abolished completely by AG490.

The IAP family proteins have been shown to inhibit active caspase-3 and caspase-7 directly and to inhibit activation of caspase-8 and caspase-9, resulting in suppression of apoptosis [42 , 47 ]. Human neutrophils possess the apoptotic protease-activating factor 1 pathway, which permits caspase-9 activation, and activation of caspase-3 and caspase-8 has been demonstrated in human neutrophils undergoing apoptosis [48 , 49 ]. Thus, it is conceivable that cIAP2, up-regulated by IFN-{alpha} or IFN-{gamma}, may contribute to the IFN-{alpha}- or IFN-{gamma}-induced antiapoptotic effect on human neutrophils by inhibiting caspase-8 and caspase-9 activation and/or caspase-3 activity. This concept is consistent with the recent findings that IFN-ß as well as G-CSF inhibits caspase activity and maintains mitochondrial integrity [5 , 50 ].

Recent studies have demonstrated that the IAP family members, notably, cIAP2 and survivin, are involved in regulation of neutrophil survival, i.e., up-regulation of cIAP2 and survivin by G-CSF and up-regulation of survivin by GM-CSF [1 , 10 ]. The present experiments demonstrate that cIAP2 is up-regulated selectively by IFN-{alpha} and IFN-{gamma} via activation of STAT3. It has also been shown that XIAP degradation may be involved in spontaneous neutrophil apoptosis, and dysregulation of cIAP2 and XIAP may be associated with prolonged neutrophil survival in chronic neutrophilic leukemia [1 , 51 ]. These findings indicate that the IAP family members play an important role in regulation of human neutrophil survival, not only in the physiological state but also in the pathological state. In addition, the results suggest that delayed neutrophil apoptosis by IFN-{alpha} or IFN-{gamma} may contribute not only to augmenting the host defense against invading microorganisms but also to increasing the tissue damage at the inflammatory sites.


   ACKNOWLEDGEMENTS
 
This work was supported by a Grant-in-Aid for Scientific Research, Japan.

Received November 26, 2004; revised March 26, 2005; accepted March 28, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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