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(Journal of Leukocyte Biology. 2001;70:783-792.)
© 2001 by Society for Leukocyte Biology

BCL-2 family expression in human neutrophils during delayed and accelerated apoptosis

Dale A. Moulding, Cahit Akgul, Mathieu Derouet, Michael R. H. White and Steven W. Edwards

The University of Liverpool, School of Biological Sciences, Life Sciences Building, Crown Street, Liverpool L69 7ZB, United Kingdom

Correspondence: Steven W. Edwards, University of Liverpool, School of Biological Sciences, Life Sciences Building, Crown Street, Liverpool L69 7ZB, United Kingdom. E-mail: sbir12{at}liv.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human neutrophil spontaneously undergoes apoptosis, but this type of cell death can be delayed or accelerated by a wide variety of agents. There are wide discrepancies in the literature regarding the expression of the Bcl-2 family of proteins in human neutrophils. Here, we show that A1, Mcl-1, Bcl-XL, and Bad are major transcripts in human neutrophils and that levels of these transcripts are cytokine regulated. However, no Bcl-XL protein was detected in Western blots. Protein levels for the proapoptotic proteins Bad, Bax, Bak, and Bik remained constant during culture, despite changes in the levels of mRNA for these gene products. These proapoptotic proteins were extremely stable, having very long half-lives. In contrast, A1 and Mcl-1 transcripts were extremely unstable (with ~3-h half-lives), and Mcl-1 protein was also subject to rapid turnover. These results indicate that neutrophil survival is regulated by the inducible expression of the short-lived Mcl-1 and possibly the A1 gene products. In the absence of their continued expression, these prosurvival gene products are rapidly turned over, and then the activity of the stable death proteins predominates and promotes apoptosis.

Key Words: A1 • Mcl-1 • Bcl-X • Bcl-2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human neutrophils spontaneously undergo apoptosis in vivo and in vitro, with a half-life of <24 h. However, neutrophils from inflammatory sites have a much longer lifespan [1 2 3 ]. There are many individual factors known to delay neutrophil apoptosis, including cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) [4 5 6 7 ], granulocyte colony-stimulating factor [5 ], interleukin (IL)-1ß [4 , 5 ], IL-2 [8 ], IL-4 [9 ], IL-6 [10 ], IL-15 [11 ], and interferon {gamma} (IFN-{gamma}) [12 ]; bacterial products such as lipopolysaccharide (LPS) [4 , 7 ]; endothelial transmigration [2 ]; and transcription activators such as glucocorticoids [13 ] and sodium butyrate [4 , 14 ]. Alternatively, neutrophil apoptosis can be accelerated by agents such as the translation and transcription inhibitors cycloheximide (CHX) and actinomycin D [14 , 15 ] and apoptotic ligands such as FasL [16 ]. The fungal metabolite gliotoxin, a potent protein synthesis inhibitor [17 ] that has recently been reported to be a nuclear factor {kappa}B (NF-{kappa}B) inhibitor [18 ], also induces neutrophil apoptosis [19 ]. The proinflammatory cytokine tumor necrosis factor {alpha} (TNF-{alpha}) has a dual action on neutrophil apoptosis, inducing apoptosis in a subpopulation of susceptible cells and delaying apoptosis in the remaining cells [20 ].

The mechanisms underlying the control of neutrophil apoptosis and cytokine-mediated rescue remain largely undefined, but recent research is identifying many of the processes involved. Synthesis of new proteins is required to delay neutrophil apoptosis [14 ], suggesting a role for labile survival proteins in controlling the lifespan of neutrophils. The Bcl-2 family is now recognized as central in the control of apoptosis in many cell types [21 ], and this family can be divided into two groups, antiapoptotic proteins (such as Bcl-2, Bcl-XL, Mcl-1, and A1/Bfl-1) and proapoptotic proteins (such as Bax, Bad, Bak, Bik, and Bid). The relative abundance of these proteins is thought to control the commitment of a cell into apoptosis or survival. However, other factors such as the localization, conformation, and phosphorylation state can also affect the function of this family [22 ]. The role of the Bcl-2 family in neutrophil apoptosis has been studied by a number of groups, but many of the data have been contradictory [23 ]. For example the Bcl-2 protein has been reported to be essential for delayed neutrophil apoptosis [24 , 25 ] or absent from mature neutrophils [4 , 26 27 28 ]. Similarly, Bcl-XL protein has been reported to be absent from mature human neutrophils [4 , 27 , 29 ], but recent reports suggest that there is both expression of Bcl-XL protein [30 ] and its involvement in controlling neutrophil apoptosis [31 ]. To date, the other Bcl-2 family members reported to be present in human neutrophils are Mcl-1 [4 , 32 ], A1/Bfl-1 [28 , 32 ], Bax [4 , 33 ], Bad [28 , 34 ], and Bak [28 , 35 36 ].

In part, some of these reported ambiguities in the expression of Bcl-2 family members in neutrophils might arise from differences in the assays used for detection. For example, great care must be taken using immunofluorescence to ensure that the signal does not arise from nonspecific binding of antibodies. Alternatively, highly sensitive techniques such as reverse transcriptase (RT)-PCR might detect signals from cells such as monocytes or eosinophils, which routinely contaminate neutrophil preparations. The aim of this study was to characterize the expression of Bcl-2 family members in human neutrophils with RNase protection assay (RPA) and Western blot analyses and to determine how this expression was regulated by agents that modulate survival. We also assessed the contribution of contaminating cells to the signals seen in neutrophil preparations. We report that neutrophils expressed transcripts for the antiapoptotic proteins Mcl-1, A1/Bfl-1, and Bcl-XL and that levels of these transcripts were cytokine regulated. However, although Mcl-1 protein was readily detected in neutrophils, no Bcl-X protein was detected. Because no commercial antibody is available that reliably detects human A1, this protein could not be measured in human neutrophils. Mcl-1 and A1 transcript levels (and Mcl-1 protein) have a very short half-life. In contrast, levels of the proapoptotic proteins Bad, Bax, Bak, and Bik were more stable and, unlike the transcript levels, were not regulated by exogenous agents. These results indicate that human neutrophils constitutively express a range of relatively stable proapoptotic proteins. Cytokine-regulated survival is thus regulated by the inducible expression of the survival proteins Mcl-1 and possibly A1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and culture of human neutrophils, peripheral blood mononuclear cells, eosinophils, Raji cells, and U937 cells
Neutrophils were isolated from heparinized venous blood of healthy volunteers by one-step centrifugation through neutrophil isolation medium (Cardinal Associates, Santa Fe, NM) according to the manufacturer’s instructions. After hypotonic lysis to remove contaminating erythrocytes, the cells were resuspended in RPMI 1640 supplemented with 5% pooled human male AB serum (Sigma, Poole, United Kingdom) at a concentration of 5 x 106 cells/mL. The culture was at 37°C with gentle agitation. The purity and viability after isolation were routinely >95% as assessed by May-Grünwald Giemsa staining and trypan blue exclusion, respectively. The cultures were supplemented with GM-CSF (Boehringer, East Sussex, United Kingdom) at a concentration of 50 ng/mL, LPS (Escherichia coli 055:B5; Sigma) at a concentration of 100 ng/mL, TNF-{alpha} (Boehringer) at a concentration of 10 ng/mL, IFN-{gamma} (Boehringer) at a concentration of 100 U/mL, gliotoxin (Calbiochem-Novabiochem Ltd., Nottingham, United Kingdom) at a concentration of 1 µg/mL, CHX (Sigma) at a concentration of 10 µg/mL, or actinomycin D (Sigma) at a concentration of 1 µM.

Peripheral blood mononuclear cells (PBMCs) were isolated as previously described [4 ]. U937 cells were cultured as previously described [37 ]. Raji cells were maintained in exponential growth in RPMI 1640 supplemented with 10% fetal calf serum (FCS). Eosinophils were isolated from venous blood of healthy volunteers by isolation of granulocytes and then immunomagnetic depletion of neutrophils as previously described [38 ], using CD16 MicroBeads from Miltenyi Biotech Ltd. (Surrey, United Kingdom).

Measurement of apoptosis
After culture, a 20-µL aliquot was diluted to 200 µL supplemented with RPMI 1640, and the cells were cytocentrifuged using a Shandon Cytospin3 (Runcorn, Cheshire, United Kingdom). May-Grünwald Giemsa staining of cytospins allowed apoptosis to be determined by morphology, as previously described [4 ]. This method correlates well with other markers of apoptosis [39 ].

RNA isolation and RNase protection assay
After culture of the cells, total RNA was extracted using Trizol reagent (Gibco BRL, Paisley, United Kingdom) according to the manufacturer’s instructions. The typical RNA yields were ~4 µg/107 neutrophils, 1.3 µg/106 eosinophils, and 2 µg/106 PBMCs. For the RPA, RNA from 5 x 107 neutrophils was used per sample. RiboQuant® MultiProbe RNase protection system and hAPO-2c template set (Pharmingen, San Diego, CA) were used for all RPAs along with [33P]UTP for probe labeling. The manufacturer’s instructions were followed for all steps of the RPA. Protected fragments were separated on 30-cm 5% acrylamide gels, and the radioactivity in fixed, dried gels was detected with a BioRad GS-363 Molecular Imager (Hemel Hempstead, Herts, United Kingdom). Protected fragments were identified by comparison with native probes and HeLa RNA supplied with the RPA kit. All bands were quantified using the Molecular Analyst software supplied with the molecular imager. Quantified data were expressed relative to signals obtained from the housekeeping gene L32.

Western blot analysis
After culture, the cells were lysed in boiling reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, immediately boiled for 5 min with occasional vortexing, and stored at -80°C until use. SDS-PAGE and electrotransfer to polyvinylidene difluoride membranes were performed as previously described [4 ]. U937 cells (2x105/lane) were used as positive controls. PBMCs (4x105 and 2x104/lane) were used to compare expression levels with those of neutrophils (106/lane). PBMCs (2x104) were used because this quantity is equivalent to the average contamination (2%) of these cells in neutrophil preparations. Membranes were blocked and incubated with primary and secondary antibodies as described elsewhere [4 ]. The primary antibodies used were as follows: Mcl-1 (13656E), Bcl-X (66461A and 65186E), Bax (13666E), Bak (66026E), Bik (67381A), and caspase-3 (65906E) from Pharmingen; Bcl-2 (OP60) from Calbiochem-NovaBiochem; Bad (AF819) from R&D Systems (Abingdon, Oxon, United Kingdom); and Bid (sc6538) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The horseradish peroxidase-conjugated secondary antisera used were donkey anti-rabbit immunoglobulin G (IgG) (Amersham, Little Chalfont, Bucks, United Kingdom), donkey anti-goat IgG (Santa Cruz Biotechnology), and sheep anti-mouse IgG (Sigma). Bound antibody was detected using Amersham’s enhanced chemiluminescence system. Densitometric analysis of blots was carried out as previously described [4 ]. Ponceau S-stained actin on membranes after electrotransfer has been shown to confirm equivalence of loading of neutrophil samples.

Statistical analysis
Statistical analysis was performed on data sets using analysis of variance. Significant differences between data sets were defined as P <= 0.05 and P <= 0.01. All data are presented as means ± SD where n equals the number of experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transcript levels of Bcl-2 family members in human neutrophils
The expression of Bcl-2 family genes in human neutrophils was analyzed with a commercially available RPA as described in Materials and Methods. The RPA was used with a template set as described in Materials and Methods that allowed the measurement of the Bcl-2 family transcripts Bcl-w, Bcl-XL, Bcl-XS, A1/Bfl-1, Bad (long and short RPA products), Bik, Bak, Bax, Bcl-2, Mcl-1, and the housekeeping genes L32 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). This system allows concurrent analysis of each of these transcripts from individual RNA samples. Figure 1A shows a representative image from an RPA gel. The native probes are shown in the right-hand lane, and the identity of the bound and protected probe fragments are marked on the left of the gel. The protected fragments are shorter than the probes because of the nature of the assay system. Neutrophils were cultured for 3 h under the conditions shown, and then total RNA was extracted and analyzed by RPA. The results of three separate experiments are summarized in Figure 1B . The major Bcl-2 family transcripts found in human neutrophils were A1/Bfl-1, Mcl-1, Bad (long RPA product), and Bcl-XL. The neutrophils showed expression of all the transcripts tested except Bcl-w. Of the transcripts detected, Bcl-2 was expressed at the lowest levels. The neutrophils expressed low levels of the housekeeping genes compared with the levels of expression seen in PBMCs and cultured Raji cells.



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  		Figure 1. Expression and regulation of Bcl-2 family transcripts in human neutrophils. Neutrophils were incubated under the conditions shown for 3 h before extraction and quantification of total RNA and subsequent RPA analysis. Equal amounts of neutrophil RNA were analyzed in each lane (5 µg). Ten micrograms of RNA were analyzed from PBMCs and Raji cells. (A) Representative RPA gel from one of three experiments performed. (B) Quantification of signals from three RPA analyses. Results were normalized to the levels of L32 signals and analyzed for significant differences by analysis of variance; {dagger}, P <= 0.05, *, P <= 0.01 compared with controls.

 
The Bcl-XL transcript signal is derived from neutrophils, not contaminating cells
The detection of high levels of Bcl-XL transcripts in neutrophils was unexpected because we previously reported a lack of expression of Bcl-X at the protein level in human neutrophils [4 ]. Our neutrophil preparations are routinely >=95% pure, with the contaminating cells typically consisting of <=2% PBMCs and 3% eosinophils. The RNA yields of neutrophils are low compared with other cell types, with ~0.4 µg recovered per 106 neutrophils, compared with ~2 µg per 106 PBMCs and <=2 µg per 106 eosinophils [38 ]. Therefore, a PBMC contamination of 2% is expected to contribute 10% of the RNA isolated, and 3% eosinophil contamination is expected to contribute <=15% of the RNA isolated from neutrophil preparations. We therefore analyzed RNA from both PBMCs and eosinophils to determine whether the Bcl-XL transcripts detected in neutrophil samples were derived from these contaminating cells in our neutrophil preparations. Bcl-XL transcript was easily detected in RNA from PBMCs, but the deliberate contamination of neutrophils (5x107) with 10% PBMCs (1.5x106) did not increase the Bcl-XL signal significantly. If the Bcl-XL signal from the neutrophil preparations was derived solely from contaminating PBMCs, then the signal should have increased five-fold when the neutrophil sample was contaminated with 10% PBMCs. Therefore, the Bcl-XL signal detected in the neutrophil samples was not derived from contaminating PBMCs (Fig. 1A) . In contrast, the deliberate contamination of the neutrophil preparations with PBMCs significantly increased the signals obtained from the housekeeping genes L37 and GAPDH, which are expressed in PBMCs at much higher levels than in neutrophils.

We next analyzed RNA from eosinophils, as shown in Figure 2A . One microgram of eosinophil RNA is equivalent to a 5–7% contamination of eosinophils in a neutrophil preparation, and 5 µg are equivalent to a 25–35% contamination, far higher than the 3% contamination we normally see. The Bcl-2 family transcript levels in 1 µg and 5 µg of eosinophil RNA were well below the levels seen in neutrophil RPAs. Based on this evidence, we concluded that contaminating eosinophils did not contribute to the neutrophil signals in these experiments. Therefore, the Bcl-XL transcripts detected in the neutrophil preparations were derived almost entirely from the neutrophils and were not derived from contaminating cells.



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  		Figure 2. Expression of Bcl-2-related transcripts in eosinophils and stability of transcripts in neutrophils. (A) Neutrophils were cultured for 0, 1, 3, or 5 h with 1 µM actinomycin D (ActD) before extraction and quantification of total RNA. Then RPA analysis was performed on 5 µg of RNA per lane. Eosinophil RNA was extracted immediately after isolation of eosinophils. The stability experiment is representative of three experiments performed. (B) Apoptosis of neutrophils after a 6-h culture under the conditions shown. (C) Quantification of Bcl-XL, A1, and Mcl-1 transcripts after treatment of neutrophils with actinomycin D.

 
Actinomycin D induces neutrophil apoptosis, accompanied by a decrease of A1 and Mcl-1 transcripts, without a decrease in Bcl-XL transcripts
To further investigate the possible role of Bcl-XL transcripts in controlling neutrophil apoptosis, we performed experiments with the RNA polymerase II inhibitor actinomycin D. Figure 2B shows the apoptosis measurements from two experiments. Actinomycin D-treated neutrophils displayed much higher rates of apoptosis than control neutrophils, in agreement with previously published data [15 ]. The inhibition of mRNA synthesis by actinomycin D resulted in a rapid loss of both Mcl-1 and A1 transcripts, but Bcl-XL transcript levels were much more stable (Fig. 2A and 2C) . Indeed, we estimate the Mcl-1 mRNA half-life to be ~2.5 h and the A1 mRNA half-life to be ~3 h. In the experiments described here, the levels of Bcl-XL mRNA did not change significantly. These results suggest that the apoptosis induced by actinomycin D (Fig. 2B) must be independent of Bcl-XL transcript levels and is much more likely to be driven by the rapid decrease in Mcl-1 and A1 transcript levels.

Regulation of Bcl-2 family mRNA levels by apoptosis-modulating agents
We then examined the effects of agents known to modulate neutrophil apoptosis on changes in expression of these genes. Figure 1B shows quantification of transcript levels from three separate experiments. The abundance of the A1 transcript was significantly increased by all the agents tested that delay neutrophil apoptosis, namely GM-CFS, LPS, TNF-{alpha}, and IFN-{gamma}. Of the apoptosis-delaying agents tested, only GM-CSF and LPS caused a significant increase in Mcl-1 mRNA levels. Notably, the use of TNF-{alpha}, which induces apoptosis in a small subset of neutrophils but delays apoptosis in the majority of neutrophils in a population [20 ], increased A1 mRNA levels but decreased Mcl-1 mRNA levels. The levels of Bcl-XL transcripts were similarly increased by apoptosis-delaying agents, but the fold increases, although statistically significant, were only slight compared with those seen for A1 and Mcl-1. Bcl-2 transcript levels were largely unaffected by apoptosis-modulating agents. The levels of proapoptotic transcripts were also increased by the apoptosis-delaying agents GM-CSF, LPS, and TNF-{alpha}, but they were not affected by IFN-{gamma}. These increases were most marked for Bad, whereas Bax, Bak, and Bik showed more modest increases in transcript levels.

Gliotoxin, which was reported to inhibit translation [17 ] and NF-{kappa}B activity [18 ], was also studied in this system. We found that gliotoxin causes a depletion of all transcripts except Bcl-XL by 3 h. Even the transcript levels of the housekeeping genes L32 and GAPDH were depleted by this treatment. This general depletion of all transcripts was not simply due to a lower RNA yield from these cells. RNA yields were quantified, and they did not deviate significantly between samples. After quantification of RNA isolated from 5 x 107 neutrophils, 5 µg of RNA were analyzed per treatment. The results presented in Figure 1B are shown as the levels of expression relative to the expression of the housekeeping genes. All Bcl-2 family transcripts tested except Bcl-XL were depleted by gliotoxin significantly more than the housekeeping genes were. The translation inhibitor CHX showed quite different results from those seen with gliotoxin, with increases in the levels of transcripts for genes such as A1 and MCL1.

Changes in the levels of Bcl-2 family proteins
Having demonstrated the expression of a variety of Bcl-2-related transcripts in human neutrophils and the modulation of mRNA levels of these genes by apoptosis-modulating agents, we next wanted to confirm the expression of these genes at the protein level. Furthermore, we wished to see whether the changes in abundance of different Bcl-2 family transcripts were reflected in the levels of Bcl-2-related proteins. We performed Western blot analyses on protein extracts from neutrophils cultured in media alone or supplemented with GM-CSF, TNF-{alpha}, or CHX. Apoptosis was assessed by morphology, as shown in Figure 3 . GM-CSF significantly delayed apoptosis at all the times measured. TNF-{alpha} accelerated apoptosis when measured at 6 h, with 18.9% (±4.3%) apoptosis compared with control cultures, which showed 6.7% (±1.6%) apoptosis. At the 22-h time point, TNF-{alpha}-treated cells showed similar levels of apoptosis to those of controls, with 44.6% (±5.6%) apoptosis in control cultures and 44.5% (±7.7%) apoptosis in cultures supplemented with TNF-{alpha}. This apparent disparity is explained by the dual action of TNF-{alpha} on neutrophil apoptosis, in which a subset of cells succumb to the TNF-{alpha} death signal, whereas the cells resistant to this signal benefit from the apoptosis-delaying action of TNF-{alpha} [20 ]. Inhibition of protein synthesis with CHX accelerated apoptosis at both 6 and 22 h. Western blot analyses for a range of Bcl-2-related proteins and caspase-3 were performed on extracts from neutrophils treated with these four agents.



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  		Figure 3. Modulation of neutrophil apoptosis by GM-CSF, TNF-{alpha}, and CHX. After culture of neutrophils under the conditions indicated for 6 or 22 h, apoptosis was assessed by morphology. (A) Results from three independent experiments, analyzed for significant differences by analysis of variance; {dagger}, P <= 0.05, *, P <= 0.01 compared with controls. (B) Representative fields from neutrophil cytospins. Arrow in upper left panel indicates early apoptotic neutrophil (vacuolization, condensation of one or more nuclear lobes); open arrows indicate apoptotic neutrophils (all nuclear lobes condensed, loss of lobes); small closed arrows indicate very late apoptotic/necrotic neutrophils (near complete loss of nuclear staining).

 
We compared the protein levels in neutrophils to those in U937 cells and PBMCs. We also measured signals in a sample of 2 x 104 PBMCs, which is equivalent to the numbers of PBMCs contaminating each lane of our neutrophil preparations, to determine whether the bands seen in neutrophil lanes are derived from contaminating cells. Figure 4A shows representative Western blots for all proteins analyzed, namely, Mcl-1, Bcl-X, Bcl-2, Bak, Bad, Bax, Bik, Bid, caspase-3, and Ponceau S-stained actin. Figure 4B shows the quantified results from three separate experiments for the levels of all proteins tested.



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  		Figure 4. Western blot analysis of Bcl-2 family proteins in neutrophils treated with apoptosis-modulating agents. Western blots were performed on proteins extracted from human neutrophils and PBMCs and U937 cells after culture under the conditions indicated: 0h, freshly isolated neutrophils; C, culture in medium only; G, GM-CSF–supplemented medium; T, TNF-{alpha}-supplemented medium; X, CHX-supplemented medium. Neutrophils were cultured for 6 or 22 h. (A) Representative Western blots from one of three experiments performed. Some blots were exposed to film for extended times to allow detection of less intense signals. Ponceau S-stained actin is shown to indicate equivalence of loading. (B) Quantification of Western blots from three experiments performed and analyzed for significant differences by analysis of variance; {dagger}, P <= 0.05, *, P <= 0.01 compared with controls.

 
The detection of Bcl-XL mRNA and the presence of very low levels of Bcl-2 mRNA in neutrophils led us to examine carefully for the presence of these proteins. We detected a strong signal for Bcl-XL protein in U937 cells and PBMCs as a band just below 29 kDa, but we could detect no Bcl-XL protein in neutrophil extracts. With longer exposures, we detected Bcl-XL signals in neutrophil preparations, but the level of expression was estimated at ~2% of that seen in PBMCs on a cell-to-cell basis. Furthermore, the intensity of the Bcl-XL signal was very similar to that detected in the lane containing 2 x 104 PBMCs, which is equivalent to the numbers of PBMCs contaminating each neutrophil lane. We could not detect Bcl-XL and Bcl-XS proteins using a different primary antibody, a polyclonal antiserum against Bcl-X proteins (results not shown). The band seen below 29 kDa in neutrophil lanes was ~24–25 kDa. This is too small to be Bcl-XL protein and, again, was seen only on very long exposures. We therefore concluded that there is no Bcl-X protein detectable by this assay in human neutrophils. Bcl-2 protein was similarly absent from neutrophils, with Bcl-2 protein detected in neutrophil samples at levels predicted if the signal was derived solely from contaminating PBMCs.

Mcl-1 protein levels are changed by apoptosis-modulating agents
The antiapoptotic protein Mcl-1 was abundantly expressed in neutrophil preparations. The levels of expression of this protein decreased as neutrophils were cultured and entered apoptosis (Fig. 4) . Furthermore, delaying apoptosis with GM-CSF caused an increase in and maintenance of Mcl-1 protein levels, as previously reported [4 ]. The increased level of apoptosis induced by TNF-{alpha} at 6 h (Fig. 3A) was not accompanied by a depletion of Mcl-1 protein at this time (Fig. 4A 4B) . This apparent anomaly might be explained by the fact that Mcl-1 protein levels might decrease in the subpopulation of cells killed by TNF-{alpha} but increase in those cells that survive. Thus, the total cellular levels in the population may remain unchanged. By 20 h, the levels of Mcl-1 protein had fallen considerably in both the control and the TNF-{alpha}-treated samples, with only GM-CSF slowing the decline in Mcl-1 levels compared with control samples. The use of CHX, which potently induced neutrophil apoptosis (Fig. 3) , allowed us to estimate the stability of the Mcl-1 protein in human neutrophils. We found that the half-life of Mcl-1 protein in this system was ~6 h. We previously found the Mcl-1 protein half-life to be nearer to 30 min for neutrophils in cultures supplemented with 10% FCS (D. A. Moulding, unpublished results) than with 5% human serum as used here, which was similar to the protein half-life seen in other systems in which FCS was used [40 , 41 ].

We also incubated PBMCs at concentrations of 2 x 104 and 1 x 105/mL in the absence and presence of GM-CSF. This was equivalent to the PBMC contamination of our neutrophil preparations by 2 and 10%, respectively. No signals were noted for Bcl-X, Bcl-2, or Mcl-1 at 6 or 22 h using exposure times that readily detected changes in Mcl-1 levels in our neutrophil preparations (D. A. Moulding, unpublished results). Thus, GM-CSF modulation of Mcl-1 protein levels was not caused by PBMC contamination.

A recently identified splicing variant of Mcl-1, Mcl-1S, has been shown to have a proapoptotic function [42 , 43 ]. The predicted molecular size of Mcl-1S is 29.4 kDa, but an in vitro-translated product runs at ~34 kDa [41 ]. The Mcl-1 antiserum used in this report was raised against a region of the protein present in both Mcl-1 and Mcl-1S [42 ]. We show that two shorter proteins were detected by these Mcl-1 antibodies in human neutrophils, a 34-kDa protein and a 29-kDa protein (Fig. 4A) . However, these proteins are much less abundant (e.g., <10%) than the full-length Mcl-1 transcript. The levels of the 29-kDa protein were parallel to the levels of full-length Mcl-1 protein. However, the levels of the 34-kDa protein remained largely unchanged in all conditions.

Levels of proapoptotic proteins remain unchanged during neutrophil culture
The proapoptotic proteins Bak, Bad, Bax, Bik, and Bid were all expressed in neutrophils. Bak and Bik were expressed at lower levels than Bad, Bax, and Bid in neutrophils compared with PBMCs and U937 cells (Fig. 4) . Despite the changes in the mRNA levels of Bad, Bax, Bak, and Bik in response to GM-CSF, LPS, and TNF-{alpha}, protein levels were unchanged by these treatments. CHX treatment showed that these proteins are remarkably stable (Fig. 4) . The most labile of these four proapoptotic proteins was Bak, whose levels decreased to 66% (±16.7%) of the levels measured in freshly isolated neutrophils after 22 h of CHX treatment.

The levels of the proapoptotic protein Bid were decreased by treatments that induced apoptosis. Bid is inactive until cleaved by caspase-8, producing an active 15-kDa protein that is not detected by the antiserum used in this study [44 ]. We showed in these experiments that full-length Bid is lost as neutrophils become apoptotic. Casapse-3 was also analyzed by Western blotting, which showed a decrease in the levels of full-length (inactive) pro-caspase-3 as cells became apoptotic, accompanied by the appearance of cleaved (active) p17 caspase-3 (Fig. 4) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we investigated the expression of Bcl-2 family members in human neutrophils. We measured both mRNA and protein levels for a range of Bcl-2 family members using RPA and Western blot analysis. We chose the two assay systems that we used because we believe the results they provide are reliable and allow us to assess the contribution of contaminating cells to signals obtained in neutrophil preparations. The sizes of proteins detected on Western blots were shown, next to positive controls, to confirm that the proteins detected by antisera were of the expected molecular mass. Previous experiments have used assays such as RT-PCR and immunofluorescence labeling and then flow cytometry to measure mRNA and protein levels in human neutrophils. The great sensitivity of RT-PCR might lead to the detection of signals from contaminating cells, so we avoided the use of that assay. Immunofluorescence labeling of intracellular antigens can also generate misleading results because antisera might react with proteins other than the intended target. Indeed, a commonly used monoclonal antibody against Bcl-2 binds strongly to 40- and 80-kDa proteins in human neutrophils but detects only Bcl-2 protein in other cell types [4 ]. Immunofluorescence, on the other hand, does have the advantage that protein levels can be detected in individual cells that can be directly related to other measures of apoptosis.

With RPA analysis, we showed the relative abundance of Bcl-2-related transcripts in human neutrophils. The assay clearly showed that A1, Mcl-1, Bad, and Bcl-XL are the most abundant Bcl-2-related transcripts in human neutrophils. This assay produces two different-sized products attributable to Bad (as described in the manufacturer’s manual). In neutrophils, the longer product is predominant (similar to that seen in PBMCs), whereas in Raji cells the shorter product predominates. The mechanism of formation and the functional significance of these two different bands are not known. They might result from alternative splicing, as has been demonstrated for Bcl-X and more recently for Mcl-1 [42 , 43 ].

The presence of Bcl-XL mRNA in human neutrophils was carefully examined by comparing their signals with those seen in PBMCs and eosinophils, cells that commonly contaminate neutrophil preparations. The strong Bcl-XL signal in PBMCs (Fig. 1A) is not solely responsible for the signal seen in neutrophils. Deliberately contaminating neutrophils with fivefold as many PBMCs (10%) as normally present in neutrophil preparations (2%) did not increase the Bcl-XL mRNA signal fivefold (Fig. 1A) . In fact, there was only a slight increase in signal intensity. Similarly, analysis of eosinophil RNA (Fig. 2A) showed that the Bcl-XL signal in neutrophils could not be from contaminating eosinophils. Therefore, Bcl-XL mRNA is expressed in human neutrophils. However, no Bcl-XL protein is present in human neutrophils, as shown in Figure 4A and 4B . The 29-kDa Bcl-XL protein can be detected in neutrophil samples, but it is present only because of contamination with PBMCs. A 24-kDa band was detected with Bcl-X antisera in human neutrophils, but this did not correspond to the size of Bcl-XL on Western blots, and the levels of expression of this protein were very low. However, the increases in Bcl-XL transcript levels in response to apoptosis-delaying agents suggests the Bcl-XL transcript might play a role in extending the neutrophil lifespan, perhaps by encoding for a protein not detected by the Bcl-X antibodies used in this study. We further investigated this possibility by inhibiting mRNA synthesis with actinomycin D. As shown in Figure 2 , the Bcl-XL mRNA signals were remarkably stable after actinomycin D treatment. These results indicate that the presence of the Bcl-XL transcript does not correlate with neutrophil apoptosis, because actinomycin D treatment rapidly induced apoptosis but had little effect on Bcl-XL transcript abundance. The rapid decrease in the levels of A1 and Mcl-1 transcripts is more likely to be the driving force behind the apoptosis induced by actinomycin D in human neutrophils.

The fungal metabolite gliotoxin is a potent protein synthesis inhibitor [17 ] and has recently been shown to inhibit NF-{kappa}B activation [18 ]. It is known to accelerate neutrophil apoptosis, which has been thought to be caused by its ability to inhibit NF-{kappa}B [19 ]. However, we have shown here that all transcripts analyzed by RPA except Bcl-XL were depleted by gliotoxin within 3 h of treatment (Fig. 1) . Although this was expected for genes such as A1, which are NF-{kappa}B responsive [45 , 46 ], it was surprising to see the housekeeping gene transcripts L32 and GAPDH depleted by gliotoxin. Furthermore, Mcl-1 transcripts were depleted by gliotoxin, but this has no NF-{kappa}B response element [47 , 48 ]. This general effect of gliotoxin of down-regulating all transcripts is not caused by its protein synthesis-inhibiting action because CHX treatment increases the abundance of some transcripts, such as Mcl-1 (Fig. 1) , as has been shown previously [49 ]. Gliotoxin also depleted levels of Mcl-1 protein by 6 h (results not shown). Therefore, gliotoxin-induced neutrophil apoptosis is unlikely to be entirely from its action on NF-{kappa}B and is probably caused by a general depletion of mRNA levels in neutrophils, perhaps in combination with gliotoxin’s ability to inhibit protein synthesis.

The up-regulation of A1 transcript levels by the apoptosis-inhibiting agents GM-CSF, LPS, TNF-{alpha}, and IFN-{gamma} suggests that the A1 protein might play an important role in the control of neutrophil apoptosis, which has been indicated previously using LPS and granulocyte-colony stimulating factor as apoptosis-delaying agents [32 ]. However, because no antiserum that reliably recognizes human A1 protein is available, no conclusions as to the role of this protein can be drawn at present. Mcl-1 transcript levels are also up-regulated by GM-CSF and LPS, but they are unaffected by IFN-{gamma} and down-regulated by TNF-{alpha}. Protein levels of Mcl-1 are also up-regulated and maintained by both GM-CSF (Fig. 4) and LPS [4 ], although TNF-{alpha} treatment had very little effect on Mcl-1 protein levels (Fig. 4) . Therefore, these results suggest that A1 and Mcl-1 functions might be differentially regulated to delay neutrophil apoptosis. The apoptosis-delaying action of TNF-{alpha} seems to act primarily through A1, and Mcl-1 appears to play a lesser role in the delay of apoptosis by this cytokine. A1 has indeed been shown to be a TNF-{alpha}-induced gene that prevents TNF-{alpha}-induced apoptosis [45 ].

The up-regulation of transcript levels for the proapoptotic Bcl-2 family members Bad, Bax, Bak, and Bik by apoptosis-delaying agents are not accompanied by increases in the levels of these proteins. One explanation for this might be the control of expression at the translation level because translation of these mRNA species is inhibited by apoptosis-delaying agents. Lack of translation of particular mRNA species can be associated with an increase in their levels of stability [50 ]. Thus, the results presented here might indicate inhibition of translation of these mRNA’s leading to mRNA accumulation because of decreased mRNA degradation. Therefore, the rise in proapoptotic mRNA levels might be a result of increased stability rather than increased transcription.

The levels of the proapoptotic proteins Bax, Bad, Bik, and Bak (but not Bid) were largely unchanged by apoptosis-modulating agents. The levels of full-length Bid decreased during neutrophil apoptosis, which is consistent with the activation of Bid by caspase-8 [44 ]. Two previous reports [30 , 31 ] showed a depletion of Bax protein levels by apoptosis-delaying agents such as GM-CSF, in contrast to the data presented here and previously by our group [4 ]. This disparity might be explained by the differences in the methods and antibodies used. Both of these studies used Bax antibodies from Santa Cruz Biotechnology, and no size was given for the detected protein. In our experiments, (D. A. Moulding, unpublished results), the antibody from Santa Cruz Biotechnology (P19) detected a protein at 24 kDa rather than 21 kDa, which is the accepted size of human Bax on SDS-PAGE [4 , 51 ]. Furthermore, the use of flow cytometry to detect changes in cellular proteins might give misleading results if the antibodies used give nonspecific binding and/or if cytoplasmic proteins are lost during permeabilization procedures. Our results clearly showed no change in p21-Bax after treatment of human neutrophils with agents such as GM-CSF.

The stability of the Bad protein is surprising because this protein has two PEST sequences [rich in proline (P), glutamic acid (E), serine (S), and threonine (T)], which are thought to be associated with the targeting of proteins for rapid turnover [52 ]. Mcl-1 also contains PEST sequences, and in the experiments shown here, the protein had a half-life of ~6 h. We considered the possibility that the Bad antiserum was detecting a protein other than human Bad, but we considered this unlikely because the band detected at ~29 kDa was the same size as the human Bad detected after SDS-PAGE [34 ]. The relatively long half-life of this protein might be explained by the reported "conditional nature of the PEST proteolytic signal" [52 ]. The PEST signals are thought to be masked in certain protein conformations or when proteins are in complexes. Thus, Bad might be protected from PEST-targeted proteolysis as a result of its association with other Bcl-2-related proteins or 14-3-3 protein [53 ]. The accessibility of PEST signals in the Bad protein might also play a role in the apparent stability of this protein in CHX-treated neutrophils. CHX induces apoptosis, and Bad might enter a stable conformation in these conditions. Conversely, the lack of an increase in the level of Bad protein, despite increased levels of Bad mRNA, after apoptosis-delaying treatments might be caused by the Bad protein adopting a less stable conformation in which the PEST sequences are accessible. We have shown that deletion of PEST sequences in Mcl-1-green fluorescent protein fusion proteins does not improve Mcl-1 protein stability [54 ]. Therefore, there are clearly other factors involved in determining the stability of Bcl-2 family proteins.

In conclusion, neutrophil apoptosis and survival might be controlled by the inducible expression of the antiapoptotic protein Mcl-1 and possibly also A1, whereas the levels of the proapoptotic proteins Bax, Bad, Bik, and Bak remain constant. Measurements of A1 protein levels are required to confirm the importance of this latter protein in human neutrophils. A1 and Mcl-1 mRNA levels are differentially regulated and might therefore act individually or in combination to delay neutrophil apoptosis. We previously showed that antisense depletion of Mcl-1 is sufficient to induce apoptosis in differentiating U937 cells without changing the levels of other Bcl-2-related proteins [36 ]. This indicates that loss of Mcl-1 alone is sufficient to trigger apoptosis. Whether loss of A1 also induces apoptosis in neutrophils or whether this protein is merely involved in resistance to TNF-{alpha}-induced cell death is unknown at present. It is also necessary to determine whether posttranslational modification of Bcl-2 family proteins plays a role in neutrophil survival.


   ACKNOWLEDGEMENTS
 
D. A. M. is supported by the Wellcome Trust, and C. A. is supported by Canakkale Onsekiz Mart Universitesi.

Received October 30, 1999; revised May 25, 2001; accepted June 18, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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