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PHAGOCYTES
From Emma Children's Hospital, Academic Medical
Center, University of Amsterdam; Central Laboratory of the Netherlands
Blood Transfusion Service (CLB) and Laboratory for Experimental and
Clinical Immunology, Academic Medical Center, University of Amsterdam,
The Netherlands; Medical Academy, Nizhnyi Novgorod, Russia.
The exact mechanism of apoptosis in neutrophils (PMNs) and the
explanation for the antiapoptotic effect of granulocyte
colony-stimulating factor (G-CSF) in PMNs are unclear. Using specific
fluorescent mitochondrial staining, immunofluorescent confocal
microscopy, Western blotting, and flow cytometry, this study found that
PMNs possess an unexpectedly large number of mitochondria, which are involved in apoptosis. Spontaneous PMN apoptosis was associated with
translocation of the Bcl-2-like protein Bax to the mitochondria and
subsequent caspase-3 activation, but not with changes in the expression
of Bax. G-CSF delayed PMN apoptosis and prevented both associated
events. These G-CSF effects were inhibited by cycloheximide. The
general caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) prevented caspase-3 activation and apoptosis in
PMNs, but not Bax redistribution. PMN-derived cytoplasts, which lack a
nucleus, granules, and mitochondria, spontaneously underwent caspase-3
activation and apoptosis (phosphatidylserine exposure), without Bax
redistribution. zVAD-fmk inhibited both caspase-3 activation and
phosphatidylserine exposure in cultured cytoplasts. Yet, G-CSF
prevented neither caspase-3 activation nor apoptosis in cytoplasts,
confirming the need for protein synthesis in the G-CSF effects. These
data demonstrate that (at least) 2 routes regulate PMN apoptosis: one
via Bax-to-mitochondria translocation and a second
mitochondria-independent pathway, both linked to caspase-3 activation.
Moreover, G-CSF exerts its antiapoptotic effect in the first, that is,
mitochondria-dependent, route and has no impact on the second.
(Blood. 2002;99:672-679) During the last decade, apoptosis, or
programmed cell death, has attracted great interest. Whole families of
molecules have been described as regulators of apoptosis. The Bcl-2
family of proteins and the family of caspase proteases are internal key regulators of the cell fate.1,2 Recent work has
demonstrated that these 2 groups of proteins are intimately connected
at the level of mitochondria: the Bcl-2 homologues govern the activity of caspases by exerting their effect through the regulation of the
mitochondrial function.3 Proapoptotic Bcl-2 proteins, such as Bax, disturb the mitochondrial membrane integrity by forming channels, which facilitates the subsequent release of cytochrome c and the activation of Apaf-1 and downstream
caspases.4-7 Bax moves from the cytosol to the
mitochondria on initiation of apoptosis.8-11 This
translocation seems to precede caspase activation.12 Some authors have suggested a critical role for Bax movement to mitochondria in the execution of the apoptotic program.11,13 Moreover,
the antiapoptotic protein Bcl-2 is believed to mediate, at least
partially, its effect through the inhibition of Bax
redistribution.11,14
A downstream event of Bax-mediated mitochondrial dysfunction is the
activation of caspases. This family of proteases executes the cleaving
of specific targets, which, finally, leads to cell disassembly and
death. Among these proteases, caspase-3 stands out for the great number
of substrates that it destroys. These targets include nuclear
proteins,15 cytoplasmic structures,16 and
cytoskeleton elements.17 Bcl-2 and its antiapoptotic
homologue, Bcl-XL, are also substrates for
caspase-3.18,19 For this reason, caspase-3 is called the
main executioner of apoptosis.
In the present study, we have investigated the apoptotic process of
mature human neutrophils (PMNs). These cells have a constitutively short life span and rapidly undergo spontaneous apoptosis within hours.20 Survival of PMNs can be extended by delaying
apoptosis with a wide variety of agents, including colony-stimulating
factors, such as granulocyte-macrophage colony-stimulating factor
(GM-CSF) and granulocyte colony-stimulating factor
(G-CSF).21-25 To date, the mechanisms of their prosurvival
effect remained unclear. Some authors have suggested that active
protein synthesis is required for agents such as GM-CSF to produce
their prosurvival effect, whereas blocking of protein synthesis
promotes PMN apoptosis and abrogates the effect of these
agents.25-27 Recently, it has been proposed that the
antiapoptotic action of these cytokines may be connected with
Bcl-2-related proteins and with regulation of caspase-3 activity.
GM-CSF has been suggested to induce expression of the antiapoptotic
Bcl-2 homologue Mcl-1,23 to maintain the level of
Bcl-XL, and to influence caspase-3
activity,28 thus preventing apoptosis. Down-regulation of
Bax expression by G-CSF and GM-CSF has been considered to delay
apoptosis.24
Regarding a role of Bcl-2 proteins in this process, one should realize
that PMNs are considered to possess no or only few rudimentary
mitochondria, which do not play a role in the active life of the
cell.29,30 Therefore, PMNs have been considered to be "a
nonmitochondrial scene" of apoptosis.23 On the other hand, mitochondria constitute a crossroad of apoptosis regulation between members of the Bcl-2 family and the caspases. In the present study, we show that PMNs do contain mitochondria, in contrast to what
was believed earlier. Also, we observed Bax movement to mitochondria
and a strong correlation of this event with caspase-3 activation and
spontaneous PMN apoptosis. The prosurvival effect of the prosurvival
factor G-CSF was demonstrated to coincide with prevention of
Bax-to-mitochondria translocation and with inhibition of caspase-3
processing in a transcription-dependent manner. At the same time, using
PMN cytoplasts, which lack mitochondria, granules, and
nuclei,31 we found a mitochondria-independent pathway of
caspase-3 activation and subsequent apoptosis. Moreover, G-CSF had no
effect on this last route of apoptosis.
PMN preparation and culturing
Cytoplast preparation and culturing
Measurement of apoptosis Apoptosis of PMNs was measured by flow cytometry with the annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis assay kit (Bender Medsystems, Vienna, Austria).34 About 105 fresh cells or cells after overnight incubation were washed once in ice-cold Hepes medium with 2.5 mM Ca++. All further steps were performed in this medium on ice. The cells were then incubated for 45 minutes with annexin-V-FITC, 1:250, which specifically binds phosphatidylserine residues on the cell membrane. During the last 10 minutes PI was added to a final concentration of 1 µg/mL. PI is a fluorescent dye that intercalates into DNA once the cell membrane has become permeable. After incubation, the cells were washed once and analyzed by FACScan (Becton Dickinson, San Jose, CA). Viable cells were defined as negative for annexin-V-FITC and PI staining. Cell survival was expressed as a percentage of viable cells in relation to the total number of counted cells. Cytoplast apoptosis was assessed in the same way, except for the PI step, with 4 × 105 cytoplasts for each preparation.Measurement of protein expression of Bcl-2 family members The expression levels of Bax, Bak, Mcl-1, and Bcl-XL were determined by FACS analysis, according to Van Vliet et al,35 with some modifications. After isolation and culture, 105 PMNs were washed once in PBS. The cells were then fixed with 2% (wt/vol) paraformaldehyde in PBS for 15 minutes at room temperature, washed twice in PBS, and resuspended in staining buffer containing 0.1% saponin (wt/vol; Calbiochem) and 1% (vol/vol) bovine serum albumin (BSA; Boseral, Organon Teknika, Eppelheim, Germany) in PBS. All further steps were performed in this solution at room temperature. The cells were distributed over a 96-well plate with conical bottoms, incubated for 10 minutes for permeabilization, and were spun down. Supernatant was removed, and PMNs were resuspended in 50 µL of the appropriate primary antibody or isotype control antibody solution. The polyclonal rabbit antibodies against human Bax, Mcl-1 (Pharmingen, San Diego, CA) or Bcl-XL (Calbiochem) were used at a final dilution of 1:250. The monoclonal mouse antihuman Bak antibody (Calbiochem) was used at a final concentration of 25 µg/mL. After 45 minutes of incubation the cells were washed twice and were resuspended in 50 µL of the secondary antibody, Alexa-488-conjugated goat-antirabbit/mouse IgG (Molecular Probes, Eugene, OR), at a final concentration of 2.5 µg/mL. Incubation with the secondary antibody took 45 minutes. After this procedure, the PMNs were washed twice and were analyzed by FACScan. Expression levels of the proteins of interest were assessed by measuring the mean fluorescence intensity (MFI) of bound antibodies.Confocal laser scanning microscopy For confocal laser scanning microscopy (CLSM) analysis, PMNs and cytoplasts were fixed and permeabilized as described for flow cytometry. To investigate the staining patterns of Bcl-2-related proteins, the cells were incubated with the appropriate primary antibodies (see above) with subsequent secondary staining with Alexa-568-conjugated goat-antirabbit/mouse IgG (final concentration 2.5 µg/mL). All incubations were performed as described above. After the last wash step, the cells were resuspended in 30 µL PBS, brought on microscope glass slides, and covered by coverslips. The slides were analyzed by a confocal laser scanning microscope (LSM510, Carl Zeiss, Heidelberg, Germany) equipped with Ar and HeNe lasers.To study the activation of caspase-3, incubations were performed with the monoclonal rabbit antibody against active human caspase-3 (Pharmingen) at a final dilution of 1.5 µg/mL, and AlexaFluor488-conjugated goat-antirabbit IgG. For the mitochondrial staining MitoTracker GreenFM (Molecular Probes) was used. About 105 fresh or cultured PMNs (or 4 × 105 cytoplasts) were incubated in IMDM for 30 minutes in a 5% CO2 incubator at 37°C with 100 nmol/L MitoTracker Green FM. Then the cells were spun down, resuspended in 30 µL stain-free medium, placed on microscope glass slides, and analyzed by CLSM. To obtain simultaneous staining of mitochondria and intracellular antigens, the cells were stained with 1 µmol/L MitoTracker Green FM, fixed, permeabilized, labeled for proteins of interest, and analyzed as described above. Western blotting The cleavage of procaspase-3 was determined by Western blotting as described elsewhere.28 Briefly, whole cell lysates were obtained by boiling 5 × 105 PMNs or 2 × 106 cytoplasts in sodium dodecyl sulfate (SDS) sample buffer with 4% mercaptoethanol for 5 minutes. Proteins were resolved on 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and were electrotransferred to nitrocellulose membrane (Hybond-ECL; Amersham, Arlington Height, IL). The blots were incubated for 1 hour with 1:1000 diluted polyclonal rabbit antihuman caspase-3 antibodies (Pharmingen), which recognize both inactive procaspase-3 and its cleavage product. Thereafter, the blots were incubated for 1 hour with swine antirabbit-IgG conjugated with horseradish peroxidase (HRP; Dako, Glostrup, Denmark), followed by band visualization with enhanced chemiluminescence as described by the manufacturer (Amersham). As a positive control, Jurkat T-cell lysates were used (Pharmingen), which contain caspase-3 as a 32-kd proenzyme.Measurement of G-CSF receptor expression in PMNs and cytoplasts The PMNs (105) or cytoplasts (4 × 105) were washed once in PBS and were incubated with a monoclonal antibody (mAb) against human G-CSF receptor (Pharmingen) at a final concentration of 5 µg/mL or with an isotype control. After a 45-minute incubation, the cells were washed twice and stained for 45 minutes with R-phycoerythrin-labeled secondary antibody (Dako) at a final dilution of 10 µg/mL. After washing, the antibody binding was assessed by FACS analysis.Statistics Where applicable, the Student t test was used to evaluate the significance of differences between the sample means. Statistical significance was defined as P less than .05.
G-CSF and zVAD-fmk effectively delay PMN apoptosis Freshly isolated PMNs contained 95.6% ± 2.7% live cells, that is, negative for both annexin-V and PI (Figure 1A). PMNs cultured overnight without additions underwent spontaneous apoptosis, with a survival not higher than 21.0% ± 3.3% (Figure 1B). In the presence of G-CSF, apoptosis was delayed, resulting in 63.2% ± 2.9% surviving cells (Figure 1C). In the presence of zVAD-fmk (Figure 1D) apoptosis was minimal, and 88.0% ± 2.3% PMNs were alive. Both agents produced a significant delay of apoptosis when compared with control incubations (P < .05).
PMNs do contain mitochondria: structural changes during apoptosis To check whether PMNs contain mitochondria, we performed mitochondrial staining with MitoTracker Green FM. In fresh PMNs (Figure 2A), the mitochondria showed a tubular shape, whereas in overnight-cultured control PMNs (Figure 2B) the mitochondria had changed into large unstructured aggregates. G-CSF (Figure 2C) prevented the mitochondrial aggregate formation, but addition of zVAD-fmk (Figure 2D) did not influence the aggregation of the mitochondria. The identity of the mitochondria was confirmed with an antibody against a component of the mitochondrial cytochrome oxidase system (mouse mAb antihuman cytochrome c oxidase complex IV, Molecular Probes; data not shown).
PMN apoptosis and caspase-3 activation: inhibition by G-CSF or zVAD-fmk We studied caspase-3 processing during spontaneous apoptosis by Western blotting and CLSM. Caspase-3 is expressed in normal PMNs as a 32-kd inactive precursor, which is proteolytically cleaved into an active form on mediation of apoptosis.36 This cleavage generates a large (17-20 kd) and a small (3-12 kd) subunit. In fresh PMNs (Figure 3, lane B) caspase-3 was found mainly as a 32-kd proenzyme. In contrast, PMNs cultured overnight without additions, undergoing spontaneous apoptosis (Figure 3, lane C), demonstrated decreased amounts of the proenzyme and a band of an approximately 17-kd cleavage product. G-CSF (Figure 3, lane D) inhibited caspase-3 activation, whereas zVAD-fmk-treated PMNs (Figure 3, lane E) showed no cleavage product but only inactive 32-kd proenzyme, indicating that caspase-3 activation was inhibited in these cells.
We also stained PMNs for active caspase-3 with a mAb against a unique
epitope of active caspase-3. This mAb does not recognize the inactive
form of the enzyme. CLSM analysis showed that fresh PMN (Figure
4A) demonstrated only very weak staining,
probably reflecting background staining or a negligible binding to
inactive enzyme. In contrast, untreated, overnight-cultured PMN (Figure 4B) showed a bright punctate staining for active caspase-3,
demonstrating a significant amount of the active form of the enzyme in
these cells. G-CSF (Figure 4C) considerably inhibited caspase-3
activation, because the cells were only slightly positive. As expected,
zVAD-fmk-treated PMNs (Figure 4D) were as dark as fresh cells,
demonstrating the absence of active caspase-3. Hence, Western blotting
and CLSM results were in compliance with each other, demonstrating a
strong correlation between PMN spontaneous apoptosis and caspase-3
activation, and a coincidence of the antiapoptotic effect of G-CSF and
zVAD-fmk with inhibition of caspase-3 activation.
Spontaneous PMN apoptosis associated with Bax-to-mitochondria translocation Next, we investigated a number of Bcl-2-related proteins. Levels of expression of Bax, Bak, Mcl-1, and Bcl-XL were studied by flow cytometry with antibodies specific for each protein to be tested. We found that the expression levels of Bax were not significantly different between fresh PMNs and overnight-incubated PMNs. Neither G-CSF nor zVAD-fmk had any effect on its expression (Figure 5). Western blotting with anti-Bax antibody confirmed the flow cytometry data; Bax levels remained unchanged under all conditions tested (data not shown). Similar results were obtained for Bak, Mcl-1, and Bcl-XL (data not shown). We also did not find a correlation between the expression of these Bcl-2 homologues and cell survival. Therefore, we conclude that the expression levels of the Bcl-2-related proteins studied by us were not subject to modulation during PMN apoptosis or its delay.
Because we did not find quantitative changes in these proteins, we
investigated possible qualitative changes. Recent studies have shown
that Bax moves from the cytosol to the mitochondria on execution of
apoptosis.8-11 Using CLSM with costaining for mitochondria
and Bax, we found that fresh PMNs (Figure
6A) showed a separate localization of Bax
(red) and mitochondria (green). (Note: due to the
fixation/permeabilization procedure, mitochondrial staining showed a
more diffuse cytoplasmic pattern than the tubular structures shown in
Figure 2, panels A and C.) In contrast, in overnight cultured,
untreated PMNs (Figure 6B), mitochondria and Bax colocalized into large
aggregates with a shift in fluorescence to yellow. G-CSF (Figure 6C)
prevented this aggregate formation and colocalization of Bax with
mitochondria, whereas zVAD-fmk (Figure 6D) did not. Bak, Mcl-1, and
Bcl-XL did not change their punctate localization separate
from mitochondria under any condition tested (data not shown).
Normal externalization of phosphatidylserine and caspase-3 activation in cytoplasts Recently, a mitochondria-independent route of caspase activation has been proposed to exist.37 To check this possibility in PMNs, we generated so-called cytoplasts, that is, enucleated PMNs that lack granules and mitochondria. Thus, cytoplasts are vesicles filled with cytoplasm, which maintain the integrity of the plasma membrane.31Cytospins stained with May-Grünwald-Giemsa stain showed
that more than 99% of the cytoplasts indeed had no nucleus. Neither MitoTracker nor the mAb against a cytochrome c oxidase
component revealed the presence of any mitochondria in cytoplasts (not
shown). Annexin-V staining was negative in 96.1% ± 3.3% of freshly
prepared cytoplasts (Figure 7A). Similar
to PMNs, during overnight culture without stimuli cytoplasts underwent
membrane "flip-flop," with exposure of phosphatidylserine residues
on the outer layer of the plasma membrane, leading to 64.5%
annexin-V+ cytoplasts (35.5% ± 3.3%
annexin-V
G-CSF effect on survival and caspase-3 activation in cytoplasts Because G-CSF needs to bind to a specific surface receptor to mediate its effects in PMNs,38 we investigated the expression of the G-CSF receptor on cytoplasts by flow cytometry. The MFI of bound anti-G-CSF receptor mAb was 193 ± 22 in PMNs, and 88 ± 10 in cytoplasts (data represent mean ± SEM of the MFI from 3 separate experiments). The difference in MFI between PMNs and cytoplasts is due to the fact that the cell surface area of PMNs is 2 to 3 times larger than that of cytoplasts.31 Moreover, the number of G-CSF receptors on the cell surface is not critical for the impact of G-CSF on cells, because the ligation of only a small fraction of the receptors is already sufficient to mediate the maximal biologic response.39 Although the expression of the G-CSF receptor on cytoplasts can be considered to be adequate, addition of G-CSF to the culture medium had no effect on cytoplast apoptosis (35.7% ± 9.0% annexin-V cells), in contrast to the
delay of PMN apoptosis by G-CSF (compare Figure 7C and Figure 1C).
Furthermore, in cytoplasts, G-CSF prevented neither caspase-3
processing (Figure 8, lane D) nor the appearance of active caspase-3
(Figure 9C), whereas G-CSF prevented caspase-3 processing and
activation in PMNs (Figure 3, lane D, and Figure 4C). At the same time,
zVAD-fmk (Figure 7D) reduced cytoplast apoptosis (91.5% ± 6.1%
annexin-V cytoplasts) and inhibited caspase-3
activation (Figure 8, lane E, and Figure 9D). Similar effects of
zVAD-fmk were found in PMNs (Figure 1D, Figure 3, lane E, and Figure
4D). Bax remained punctate under either treatment (not shown). We
suppose that the G-CSF prosurvival effect and caspase-3
inhibiting action in PMNs are transcription-dependent events.
CHX abolishes the antiapoptotic effect of G-CSF in intact PMNs To support a protein synthesis-dependent character of the G-CSF prosurvival effect, we used CHX, a well-known inhibitor of protein synthesis. The overnight survival of untreated PMNs, which was 23.5% ± 2.5% in this set of experiments (Figure 10A), was not disturbed by addition of the small dose of CHX (5 µg/mL; Figure 10B; 23.1% ± 2.0% surviving PMNs). G-CSF protected 59.7% ± 5.5% PMNs from apoptosis (Figure 10C). At the same time, when coincubated overnight, CHX completely abrogated the prosurvival effect of G-CSF, with 28.7% ± 5.3% surviving cells (Figure 10D). Hence, this provides additional evidence that the G-CSF prosurvival effect is a strictly protein synthesis-dependent event in PMNs.
In our study, we examined the mechanisms of apoptosis in PMNs. Using a specific fluorescent mitochondrial stain and a mAb against a mitochondrial cytochrome c oxidase component, we found that PMNs do contain mitochondria. One of the possible routes of apoptosis in PMNs is mitochondria dependent and includes Bax-to-mitochondria translocation with subsequent executioner caspase activation. Our findings demonstrate that the G-CSF prosurvival effect coincides with prevention of mitochondrial clustering and Bax translocation, with concomitant inhibition of caspase-3 activity and retardation of apoptosis. This might be a possible route of G-CSF prosurvival action. At the same time, the general caspase inhibitor zVAD-fmk was found to inhibit caspase-3 and to delay PMN apoptosis without blocking the Bax-to-mitochondria translocation. In previous work with a cell-free apoptosis system40 and human embryonic kidney cells,41 it has also been shown that zVAD-fmk, despite caspase inhibition and prevention of apoptosis, was not able to prevent mitochondrial dysfunction and cytochrome c release. Apparently, Bax redistribution and mitochondrial dysfunction may be required but are not sufficient to induce apoptosis. Only if Bax translocation results in caspase-3 activation, as in untreated, overnight-cultured PMNs, does apoptosis of PMNs occur. This statement is supported by the finding that neither mitochondria nor Bax individually induce proteolytic processing and activation of caspases.42 Thus, our results support a dominant role of the executioner caspases in the downstream events of the apoptotic process in PMNs. Experiments with PMN cytoplasts revealed additional features of the PMN apoptotic machinery and the G-CSF antiapoptotic effect in PMN. In the absence of a nucleus and mitochondria, cytoplasts underwent apoptotic changes, that is, phosphatidylserine exposure and caspase-3 activation, reminiscent of intact PMNs. Furthermore, even in "apoptotic" cytoplasts, Bax preserved a punctate distribution, showing no clustering as seen in PMNs. This underscores that in PMNs mitochondria serve as "docking sites" for Bax, and Bax movement to mitochondria is an important step in the activation of the mitochondria-dependent route of apoptosis in PMNs. In addition, there must be a second, mitochondria-independent route of PMN apoptosis, which was evident in cytoplasts. Activation of this alternative route may explain the observation that G-CSF is unable to completely prevent apoptosis in intact PMNs. In cytoplasts, G-CSF did not prevent caspase-3 activation and phosphatidylserine "flip-flop," and thus it showed no prosurvival effect. In PMNs, where the protein synthesis apparatus is intact, G-CSF had a prominent antiapoptotic effect. This indicates that agents like G-CSF, in line with previous findings, require de novo protein synthesis to produce a prosurvival effect.26,27 Experiments with CHX, in which this inhibitor of protein synthesis prevented the prosurvival effect of G-CSF, gave additional support for this idea. On the other hand, considering the absence of mitochondria in PMN-derived cytoplasts, these data also indicate a mitochondria-dependent character of the G-CSF antiapoptotic action in intact PMNs. Which protein(s) are needed for the antiapoptotic effect of G-CSF remain to be determined. A logical suggestion would be that an antiapoptotic Bcl-2 family member is involved, especially because Bcl-2 itself has been shown to prevent Bax-to-mitochondria translocation.11,14 But PMNs, either when freshly isolated or apoptotic, do not express Bcl-2,23,24,43 and the antiapoptotic Bcl-2-related proteins tested in our study, Mcl-1 and Bcl-XL, were found not to change their expression levels on execution of apoptosis. Probably, G-CSF is responsible for the synthesis of another Bcl-2-related candidate, or some different protein, for example, the upstream caspase-8 inhibitor FLIP, which has been shown in several recent publications to be up-regulated in endothelial cells,44,45 macrophages,46 T cells,47 and B cells48 under prosurvival conditions. The other apoptosis route, as observed in PMN-derived cytoplasts, does not require mitochondria and Bax translocation. Nevertheless, caspase-3 activation is also involved in this route, being a final executioner of cell fate. Evidently, G-CSF has no effect on this mitochondria-independent pathway of apoptosis. In conclusion, PMNs share with other cell types the ability to
translocate Bax to mitochondria during apoptosis. However, we have
demonstrated that apoptosis in PMNs is not strictly dependent on Bax
translocation, as is supported by studies in Bax
Submitted March 6, 2001; accepted September 20, 2001.
Supported by a grant for doctoral student from the President of the Russian Federation (N.A.M.). T.W.K. is a research fellow of the Royal Dutch Academy of Sciences.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Taco W. Kuijpers, Central Laboratory of the Netherlands Blood Transfusion Service, Dept of Experimental Immunohematology, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands; e-mail: t_kuijpers{at}clb.nl.
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  A. Drewniak, B. J. van Raam, J. Geissler, A. T.J. Tool, O. R.F. Mook, T. K. van den Berg, F. Baas, and T. W. Kuijpers Changes in gene expression of granulocytes during in vivo granulocyte colony-stimulating factor/dexamethasone mobilization for transfusion purposes Blood, June 4, 2009; 113(23): 5979 - 5998. [Abstract] [Full Text] [PDF]
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S. A. Didichenko, N. Spiegl, T. Brunner, and C. A. Dahinden IL-3 induces a Pim1-dependent antiapoptotic pathway in primary human basophils Blood, November 15, 2008; 112(10): 3949 - 3958. [Abstract] [Full Text] [PDF]
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 D. El Kebir, L. Jozsef, and J. G. Filep Opposing regulation of neutrophil apoptosis through the formyl peptide receptor-like 1/lipoxin A4 receptor: implications for resolution of inflammation J. Leukoc. Biol., September 1, 2008; 84(3): 600 - 606. [Abstract] [Full Text] [PDF]
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B. J. van Raam, A. Drewniak, V. Groenewold, T. K. van den Berg, and T. W. Kuijpers Granulocyte colony-stimulating factor delays neutrophil apoptosis by inhibition of calpains upstream of caspase-3 Blood, September 1, 2008; 112(5): 2046 - 2054. [Abstract] [Full Text] [PDF]
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 A. Nagarsekar, R. S. Greenberg, N. G. Shah, I. S. Singh, and J. D. Hasday Febrile-Range Hyperthermia Accelerates Caspase-Dependent Apoptosis in Human Neutrophils J. Immunol., August 15, 2008; 181(4): 2636 - 2643. [Abstract] [Full Text] [PDF]
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 A. Drewniak, J.-J. Boelens, H. Vrielink, A. T.J. Tool, M. C.A. Bruin, M. van den Heuvel-Eibrink, L. Ball, M. D. van de Wetering, D. Roos, and T. W. Kuijpers Granulocyte concentrates: prolonged functional capacity during storage in the presence of phenotypic changes Haematologica, July 1, 2008; 93(7): 1058 - 1067. [Abstract] [Full Text] [PDF]
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 S. Conus, R. Perozzo, T. Reinheckel, C. Peters, L. Scapozza, S. Yousefi, and H.-U. Simon Caspase-8 is activated by cathepsin D initiating neutrophil apoptosis during the resolution of inflammation J. Exp. Med., March 17, 2008; 205(3): 685 - 698. [Abstract] [Full Text] [PDF]
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D. El Kebir, L. Jozsef, T. Khreiss, W. Pan, N. A. Petasis, C. N. Serhan, and J. G. Filep Aspirin-Triggered Lipoxins Override the Apoptosis-Delaying Action of Serum Amyloid A in Human Neutrophils: A Novel Mechanism for Resolution of Inflammation J. Immunol., July 1, 2007; 179(1): 616 - 622. [Abstract] [Full Text] [PDF]
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G. Seumois, M. Fillet, L. Gillet, C. Faccinetto, C. Desmet, C. Francois, B. Dewals, C. Oury, A. Vanderplasschen, P. Lekeux, et al. De novo C16- and C24-ceramide generation contributes to spontaneous neutrophil apoptosis J. Leukoc. Biol., June 1, 2007; 81(6): 1477 - 1486. [Abstract] [Full Text] [PDF]
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T. W. Kuijpers, R. v. Bruggen, N. Kamerbeek, A. T. J. Tool, G. Hicsonmez, A. Gurgey, A. Karow, A. J. Verhoeven, K. Seeger, O. Sanal, et al. Natural history and early diagnosis of LAD-1/variant syndrome Blood, April 15, 2007; 109(8): 3529 - 3537. [Abstract] [Full Text] [PDF]
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A. D. Gregory, L. A. Hogue, T. W. Ferkol, and D. C. Link Regulation of systemic and local neutrophil responses by G-CSF during pulmonary Pseudomonas aeruginosa infection Blood, April 15, 2007; 109(8): 3235 - 3243. [Abstract] [Full Text] [PDF]
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S. A. Renshaw, C. A. Loynes, D. M.I. Trushell, S. Elworthy, P. W. Ingham, and M. K.B. Whyte A transgenic zebrafish model of neutrophilic inflammation Blood, December 15, 2006; 108(13): 3976 - 3978. [Abstract] [Full Text] [PDF]
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 P. S. D. Weber, S. A. Madsen-Bouterse, G. J. M. Rosa, S. Sipkovsky, X. Ren, P. E. Almeida, R. Kruska, R. G. Halgren, J. L. Barrick, and J. L. Burton Analysis of the bovine neutrophil transcriptome during glucocorticoid treatment Physiol Genomics, December 13, 2006; 28(1): 97 - 112. [Abstract] [Full Text] [PDF]
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L. E. Kilpatrick, S. Sun, D. Mackie, F. Baik, H. Li, and H. M. Korchak Regulation of TNF mediated antiapoptotic signaling in human neutrophils: role of {delta}-PKC and ERK1/2 J. Leukoc. Biol., December 1, 2006; 80(6): 1512 - 1521. [Abstract] [Full Text] [PDF]
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 S. A. Madsen-Bouterse, G. J. M. Rosa, and J. L. Burton Glucocorticoid Modulation of Bcl-2 Family Members A1 and Bak during Delayed Spontaneous Apoptosis of Bovine Blood Neutrophils Endocrinology, August 1, 2006; 147(8): 3826 - 3834. [Abstract] [Full Text] [PDF]
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 R. Tehranchi, B. Fadeel, J. Schmidt-Mende, A.-M. Forsblom, E. Emanuelsson, M. Jadersten, B. Christensson, R. Hast, R. B. Howe, J. Samuelsson, et al. Antiapoptotic Role of Growth Factors in the Myelodysplastic Syndromes: Concordance Between In vitro and In vivo Observations Clin. Cancer Res., September 1, 2005; 11(17): 6291 - 6299. [Abstract] [Full Text] [PDF]
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S. von Gunten, S. Yousefi, M. Seitz, S. M. Jakob, T. Schaffner, R. Seger, J. Takala, P. M. Villiger, and H.-U. Simon Siglec-9 transduces apoptotic and nonapoptotic death signals into neutrophils depending on the proinflammatory cytokine environment Blood, August 15, 2005; 106(4): 1423 - 1431. [Abstract] [Full Text] [PDF]
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E. Sakamoto, F. Hato, T. Kato, C. Sakamoto, M. Akahori, M. Hino, and S. Kitagawa Type I and type II interferons delay human neutrophil apoptosis via activation of STAT3 and up-regulation of cellular inhibitor of apoptosis 2 J. Leukoc. Biol., July 1, 2005; 78(1): 301 - 309. [Abstract] [Full Text] [PDF]
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R. Tehranchi, R. Invernizzi, A. Grandien, B. Zhivotovsky, B. Fadeel, A.-M. Forsblom, E. Travaglino, J. Samuelsson, R. Hast, L. Nilsson, et al. Aberrant mitochondrial iron distribution and maturation arrest characterize early erythroid precursors in low-risk myelodysplastic syndromes Blood, July 1, 2005; 106(1): 247 - 253. [Abstract] [Full Text] [PDF]
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T. W. Kuijpers, M. Alders, A. T. J. Tool, C. Mellink, D. Roos, and R. C. M. Hennekam Hematologic abnormalities in Shwachman Diamond syndrome: lack of genotype-phenotype relationship Blood, July 1, 2005; 106(1): 356 - 361. [Abstract] [Full Text] [PDF]
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A. Bruno, S. Conus, I. Schmid, and H.-U. Simon Apoptotic Pathways Are Inhibited by Leptin Receptor Activation in Neutrophils J. Immunol., June 15, 2005; 174(12): 8090 - 8096. [Abstract] [Full Text] [PDF]
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M. Pederzoli, C. Kantari, V. Gausson, S. Moriceau, and V. Witko-Sarsat Proteinase-3 Induces Procaspase-3 Activation in the Absence of Apoptosis: Potential Role of this Compartmentalized Activation of Membrane-Associated Procaspase-3 in Neutrophils J. Immunol., May 15, 2005; 174(10): 6381 - 6390. [Abstract] [Full Text] [PDF]
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N. A. Maianski, D. Roos, and T. W. Kuijpers Bid Truncation, Bid/Bax Targeting to the Mitochondria, and Caspase Activation Associated with Neutrophil Apoptosis Are Inhibited by Granulocyte Colony-Stimulating Factor J. Immunol., June 1, 2004; 172(11): 7024 - 7030. [Abstract] [Full Text] [PDF]
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F. Altznauer, S. Martinelli, S. Yousefi, C. Thurig, I. Schmid, E. M. Conway, M. H. Schoni, P. Vogt, C. Mueller, M. F. Fey, et al. Inflammation-associated Cell Cycle-independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils J. Exp. Med., May 17, 2004; 199(10): 1343 - 1354. [Abstract] [Full Text] [PDF]
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T. W. Kuijpers, N. A. Maianski, A. T. J. Tool, K. Becker, B. Plecko, F. Valianpour, R. J. A. Wanders, R. Pereira, J. Van Hove, A. J. Verhoeven, et al. Neutrophils in Barth syndrome (BTHS) avidly bind annexin-V in the absence of apoptosis Blood, May 15, 2004; 103(10): 3915 - 3923. [Abstract] [Full Text] [PDF]
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A. Bouchard, C. Ratthe, and D. Girard Interleukin-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors: role of Mcl-1 and decreased activity of caspase-3 and caspase-8 J. Leukoc. Biol., May 1, 2004; 75(5): 893 - 900. [Abstract] [Full Text] [PDF]
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 F. Altznauer, S. Conus, A. Cavalli, G. Folkers, and H.-U. Simon Calpain-1 Regulates Bax and Subsequent Smac-dependent Caspase-3 Activation in Neutrophil Apoptosis J. Biol. Chem., February 13, 2004; 279(7): 5947 - 5957. [Abstract] [Full Text] [PDF]
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 R. BAUMANN, C. CASAULTA, D. SIMON, S. CONUS, S. YOUSEFI, and H.-U. SIMON Macrophage migration inhibitory factor delays apoptosis in neutrophils by inhibiting the mitochondria-dependent death pathway FASEB J, December 1, 2003; 17(15): 2221 - 2230. [Abstract] [Full Text] [PDF]
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T. W. Kuijpers, N. A. Maianski, A. T. J. Tool, G. P. A. Smit, J. P. Rake, D. Roos, and G. Visser Apoptotic neutrophils in the circulation of patients with glycogen storage disease type 1b (GSD1b) Blood, June 15, 2003; 101(12): 5021 - 5024. [Abstract] [Full Text] [PDF]
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M. Miyamoto, M. Emoto, Y. Emoto, V. Brinkmann, I. Yoshizawa, P. Seiler, P. Aichele, E. Kita, and S. H. E. Kaufmann Neutrophilia in LFA-1-Deficient Mice Confers Resistance to Listeriosis: Possible Contribution of Granulocyte-Colony-Stimulating Factor and IL-17 J. Immunol., May 15, 2003; 170(10): 5228 - 5234. [Abstract] [Full Text] [PDF]
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A. Villunger, C. Scott, P. Bouillet, and A. Strasser Essential role for the BH3-only protein Bim but redundant roles for Bax, Bcl-2, and Bcl-w in the control of granulocyte survival Blood, March 15, 2003; 101(6): 2393 - 2400. [Abstract] [Full Text] [PDF]
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B. M. Murphy, A. J. O'Neill, C. Adrain, R. W. G. Watson, and S. J. Martin The Apoptosome Pathway to Caspase Activation in Primary Human Neutrophils Exhibits Dramatically Reduced Requirements for Cytochrome c J. Exp. Med., March 3, 2003; 197(5): 625 - 632. [Abstract] [Full Text] [PDF]
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N. A. Maianski, D. Roos, and T. W. Kuijpers Tumor necrosis factor alpha induces a caspase-independent death pathway in human neutrophils Blood, March 1, 2003; 101(5): 1987 - 1995. [Abstract] [Full Text] [PDF]
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G. Fossati, D. A. Moulding, D. G. Spiller, R. J. Moots, M. R. H. White, and S. W. Edwards The Mitochondrial Network of Human Neutrophils: Role in Chemotaxis, Phagocytosis, Respiratory Burst Activation, and Commitment to Apoptosis J. Immunol., February 15, 2003; 170(4): 1964 - 1972. [Abstract] [Full Text] [PDF]
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R. Tehranchi, B. Fadeel, A.-M. Forsblom, B. Christensson, J. Samuelsson, B. Zhivotovsky, and E. Hellstrom-Lindberg Granulocyte colony-stimulating factor inhibits spontaneous cytochrome c release and mitochondria-dependent apoptosis of myelodysplastic syndrome hematopoietic progenitors Blood, February 1, 2003; 101(3): 1080 - 1086. [Abstract] [Full Text] [PDF]
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T. Hasegawa, K. Suzuki, C. Sakamoto, K. Ohta, S. Nishiki, M. Hino, N. Tatsumi, and S. Kitagawa Expression of the inhibitor of apoptosis (IAP) family members in human neutrophils: up-regulation of cIAP2 by granulocyte colony-stimulating factor and overexpression of cIAP2 in chronic neutrophilic leukemia Blood, February 1, 2003; 101(3): 1164 - 1171. [Abstract] [Full Text] [PDF]
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C. Pisano, P. Kollar, M. Gianni, Y. Kalac, V. Giordano, F. F. Ferrara, R. Tancredi, A. Devoto, A. Rinaldi, A. Rambaldi, et al. Bis-indols: a novel class of molecules enhancing the cytodifferentiating properties of retinoids in myeloid leukemia cells Blood, November 15, 2002; 100(10): 3719 - 3730. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-mediated regulation of apoptosis of human neutrophils via caspase-3.
Blood.
1999;93:3106-3115