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Originally published online as doi:10.1189/jlb.0507282 on October 26, 2007

Published online before print October 26, 2007
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(Journal of Leukocyte Biology. 2008;83:393-400.)
© 2008 by Society for Leukocyte Biology

Terminally differentiated neutrophils predominantly express Survivin-2{alpha}, a dominant-negative isoform of Survivin

Huiyuan Hu*,{dagger}, Yayoi Shikama*,1, Isao Matsuoka*,{ddagger} and Junko Kimura*

* Department of Pharmacology, Fukushima Medical University School of Medicine, Fukushima, Japan;
{dagger} Department of Pharmacology, China Medical University, Shenyang, China; and
{ddagger} Laboratory of Pharmacology, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan

1 Correspondence: Department of Pharmacology, Fukushima Medical University, 1 Hikarigaoka, Fukushima, 960-1295, Japan. E-mail: yayois{at}fmu.ac.jp


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ABSTRACT
 
Survivin, which is a member of the inhibitor of apoptosis protein family, was found originally in immature cells and cancer cells but not in non-neoplastic adult tissues. The subsequent identification of four other alternative splice variants that possess distinct functions and localizations suggested the diverse roles of survivin isoforms. An unspecified isoform of survivin was found recently to be induced in terminally differentiated neutrophils by cytokines that prolong the neutrophil lifespan, such as GM-CSF and G-CSF, suggesting the importance of survivin in blocking apoptosis in neutrophils. To examine the mechanism by which survivin inhibits neutrophil apoptosis, we attempted to induce survivin by GM-CSF/G-CSF in an HL60 cell line that was differentiated into neutrophils by all-trans retinoic acid and DMSO and freshly isolated human neutrophils. The antiapoptotic isoform "Survivin," which was decreased during differentiation, was re-induced by GM-CSF in neutrophil-like, differentiated HL60. In contrast, in normal neutrophils, survivin mRNA was observed to increase spontaneously after 24 h incubation, and no additional elevation was induced by GM-CSF/G-CSF, which exerted their antiapoptotic effects on the neutrophils in 6 h, despite the lack of survivin induction. PCR and Western blotting detected Survivin-2{alpha}, a dominant-negative of antiapoptotic Survivin, with no other isoforms in the freshly isolated or incubated neutrophils. Our study revealed that the expressed isoforms and the response to GM-CSF were different between the HL60-derived and normal neutrophils, which predominantly expressed Survivin-2{alpha}, not likely involved in apoptosis inhibition by GM-CSF/G-CSF.

Key Words: splice variant • apoptosis inhibition • neutrophilic granulocytes • cytokines


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INTRODUCTION
 
Neutrophils have a short lifespan and undergo spontaneous apoptosis rapidly to facilitate normal cell turnover and immune system homeostasis [1 ]. Inflammatory cytokines, such as GM-CSF and G-CSF, are known to extend the survival of neutrophils by delaying apoptosis [2 , 3 ]. Although the neutrophil-protection mechanisms of these cytokines have been shown to involve the Bcl-2 family members, including Mcl-1 [4 , 5 ], A1 [6 , 7 ], and Bax [8 , 9 ], and the inhibitor of apoptosis protein (IAP) family members, such as cellular IAP-2 [10 ], the antiapoptotic mechanisms are still not understood fully.

Survivin is a member of the IAP family, characterized by the highly conserved baculovirus IAP repeat domains [11 ]. Survivin has been found primarily in fetal tissues [12 , 13 ] and most human cancer cells but not in non-neoplastic adult tissues [12 ]. The cells in the G2/M phase show an especially enhanced expression of survivin, and survivin regulates their proliferation as a spindle-associated protein [14 15 16 ]. Further functional studies have shown that survivin inhibits apoptosis via blockage of the caspase activities in cancer cells treated with various apoptotic stimuli [17 18 19 20 ]. To date, five survivin isoforms have been identified in humans as the originally found isoform "Survivin" consisting of four exons, Survivin-2B, Survivin-{Delta}Ex3, Survivin-3B, and Survivin-2{alpha}, which have diverse effects on apoptosis regulation [21 22 23 24 25 ]. The functions described above are ascribed to Survivin. Survivin-{Delta}Ex3, which lacks exon 3, has antiapoptotic properties [26 , 27 ], and Survivin-2B, which retains a part of intron 2 as a cryptic exon, exon 2B, is proapoptotic [23 , 27 , 28 ]. Survivin-2{alpha}, which is composed of exon 1, exon 2, and 3' 197 nucleotides derived from intron 2, providing a stop codon and a unique 3' untranslated region (UTR), interacts physically with Survivin and acts as a natural dominant-negative of Survivin’s antiapoptotic activities in several malignant cell lines and primary tumor cells [22 ]. No function has yet been ascribed to Survivin-3B, an isoform resulting from an insertion of a novel exon, exon 3B [29 ].

Although the majority of these studies has been conducted on cancer cells so far, recent studies have shown that survivin isoforms are expressed in normal adult cells, particularly in cells that preserve proliferating potentials [30 31 32 ]. 3' UTR of survivin mRNA was detected by RT-PCR in CD34+ cells isolated from adult bone marrow [30 ]. The inverse correlation between survivin expression and apoptosis accompanied by the up-regulation of caspase 3 in CD34+ cells indicated the involvement of survivin in apoptosis inhibition in normal hematopoietic stem cells [30 , 33 ]. T lymphocytes express survivin, which is crucial for T cell maturation [31 ]. Survivin-{Delta}Ex3 has been identified in vascular endothelial cells, suggesting that it may play a novel role in angiogenesis [32 ].

Recently, survivin was detected in terminally differentiated neutrophils isolated from patients with cystic fibrosis, a chronic inflammatory disease [34 ]. The study also showed that proinflammatory cytokines that prolong the neutrophil lifespan, GM-CSF and G-CSF, induced survivin in normal neutrophils, which lost proliferative potential, suggesting the contribution of survivin to cytokine-induced antiapoptosis. However, the isoform that was expressed in the normal neutrophils was not clarified carefully, and the mechanism by which survivin blocks the apoptosis of neutrophils remains unclear.

To investigate the mechanism, we attempted to confirm the involvement of survivin isoforms in apoptosis inhibition by GM-CSF/G-CSF in terminally differentiated neutrophils using freshly isolated neutrophils and HL60 differentiated by all-trans retinoic acid (ATRA) and DMSO. However, unexpectedly, neither GM-CSF nor G-CSF increased any isoforms of survivin in normal neutrophils, and the GM-CSF-dependent elevation of an isoform "Survivin" was observed in the differentiated HL60. The survivin isoform expressed predominantly in the normal neutrophils was identified as Survivin-2{alpha}, a dominant negative of antiapoptotic Survivin, and none of the other isoforms were detectable. Our findings suggest that survivin is likely not involved in cytokine-induced neutrophil protection.


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MATERIALS AND METHODS
 
Cell preparation
A human promyelocytic leukemia cell line, HL60, was purchased from Riken BRC Cell Bank (Japan). The cells were grown in RPMI 1640 (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% (v/v) heat-inactivated FBS (Sigma Chemical Co.) and maintained at 37ºC in a humidified atmosphere with 5% CO2. To induce differentiation toward the neutrophilic granulocytes, the HL60 cells were cultured in the presence of 1 µmol/L ATRA (Sigma Chemical Co.) plus 1.25% of DMSO (Nakarai Tesque Inc., Kyoto, Japan) for 4 days. For the analysis of survivin induction by GM-CSF in the differentiated HL60 cells, recombinant human GM-CSF (PeproTech EC, London, UK) was applied after a 4-day culture with ATRA and DMSO.

Citrated blood was obtained by a venopuncture from 10 healthy volunteers after obtaining informed consent and approval from the Institutional Human Research Committee. Polymorphonuclear leukocytes (PMN) were isolated by centrifugation over LymphoprepTM (l.077 g/mL, Axis-Shield PoC AS, Oslo, Norway). After a hypotonic lysis of the erythrocytes, the neutrophils were found to be ≥98% in the PMN fraction, as determined by May-Grünwald and Giemsa (Merck, Darmstadt, Germany) staining. The viabilities of the freshly isolated neutrophils were found to be more than 97% by the trypan blue exclusion technique. The cells were cultured at a concentration of 1 x 106 cells/mL in RPMI 1640 containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin, with or without GM-CSF or G-CSF (Sigma Chemical Co.). Polymyxin B (50 µg/mL; Sigma Chemical Co.) was included in the medium in some experiments.

RNA extraction, reverse transcription, and real-time PCR
Total cellular RNA was extracted from HL60 and neutrophils by the acid-guanidine thiocyanate/phenol/chloroform method [35 ] and digested by DNase I (TaKaRa Bio Inc., Otsu, Japan). First-strand cDNA primed by random hexamers (TaKaRa Bio Inc.) was prepared from the total RNA (1 µg) using Moloney murine leukemia virus RT (Invitrogen, Carlsbad, CA, USA). For quantification of survivin by real-time PCR, two different primer pairs, 1S/1A and 2S/2A, were designed. Their sequences are shown in Table 1 . Primers 1S and 1A are complementary to the sequences in exon 3 and exon 4, respectively, and the complementary sequences to 2S and 2A reside in exon 1 and exon 2, both of which are shared by all five splice isoforms of survivin (Fig. 1A ). The reverse-transcribed cDNA was amplified for survivin and β-actin, which served as an internal control, using SYBR Premix Ex TaqTM (TaKaRa Bio Inc.) in a LightCycler system (Roche Diagnostics GmbH, Mannheim, Germany). The sense and antisense primers for β-actin were 5'-CAAGAGATGGCCACGGCTGCT-3' (746–766) and 5'-TCCTTCTGCATCCTGTCGGCA-3' (1020–1000), respectively (GenBank Accession Number NM001101). To verify the reliability of the quantification, real-time PCR products after 40-cycle amplification were visualized on agarose gels, and the sizes of the target fragments and the absence of other nonspecific products were confirmed in each experiment.


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  		Table 1. Primer Sequences


Figure 1
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  		Figure 1. Determination of PCR primers. (A) Five survivin isoforms and location of survivin primers. Boxes represent exons, and shaded parts are 3' UTR. The hatched box in Survivin-2{alpha} is 197 bp, derived from intron 2, generating a new stop codon and a unique 3' UTR. The arrowheads indicate the positions of stop codons, and arrows show primer-annealing sites. (B) Products of real-time PCR. The upper and lower panels show real-time PCR products after 40-cycle amplification using 1S/1A and 2S/2A primers, respectively.

Cell cycle analysis
The cell cycle distribution of the HL60 cells undergoing differentiation was assessed based on their DNA contents. Washed cells were resuspended in hypotonic solution [0.1% sodium citrate, 0.2% Nonidet P-40 (NP-40)], including 50 µg/mL propidium iodide (PI; Sigma Chemical Co.) and 0.25 mg/mL RNase A (Roche Applied Science, Indianapolis, IN, USA), and incubated at 4ºC for 30 min in the dark. Following further incubation at 37ºC for 15 min, the DNA contents were analyzed using flow cytometry (FACSCalibur 4A, BD Biosciences, Mountain View, CA, USA).

Western blot analysis
HL60 cells (4x106) and 1 x 107 neutrophils were lysed by ice-cold NP-40 lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 250 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L NaF, and 0.5% NP-40) containing a protease inhibitor cocktail (5 mmol/L PMSF, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 2 µg/mL aprotinin, 50 µg/mL each chymostatin and {alpha}-antitripsin). The cell lysate was mixed with Laemmeli sample buffer containing 2% 2-ME, boiled, and electrophoresed on a 15% SDS-polyacrylamide gel. The separated proteins were then transferred onto an Immobilon-P polyvinylidene fluoride membrane (Millipore Corp., Billerica, MA, USA) using a semi-dry Western transfer apparatus (Atto, Amherst, NY, USA). After blocking with 2% nonfat powdered milk dissolved in TBS (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl) with 0.1% Tween-20 (TBS-T) for 1 h at room temperature, the membrane was incubated with affinity-purified rabbit polyclonal anti-human survivin (R&D Systems, Inc., Minneapolis, MN, USA) or rabbit anti-survivin N terminus (Chemicon International, Inc., Temecula, CA, USA) at 4ºC overnight. After washing with TBS-T three times, the membrane was then incubated with HRP-conjugated donkey anti-rabbit IgG (1:10,000, Amersham Biosciences, Little Chalfont, UK) in TBS-T for 1 h at room temperature and developed by an ECL system (ECLTM, Amersham Biosciences). To confirm the amounts of the loaded proteins, the membrane was reprobed with rabbit anti-human actin (1:200, Sigma Chemical Co.).

Determination of apoptosis
The neutrophils undergoing apoptosis were determined by the Annexin V-FITC/PI apoptosis assay kit (Beckman Coulter, Fullerton, CA, USA), according to the manufacturer’s instructions. Briefly, the cells washed with PBS were resuspended in ice-cold binding buffer (10 mmol/L HEPES, pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L CaCl2) and incubated with Annexin V-FITC and PI for 10 min prior to flow cytometry (FACSCalibur 4A) analysis.

PCR and sequencing of PCR products
cDNA was synthesized from the total RNA isolated from the HL60 cells and neutrophils and subjected to PCR amplification by Taq DNA polymerase (TaKaRa Bio Inc.) with primer pairs 3S/3A or 3S/4A (Table 1) on a thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). Primer 3S corresponded to a sequence in exon 2 that is included by all five isoforms. The complementary sequences of 3A resided in 3' UTR shared by four isoforms except Survivin-2{alpha}, and primer 4A corresponded to 3' UTR, which was unique to Survivin-2{alpha} (Fig. 1A) . After 28-cycle amplification, the products were subcloned directly into pCR4-TOPO plasmid vectors (Invitrogen) using a TOPO TA cloning kit (Invitrogen) and transformed into DH5{alpha} cells (TaKaRa Bio Inc.). The residual PCR products were then separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Five ampicillin-resistant colonies were picked up from each PCR sample, and plasmid DNA was extracted using UltraCleanTM 6-minute mini plasmid prep kitTM (Mo Bio Laboratories, Inc., Carlsbad, CA, USA) following the manufacturer’s instructions. The sequences of subcloned PCR products were determined by the dideoxy chain-termination method using a Thermo SequenaseTM cycle sequencing kit (Amersham Biosciences) and IRD-800-labeled T3 or T7 primers (Li-Cor, Inc., Lincoln, NE, USA) in an automated DNA sequencing system (Aloka Co., Tokyo, Japan).

Statistical analysis
Student’s t-test was used for comparisons of the mean values between two groups. For a multiple data comparison, one-way ANOVA with Scheffe’s test was used. The data were expressed as the mean ± SEM, and differences were considered to be significant when the P value was less than 0.05.


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RESULTS
 
Primer determination for real-time PCR
To determine if the 1S/1A and 2S/2A primers are appropriate for the quantification by the real-time RT-PCR SYBR Green method, the products of the 40-cycle amplification were visualized on agarose gels (Fig. 1B) . From the untreated and ATRA/DMSO-treated HL60 samples, PCR with 1S/1A generated 161-bp fragments that could be derived from two isoforms, Survivin and Survivin-2B, and 2S/2A primers detected 136-bp fragments that could be derived from all five isoforms. The absence of any other nonspecific products indicated that survivin mRNA was amplified specifically. As the quantification of survivin mRNA levels in HL60 by real-time PCR using 1S/1A primers was highly correlated with the results using 2S/2A primers (r=0.945, P=0.002), the 1S/1A primer pair was used for the following analyses of survivin expression in the HL60 cells. In contrast, in the normal neutrophils, 1S/1A failed to detect survivin mRNA, and 2S/2A generated the 136-bp band with no other visible fragments. Therefore, 2S and 2A primers were used for the following analyses of the neutrophils.

Decrease in survivin during differentiation of HL60
To induce neutrophilic differentiation, the HL60 cell line was cultured in the presence of ATRA and DMSO for 4 days. The ATRA/DMSO-treated cells obtained smaller cell sizes and neutrophil-like nuclear segmentation (Fig. 2A ) with a loss of S/G2/M cell cycle population (Fig. 2B) .


Figure 2
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  		Figure 2. Decrease in the survivin expression during the differentiation of HL60. (A) Cell morphology. Untreated HL60 and HL60 cultured with ATRA and DMSO for 4 days were stained by May-Grünwald and Giemsa solution prior to observation under microscopy. (B) Cell cycle distribution. Untreated HL60 and ATRA/DMSO-treated HL60 were permeabilized and stained with PI. The populations of the cells at the S and G2/M phases were calculated. (C) Survivin mRNA levels during culture with ATRA and DMSO. Survivin mRNA levels were analyzed by real-time RT-PCR with primer pair 1S/1A. The relative amounts of survivin/β-actin in comparison with the initial level are presented. **, P < 0.01, in comparison with the level at Day 0. (D) Protein expression. Cell lysates of HL60 cultured with ATRA and DMSO for 0, 2, and 4 days were loaded onto 15% polyacrylamide gels. Separated proteins were transferred onto a membrane and blotted with rabbit polyclonal antibodies against survivin or actin. The 16.5-kDa Survivin and 42-kDa actin were indicated. The results are representative of three independent experiments.

During the ATRA/DMSO-induced differentiation, the survivin mRNA levels decreased (Fig. 2C) . The mRNA level became 24% of the initial level (survivin/β-actin; 3.45x10–2±0.50x10–2) on Day 2 and less than 1% on Day 4. Consistent with this change in mRNA levels, the highly expressed 16.5-kDa survivin protein became undetectable on Day 4 (Fig. 2D) . The molecular weight was compatible with the size of an isoform consisting of four exons—Survivin. In the following experiments, the HL60 cells cultured with ATRA and DMSO for 4 days were used as "differentiated HL60."

Induction of survivin by GM-CSF in HL60 differentiated by ATRA and DMSO
To examine if GM-CSF induces survivin expression in differentiated cells, the differentiated HL60 cells were stimulated with 10 ng/mL GM-CSF. The survivin mRNA level started to increase after 12 h, and a significant increase was observed at 24 h (5.79±1.55 folds, P=0.002, Fig. 3A ). When stimulated with various concentrations of GM-CSF for 24 h, the mRNA level in the differentiated HL60 cells increased dose-dependently (Fig. 3B) . The maximum increase of survivin was obtained at 1 ng/mL (12.38±3.14 folds, P=0.003, in comparison with 0 ng/mL), which was similar to the effect of 10 ng/mL GM-CSF (11.51±1.89 folds). Consistent with the mRNA increase, the 16.5-kDa Survivin, which was undetectable in the differentiated HL60 cells, was re-induced when stimulated with 10 and 50 ng/mL GM-CSF for 24 h (Fig. 3C) .


Figure 3
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  		Figure 3. The induction of survivin by GM-CSF in differentiated HL60 cells. (A) Time course of survivin mRNA induction by GM-CSF in differentiated HL60 cells, which when cultured with ATRA and DMSO for 4 days, were stimulated with 10 ng/mL GM-CSF. Survivin mRNA levels were analyzed by real-time RT-PCR with primer pair 1S/1A. The relative amounts of survivin/β-actin mRNA in comparison with that at 0 h are plotted. The results are expressed as the mean ± SEM of four to 10 experiments. **, P < 0.01, in comparison with 0 h. (B) Dose response of survivin mRNA induction. Differentiated HL60 cells were stimulated with various doses of GM-CSF for 24 h. The survivin mRNA levels were analyzed by real-time RT-PCR with primer pair 1S/1A. Data are shown as relative amounts of survivin/β-actin in comparison with that of 0 ng/mL GM-CSF. *, P < 0.05; **, P < 0.01, in comparison with 0 ng/mL. Results are expressed as the mean ± SEM of four to seven experiments. (C) Protein expression. Undifferentiated and differentiated HL60, incubated with or without GM-CSF, was subjected to Western blotting using rabbit polyclonal anti-survivin. A representative result from three different experiments is shown.

No survivin induction by GM-CSF/G-CSF in terminally differentiated normal neutrophils
To examine whether inflammatory cytokines induce survivin isoforms in normal cells, neutrophils that were freshly isolated from the peripheral blood of healthy volunteers were stimulated with 10 ng/mL GM-CSF, which was sufficient for survivin induction in the ATRA/DMSO-treated HL60 cells and 50 ng/mL GM-CSF. As shown in Figure 4 , no significant change was observed in the mRNA levels after 6 h in comparison with the initial level (survivin/β-actin; 6.11x10–5±0.09x10–5). It is surprising that in the absence of cytokines, survivin mRNA increased to 6.55 ± 0.85 folds (P<0.05) after 24 h. No additional increase was induced by stimulation with GM-CSF. There was no statistically significant difference in the mRNA levels among the three groups incubated for 24 h, with or without cytokines. The addition of polymyxin B into the medium, which blocks the effects of LPS, possibly contaminated, which could induce survivin [36 ], did not affect the spontaneous increase of survivin significantly (7.03±2.33 folds in the absence of polymyxin B vs. 10.03±1.91 folds in the presence of polymyxin B). The spontaneous increase of survivin mRNA and the lack of a further elevation by GM-CSF were reproducible in the culture medium containing different fetal bovine sera in the presence of polymyxin B as shown in Table 2 . We also tested another antiapoptotic cytokine—G-CSF. No significant increase was observed in the neutrophils stimulated with 25 ng/mL G-CSF in comparison with unstimulated cells at 6 and 24 h (Fig. 4) .


Figure 4
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  		Figure 4. The effects of GM-CSF and G-CSF on the survivin mRNA levels in normal neutrophils. Freshly isolated neutrophils were incubated with no cytokines (open bars), 10 ng/mL (double-hatched bars), or 50 ng/mL (dotted bars) GM-CSF or 25 ng/mL (hatched bars) G-CSF. The survivin mRNA levels at 6 and 24 h were quantified by real-time RT-PCR with primer pair 2S/2A. The survivn/β-actin mRNA values were compared with that at 0 h. The data represent the mean ± SEM of four to 10 different experiments. *, P < 0.05, in comparison with the 0-h control.


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  		Table 2. Fold Increase of Survivin mRNA in Neutrophils Cultured in the Presence of Different Serum

Antiapoptotic effects of GM-CSF/G-CSF prior to survivin induction in neutrophils
The cytoprotective effects of GM-CSF and G-CSF in neutrophils were verified (Fig. 5 ). In the absence of cytokines, culture for 6 h resulted in 10–20% of AnnexinV-positive/PI-negative, early apoptotic cell population and 75–85% of AnnexinV/PI-double-negative, viable cells. The presence of GM-CSF or G-CSF reduced the apoptotic populations (5–10%) and enlarged the viable cell fractions (85–95%). After 24 h, the differences in the sizes of the viable cell fractions between the absence and presence of cytokines became larger. GM-CSF and G-CSF preserved 35–50% of neutrophils alive for 24 h, and only 15–25% of the untreated cells were viable. The apoptotic populations, which were 45–65% in the absence of cytokines, were reduced to 30–45% by GM-CSF or G-CSF. These data suggested that the antiapoptotic effects of GM-CSF/G-CSF were not accompanied by an elevation of survivin expression in normal neutrophils.


Figure 5
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  		Figure 5. The antiapoptotic effects of GM-CSF and G-CSF on neutrophils. Freshly isolated neutrophils were incubated with no cytokines, 10 ng/mL GM-CSF, or 25 ng/mL G-CSF. After 6 and 24 h, the cells were incubated with FITC-labeled AnnexinV and PI, followed by flow cytometry analyses. The double-negative, AnnexinV-positive/PI-negative, and double-positive cells were counted as viable, early apoptotic, and dead cells, respectively. The results are representative of three separate experiments.

Identification of survivin isoform in neutrophils
To determine the survivin isoform expressed in neutrophils, the survivin was amplified using primer 3S, complementary to a sequence in exon 2 shared by all five isoforms, and primer 3A, corresponding to a sequence in 3' UTR, common to four isoforms except Survivin-2{alpha}. The survivin isoforms were distinguishable by PCR with these primers, as the length amplified by the 3S and 3A primers was different among the isoforms. The expected product sizes are 665 bp in Survivin, 734 bp in Survivin-2B, 547 bp in Survivin-{Delta}Ex3, and 830 bp in Survivin-3B. As shown in Figure 6A , the most prominent product in the HL60 cells was Survivin-derived 665 bp, and none of the isoforms were detectable in the neutrophils, thus suggesting that the neutrophils expressed neither Survivin, Survivin-3B, Survivin-{Delta}Ex3, nor Survivin-2B.


Figure 6
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  		Figure 6. Identification of survivin isoform expressed in neutrophils. (A) Isoform determination by different PCR primers. cDNA derived from HL60, freshly isolated neutrophils, and neutrophils cultured with or without 10 ng/mL GM-CSF for 24 h was subjected to PCR with 3S/3A or 3S/4A primers. The 28-cycle amplification products were visualized on a 2% agarose gel. (B) Sequence of PCR products derived from neutrophils. The 137-bp products of PCR with 3S/4A obtained from freshly isolated neutrophils and neutrophils incubated for 24 h, with or without GM-CSF, were sequenced, and the result from freshly isolated neutrophils is presented. The arrows indicate primer-annealing sites. The sequence belonging to exon 2 is surrounded by a box. The same sequences were exhibited in both of the 137-bp fragments derived from the neutrophils incubated with or without GM-CSF. (C) Protein expression. The cell lysates of HL60, untreated neutrophils, and neutrophils cultured with or without 10 ng/mL GM-CSF for 24 h were loaded on a 15% SDS-polyacrylamide gel and subjected to an immunoblot analysis with a rabbit polyclonal antibody against survivin N terminus. Only 8.5 kDa Survivin-2{alpha} was detectable in the neutrophils, and the HL60 cells expressed 16.5 kDa Survivin.

When the Survivin 2{alpha}-specific 4A primer was used instead of 3A, the 137-bp product was amplified from freshly isolated neutrophils and incubated neutrophils. The sequences of the 137-bp PCR products were confirmed to be identical to that between exon 2 and 3' UTR, unique to Survivin-2{alpha} (Fig. 6B) . The molecular weight of the protein detected in the freshly isolated and cultured neutrophils by immunoblotting using anti-survivin N terminus was 8.5 kDa, which was compatible with the size of Survivin-2{alpha}. These results therefore indicated that the spontaneously increased survivin isoform in neutrophils was Survivin-2{alpha}.


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DISCUSSION
 
The present study demonstrated that none of the survivin isoforms were induced by the antiapoptotic cytokines GM-CSF and G-CSF in terminally differentiated, normal neutrophils, unlike differentiated HL60 cells, in which the expression levels of Survivin increased in response to GM-CSF. These data challenge the importance of survivin shown previously in the regulation of neutrophil apoptosis [34 ]. The identification of Survivin-2{alpha} as an isoform predominantly expressed in neutrophils indicated further that none of the survivin isoforms contributed to apoptosis inhibition by GM-CSF/G-CSF in the normal neutrophils.

The discrepant results of PCR with 1S/1A between the HL60 cells and the neutrophils suggested that these cells expressed different isoforms of survivin. The 161-bp fragment in the HL60 cells could be generated from Survivin or Survivin-2B. The 16.5-kDa proteins detected in the untreated and differentiated HL60 cells were the translated products of an isoform Survivin consisting of four exons. Therefore, we concluded that the HL60 cells expressed Survivin throughout their differentiation. In contrast, the survivin isoform induced in neutrophils appears to be Survivin-{Delta}Ex3 or Survivin-2{alpha}, as primers 1S/1A generated neither 161 bp nor Survivin-3B-derived 326 bp fragments that contained 165 bp exon 3B.

Survivin expression, which was decreased in ATRA/DMSO-induced differentiation, was re-induced by GM-CSF in the differentiated HL60 cells. The reduction of survivin levels during differentiation may be common in normal and malignant hematopoietic cells. Normal CD34+ hematopoietic stem cells expressed higher levels of survivin than lineage-committed progenitor cells or circulating cells, although the isoform was not specified [30 ]. In contrast, survivin induction by GM-CSF was different between the neutrophil-like HL60 cells and the normal neutrophils. GM-CSF increased the mRNA and protein levels of survivin significantly in the ATRA/DMSO-treated HL60 cells but not in the neutrophils. The difference was not a result of the difference in their cell-cycle phases, as the differentiated HL60 cells remained in the G1 phase with no increase in the G2/M population when stimulated with GM-CSF (data not shown). This may reflect a distinct molecular basis between neoplasm and normal cells. The survivin induction by GM-CSF could be a marker that distinguishes neutrophils derived from leukemic clones from normal clone-derived neutrophils.

The lack of survivin induction by GM-CSF/G-CSF in the normal neutrophils and the spontaneous increase in survivin mRNA in 24 h were unanticipated findings. A previous study reported that GM-CSF and G-CSF increase survivin mRNA and protein significantly in 4 h and 12 h in normal neutrophils, respectively [34 ]. As the report had no descriptions of real-time PCR primer sequences and gel concentration for immunoblotting, which may not have been high enough to distinguish the subtle difference in the molecular weights of isoforms, it was unclear which isoform was quantified. Our real-time PCR primers, which corresponded to exons 1 and 2, were capable of detecting all five known isoforms. It is possible that some components included or contaminated in our FBS affected the expression levels. However, the absence of the mitigation of survivin levels in the presence of polymyxin B suggested that the increase of survivin is not a result of the contamination of LPS, a possible survivin inducer. Furthermore, three different FBS tested by us reproduced the spontaneous survivin increase and a lack of further induction by GM-CSF. Therefore, it is unlikely that the observed phenomenon depends on FBS used. The elevation of mRNA levels generally reflects a reduced degradation rate or an increased transcription rate. A possible hypothesis to be studied is that Survivin-2{alpha} mRNA degradation was down-regulated in the process of neutrophil apoptosis and that GM-CSF did not accelerate the transcription of survivin significantly. It is interesting that another study that quantified survivin mRNA levels by quantitative PCR using a primer pair corresponding to 3' UTR, common to four isoforms except Survivin-2{alpha}, showed that a combination of thrombopoietin, flt3 ligand, and stem cell factor induced survivin in G0/G1- and G2/M-phase CD34+ hematopoietic stem cells but not in CD34-negative, lineage-committed cells [30 ]. This observation suggests that the survivin-inducible effects of the cytokines decreased in the normally differentiating cells, which is compatible with our findings.

It is unlikely that survivin functions as an antiapoptotic molecule in normal neutrophils. The expectation of an antiapoptotic function of survivin has been based on its structure, which belongs to the IAP family [12 ], an enhanced survival of transformed cells that highly expresses survivin [37 38 39 40 ], or an inverse correlation between survivin levels and sizes of apoptotic fractions with active caspase-3 [30 ]. However, the neutrophil-protective effects of GM-CSF/G-CSF, which became obvious within 6 h, were not accompanied by an up-regulation of survivin, and a spontaneous increase in survivin mRNA was detected along with the reduction in the viable neutrophil fraction after 24 h. The identification of the predominant expression of Survivin-2{alpha}, which is known as a dominant-negative isoform [22 , 23 ], further diminished the possibility that survivin is involved in antiapoptosis by GM-CSF/G-CSF in normal neutrophils.

The roles of Survivin-2{alpha} in neutrophils remain unclear. Survivin-2{alpha} was previously shown to attenuate the antiapoptotic activities of Survivin via physical interaction with Survivin. However, none of the other isoforms were detectable in the neutrophils in our study, thus suggesting that Survivin-2{alpha} functions alone with no requirement of a dominant-negative mechanism. Based on the spontaneous increase of Survivin-2{alpha}, along with the growing apoptotic fractions, Survivin-2{alpha} by itself may have a proapoptotic role, which could be related to the short lifespan of neutrophils. In transiently transfected Daoy cells, Survivin-2{alpha} enlarged the apoptotic fraction [22 ]. It is also possible that Survivin-2{alpha} has some unknown functions, which are different from apoptosis regulation in neutrophils. The functions of survivin isoforms expressed in adult tissues are not limited to apoptosis regulation, as Survivin in T lymphocytes and Survivin-{Delta}Ex3 in vascular endothelial cells are required for T cell maturation and angiogenesis, respectively [31 , 32 ].

In conclusion, our study demonstrated that normal neutrophils predominantly expressed Survivin-2{alpha} but not antiapoptotic survivin isoforms, thereby suggesting that the expressed survivin was not involved in cytokine-induced antiapoptosis.


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ACKNOWLEDGEMENTS
 
This work was partially supported by project research grants from Fukushima Medical University to H. H. and Y. S. (Japan) and a grant from the Smoking Research Foundation (Japan). We thank Dr. Sachiko Maeda and Dr. Tomoyuki Ono for technical assistance and Dr. Kazuho Sakamoto and Ms. Sanae Sato for assisting data analyses.

Received May 6, 2007; revised September 3, 2007; accepted September 20, 2007.


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