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Originally published online as doi:10.1189/jlb.0703317 on February 3, 2004

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(Journal of Leukocyte Biology. 2004;75:836-843.)
© 2004 by Society for Leukocyte Biology

Identification of caspase-10 in human neutrophils and its role in spontaneous apoptosis

Friederike Goepel*, Pamela Weinmann*, Jürgen Schymeinsky{dagger} and Barbara Walzog{dagger},1

* Department of Physiology, Freie Universität, Berlin, Germany; and
{dagger} Department of Physiology, Ludwig-Maximilians-Universität, München, Germany

1Correspondence: Department of Physiology, Ludwig-Maximilians-Universität München, Schillerstrasse 44, D-80336 München, Germany. E-mail: walzog{at}lrz.uni-muenchen.de


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ABSTRACT
 
In the present study, we investigated the molecular mechanisms of spontaneous and tumor necrosis factor {alpha} (TNF-{alpha})-mediated apoptosis of human polymorphonuclear neutrophils (PMN). Whereas TNF-{alpha}-mediated apoptosis was almost absent in the presence of the caspase-8 inhibitor Z-Ac-Ala-Glu-Val-Asp-7-fluoromethyl ketone (Z-AEVD-FMK), the inhibitor had no effect on spontaneous apoptosis, suggesting that spontaneous apoptosis was independent of caspase-8. Subsequently, we identified different isoforms of caspase-10 in human PMN and found high expression of caspase-10/b and/or -10/d and low expression of caspase-10/a and -10/c at the mRNA level. At the protein level, freshly isolated PMN showed high expression of caspase-10/b and -10/d as well as moderate expression of caspase-10/a and -10/c. Upon spontaneous apoptosis, caspase-10/b was down-regulated, which was accompanied by the appearance of a specific 47-kDa caspase-10/b cleavage product and an increased caspase-10 activity. In contrast, no down-regulation of caspase-10/a, -10/c, or -10/d was observed, suggesting that spontaneous apoptosis was associated with a differential activation of caspase-10/b. This was confirmed by the finding that spontaneous apoptosis was inhibited in the presence of Z-Ile-Glu-Thr-Asp (Z-IETD)-FMK, which blocks caspase-10. However, no down-regulation of caspase-10 isoforms was observed in the presence of TNF-{alpha}, suggesting that caspase-10 was not involved in TNF-{alpha}-induced apoptosis. Taken together, our study demonstrates that spontaneous and TNF-{alpha}-mediated apoptosis of PMN have different molecular requirements. Whereas TNF-{alpha}-mediated apoptosis depends on the activation of caspase-8, spontaneous apoptosis requires the activation of caspase-10/b. This finding may reveal that PMN apoptosis in different (patho-) physiological settings results from distinct molecular mechanisms.

Key Words: polymorphonuclear neutrophil • tumor necrosis factor {alpha} • Z-AEVD-FMK • Z-IETD-FMK • inflammation


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INTRODUCTION
 
Apoptosis is an active and well-regulated process, which is characterised by specific phenomena such as cell shrinkage, chromatin condensation, internucleosomal DNA fragmentation, membrane blebbing, and finally, the decay into apoptotic bodies [1 , 2 ]. Mature human polymorphonuclear neutrophils (PMN) undergo apoptosis spontaneously within hours to days, and this is thought to contribute to the high turnover of these cells in the circulation [3 ]. The lifespan of circulating PMN is relatively short when compared with other leukocytes, but it can be further shortened by inflammatory mediators such as tumor necrosis factor {alpha} (TNF-{alpha}), which accelerates apoptosis of human PMN [4 ]. In contrast, other mediators such as granulocyte macrophage-colony stimulating factor (GM-CSF) or G-CSF prolong the lifetime of PMN by inhibiting apoptosis [4 , 5 ].

Growing evidence supports the concept that the control of PMN apoptosis contributes to the maintenance of PMN homeostasis in the circulation [6 ]: Shi et al. [7 ] demonstrated that apoptosis is critical for the clearance of PMN from the peripheral blood by Kupffer cells in the liver. Other reports show that cytokine-mediated neutrophilia is not simply a result of enhanced hematopoiesis. In patients with severe burns, circulating PMN showed impaired apoptosis as a result of plasma factors, which up-regulated GM-CSF levels [5 ]. A cytokine-mediated delay of apoptosis in peripheral PMN was also observed in inflammatory diseases associated with neutrophilia such as cystic fibrosis and pneumonia [8 ]. Neutropenia in large granular lymphocyte leukemia has been reported to involve an enhancement of PMN apoptosis associated with high levels of circulating Fas ligand in the serum [9 ]. Taken together, a body of evidence exists showing that the dysregulation of PMN apoptosis may contribute to the expansion or the reduction of the peripheral PMN pool under pathological conditions. In addition, PMN apoptosis plays an important role in inflammatory processes, where it allows the nonphlogistic elimination of the emigrated PMN in the tissue and thereby contributes to graduation and final resolution of acute inflammation [10 , 11 ]. However, the molecular requirements for the induction of PMN apoptosis in these different (patho-) physiological settings are undefined.

The caspases, a group of cysteine-dependent aspartate-specific proteases [12 ], are well-known to be critical molecular components of the apoptotic machinery. Caspases are synthesized as inactive proenzymes bearing a prodomain. Proteolytic cleavage of this domain as well as the cleavage into a large and a small subunit are required to gain full, functional activity of the enzyme, which is composed of a tetrapeptide with two small and two large subunits [13 ]. Caspases recognize a tetrapeptide motif and cleave their substrates at the carboxyl side of an aspartate residue (P1 site). The different caspases show distinct substrate specificities, which are determined by the amino acid sequence within the tetrapeptide motif [14 ]. Based on their substrate preferences, three different subgroups are defined: group I (caspase-1, -4, -5) with WEXD as preferred substrate; group II (caspase-2, -3, -7), which is highly selective for DEXD; and group III (caspase-6, -8, -9, -10), which prefers the (L/V)EXD motif. Whereas group I caspases are mainly involved in inflammation by generating inflammatory cytokines, groups II and III caspases play a pivotal role in apoptosis. With respect to their biological function, the caspases involved in apoptosis are divided into effector caspases, which finally execute the apoptotic program and initiator caspases or "instigators", which incite the proteolytic cascade [15 ].

Effector caspases such as caspase-3, -6, and -7 are located downstream in the death signal-transduction pathways and cleave substrates that are involved in the execution of the apoptotic program, e.g., gelsolin and lamin [16 , 17 ]. Initiator caspases such as caspase-8, -9, and -10 are located upstream in death-signaling pathways and transduce or amplify apoptotic signals via proteolytic cleavage and activation of effector and/or initiator caspases [15 ]. Among the effector caspases, expression of caspase-7 has been found at the mRNA level in human PMN [18 ], but it was not detectable at the protein expression level [19 ]. In contrast, caspase-3 has been shown to be critically involved in the final execution of the apoptotic program in human PMN [4 ]. Moreover, a recent report presented evidence for an unanticipated role of caspase-1 (group I caspase) in neutrophil apoptosis [20 ]. Among the initiator caspases, caspase-8 has been reported to be involved in the TNF-{alpha}-mediated apoptosis of PMN [19 ], and caspase-9 has been found to play a role in activation of caspase-3 and -8 in human PMN [21 ]. Caspase-10 is known to initiate TNF-{alpha}-mediated apoptosis in T cells [22 ], but there are no data to show the protein expression of caspase-10 in PMN or its involvement in PMN apoptosis.

PMN apoptosis contributes to the maintenance of PMN homeostasis in the blood and also in tissues during inflammatory processes. Therefore, the elucidation of the molecular mechanisms for PMN apoptosis in the context of both settings is important for our understanding of the resolution of inflammation. Although progress has been made, the involvement of specific caspases in PMN apoptosis in these settings remains undefined. The present study was undertaken to elucidate the putative involvement of the upstream initiator caspase-8 and -10 in the induction of spontaneous and/or cytokine-mediated apoptosis of human PMN.


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MATERIALS AND METHODS
 
Isolation of human PMN and cell culture
PMN were isolated from heparinized venous blood (10 I.E./ml) of healthy adult volunteers according to the institutional guidelines of the Ludwig-Maximilians-University (Munich, Germany). After erythrocyte sedimentation in the presence of 40% (v/v) autologous plasma, the leukocyte-rich plasma was layered onto a discontinuous PercollTM gradient as described [23 ] and centrifuged at 600 g for 20 min. The PMN-containing band was collected and washed in Dulbecco’s phosphate-buffered saline (PBS). PMN were suspended in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS). PMN viability was >99%, as assessed by the trypan blue exclusion test; purity was >99%, as analyzed by microscopy using HemacolorTM staining (Merck, Darmstadt, Germany). The human leukemia cell lines HL-60 and Jurkat were grown in RPMI-1640 medium supplemented with 10% FCS and antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin) in 5% CO2 at 37°C.

Analysis of nuclear morphology
PMN (5x105/50 µl) were incubated with acridine orange in a final concentration of 5 µg/ml for 5 min at room temperature. Cells (50–100 per sample) were analyzed on a Nikon microscope using a 40/0.6 objective.

Internucleosomal DNA fragmentation assay
PMN (3x107) were lysed for 20 min on ice in 600 µl hypotonic lysis buffer (10 mM EDTA, 0.2% Triton X-100, 10 mM Tris, pH 7.5). After centrifugation for 20 min at 4°C at 13000 g, DNA was isolated by phenol/chloroform extraction and subsequent precipitation with 2 vol ethanol containing 0.1 M NaCl overnight at –20°C. After centrifugation at 13,000 g for 20 min at 4°C, the pellets were washed in 70% ethanol, dried, and resuspended in 20 µl H2O. After treatment with DNase-free RNase in a final concentration of 0.8 mg/ml for 30 min at 37°C, samples were subjected to gel electrophoresis in 1.8% agarose and visualized with ethidium bromide under UV light.

Analysis of DNA content
DNA content was analyzed by flow cytometry (FACScan, Becton Dickinson, San Jose, CA) using propidium iodide (PI). Briefly, PMN (5x105/100 µl) were washed with PBS, supplemented with 1 mM EDTA, and resuspended in 70% ethanol. PMN were permeabilized overnight at –20°C, washed with PBS, supplemented with 1 mM EDTA, and suspended in 250 µl PBS with 1 mM EDTA. After addition of 20 µg/ml DNase-free RNase and 50 µg/ml PI (final concentrations), samples were incubated for 15 min at room temperature and kept at 4°C until flow cytometric analysis. In each sample, 104 cells were counted and analyzed using Cell Quest software.

Reverse transcription (RT) and polymerase chain reaction (PCR)
Total RNA was isolated using the guanidine isothiocyanate method [24 ] using TRIZOLTM (Life Technologies, Eggenstein, Germany). RNA (0.75 µg) was transcribed into cDNA using oligo(dT) primers (Amersham Pharmacia Biotech, Freiburg, Germany) and 50 U RT Moloney murine leukemia virus (Promega, Mannheim, Germany). PCR amplification was performed using a specific primer set, which was described previously [25 ] (TIB MOLBIOL, Berlin, Germany) for the different caspase-10 isoforms (upstream primer 5'-CCG-AGT-CGT-ATC-AAG-GAG-AGG-AAG-AAC, downstream primer 5'-TAT-ATG-CAC-TGT-GAA-CCC-AAG-CCA), which yield a 350-bp product (caspase-10/b and -10/d), a 260-bp product (caspase-10/c), and a 230-bp product (caspase-10/a). For positive control, a specific primer set (MWG Biotech, Ebersberg, Germany) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; upstream primer 5'-GGT CGG AGT CAA CGG ATT TGG T, downstream primer 5'-TGT GGG CCA TGA GGT CCA CCA C) was used, which yields a 977-bp product (data not shown). PCR (36 cycles for caspase-10 and 30 cycles for GAPDH: 55 s, 94°C; 55 s, 60°C; 55 s, 72°C) was performed using 1.25 U AmpliTaq DNA polymerase (Promega). PCR products were analyzed by agarose gel electrophoresis in 2% agarose and visualized with ethidium bromide under UV light.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting
PMN (2x106) were pelleted and lysed in 1 x Laemmli buffer [2% (w/v) SDS, 6% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, and a trace amount of bromphenol blue in 200 mM Tris-HCl, pH 7.5]. The samples were immediately heated for 5 min at 100°C. Total cell lysates (106 cells/sample) were subjected to SDS-PAGE on gels containing 10% (w/v) acrylamide under reducing conditions. Separated proteins were transferred to nitrocellulose filters using a semi-dry technique at 150 mA for 1.5 h. All blots were tested for loading of equal amounts of protein in each lane by Ponceau S staining (data not shown). Filters were blocked by treatment with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 1 h and subsequently incubated with the anticaspase-10 monoclonal antibody (mAb; R&D Systems, Wiesbaden, Germany) or the anticaspase-8 mAb (Oncogene, San Diego, CA) in a final concentration of 1 µg/ml for 1 h in TBS supplemented with 0.1% BSA. After three washes in TBS containing 0.1% Tween-20, filters were incubated with (final dilution, 1:1000) the peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG; Sigma, Deisenhofen, Germany) in TBS supplemented with 0.1% BSA for 1 h and subsequently washed as described above. Detection was performed by chemiluminescence using an enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech) and subsequent autoluminography by exposure to X-ray films (XOMAT-AR, Kodak, Germany).

Immunoprecipitation of caspase-10
PMN (2x107) were lysed for 10 min on ice with 500 µl lysis buffer (pH 7.5) containing 5 mM HEPES, 10% sucrose, 0.1% 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate (CHAPS), and 10 mM dithiothreitol (DTT). The lysates were precleared by centrifugation for 15 min at 4°C and 12,000 g. The supernatant was incubated for 1 h at 4°C with 5 µg anticaspase-10 mAb coupled to an agarose-conjugated goat anti-mouse IgG and centrifuged. The immunoprecipitate was washed twice with ice-cold lysis buffer.

Measurement of caspase-10 activity
Caspase activity was measured in the whole cell lysates of PMN as well as in the immunoprecipitates and the immunodepletates. The protein concentration in the whole cell lysates and in the immunodepletates was determined using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) according to the supplier’s instructions. For measurement of caspase-10 activity, an aliquot of the whole cell lysates and the immunodepletates (50 µg protein each) and the complete immunoprecipitates was diluted to a final volume of 100 µl using lysis buffer. Each sample was supplemented with 50 µM fluorogenic substrate Z-Ile-Glu-Thr-Asp-7-amino-4-(trifluoromethyl)coumarin (Z-IETD-AFC) and incubated for 90 min at 37°C. The measurement was performed at an excitation wavelength of 400 nm and an emission wavelength of 505 nm using a luminescence spectrometer (LS 50B, Perkin Elmer, Wellesley, MA). Blanks were measured in the absence of cell lysates to determine background fluorescence.

Antibodies
The specific anti-human caspase-10 mAb (clone 63131.111) was obtained from R&D Systems. The specific anti-human caspase-8 mAb (clones 1–3) was purchased from Oncogene. The peroxidase-conjugated goat anti-mouse IgG and the agarose-conjugated goat anti-mouse IgG were obtained from Sigma. The fluorescein isothiocyanate (FITC)-conjugated anti-human CD16 (Fc{gamma} receptor type III) mAb (clone DJ130c) and the FITC-conjugated, nonbinding isotype-matched control mAb (IgG1 subclass) were purchased from Dako Diagnostica (Hamburg, Germany).

Reagents
BSA, CHAPS, dimethyl sulfoxide, DTT, ovalbumin, penicillin, Percoll, Ponceau S, PI, streptomycin, sucrose, Triton X-100, TNF-{alpha}, and Tween-20 were obtained from Sigma. ECL Western blotting detection kit (RPN 2106) and electrophoresis calibration standards for molecular mass determination were purchased from Amersham Pharmacia Biotech. The fluorogenic substrate Z-IETD-AFC and the inhibitor Z-IETD-fluoromethyl ketone (FMK) were obtained from Calbiochem (Schwalbach, Germany). The inhibitor Z-Ac-Ala-Glu-Val-Asp (Z-AEVD)-FMK was purchased from R&D Systems. Buffers, cell culture media, and FCS were obtained from Biochrom (Berlin, Germany).

Statistical analysis
Data shown represent mean ± SD where applicable. Statistical significance was determined using Student’s t-test; P < 0.05 was considered statistically significant.


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RESULTS
 
Activation of caspase-8 is critical for TNF-{alpha}-induced apoptosis
It has previously been shown that spontaneous apoptosis of PMN is accelerated upon stimulation of PMN by TNF-{alpha} [4 , 26 ]. To elucidate the role of caspase-8 in the induction of the apoptotic cell death of PMN, apoptosis was measured in the presence of the caspase-8 inhibitor Z-AEVD-FMK [27 ] by detecting the nuclear morphology using fluorescence microscopy of acridine orange-stained PMN. As shown in Figure 1A , the majority of unstimulated PMN showed segmented nuclei within 4 h after the onset of culture, which is typical for mature, human PMN. Stimulation of PMN with TNF-{alpha} induced a condensation of the nuclei within this time period, a well-known marker for apoptotic PMN. The quantitative analysis showed that 15.0% of the unstimulated PMN showed condensed nuclei within 4 h after the onset of the incubation period, whereas TNF-{alpha} induced a substantial increase of the percentage of apoptotic cells when compared with unstimulated PMN and led to nuclear condensation in 26.0% of the cells (Fig. 1B) . Moreover, the TNF-{alpha}-induced increase of apoptosis was abrogated in the presence of the Z-AEVD-FMK inhibitor, demonstrating that caspase-8 activation is required for the TNF-{alpha}-stimulated acceleration of apoptosis. In contrast, the Z-AEVD-FMK inhibitor did not significantly affect spontaneous apoptosis. This suggests that spontaneous apoptosis and TNF-{alpha}-induced apoptosis may result from different molecular mechanisms.



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  		Figure 1. TNF-{alpha}-mediated apoptosis but not spontaneous apoptosis requires activation of caspase-8 as detected by nuclear morphology. PMN were treated with 100 µM caspase inhibitor Z-AEVD-FMK (+AEVD) or vehicle (–AEVD) for 1 h and were stimulated for 4 h with TNF-{alpha} (300 U/ml) or left unstimulated to measure spontaneous apoptosis (unstimulated). (A) Fluorescence microscopy of acridine orange-stained PMN. Arrows indicate apoptotic PMN. (B) Data represent apoptotic PMN with condensed nuclei in percent of total cell number. Mean ± SD, n = 3. ***, P < 0.05; n.s., not significant.

To further prove this hypothesis, apoptosis was measured in the presence of the Z-AEVD-FMK inhibitor by detecting degradation of DNA (Fig. 2 ). Within 6 h after the onset of the experiment, no substantial DNA laddering was observed in unstimulated PMN, demonstrating that DNA fragmentation did not occur spontaneously at this early time point. In contrast, stimulation of the PMN with TNF-{alpha} induced strong DNA laddering at 6 h of culture, indicating that these cells underwent apoptosis. Moreover, the presence of the Z-AEVD-FMK inhibitor substantially decreased DNA laddering to basal levels, demonstrating that the TNF-{alpha}-induced acceleration of apoptosis was dependent on caspase-8. Within 22 h after the onset of the experiment, unstimulated control cells also showed DNA laddering. However, the Z-AEVD-FMK inhibitor was not able to inhibit the fragmentation of DNA, suggesting that spontaneous apoptosis was independent of the activation of caspase-8.



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  		Figure 2. Different molecular requirements for spontaneous and TNF-{alpha}-induced apoptosis are also apparent upon detection of DNA laddering. Agarose gel of low molecular weight DNA of PMN, which were treated with 100 µM caspase inhibitor Z-AEVD-FMK (+) or vehicle (–) for 1 h and were stimulated for indicated times with TNF-{alpha} (300 U/ml) or left unstimulated to measure spontaneous apoptosis (unstimulated). Results are representative of three independent experiments.

This finding was further confirmed by measuring the loss of DNA content using PI-stained PMN (Fig. 3 ). Again, only TNF-{alpha}-induced apoptosis was found to be significantly reduced from 33.8% to 14.9% in the presence of the Z-AEVD-FMK inhibitor within 2 h after the onset of the experiment. In contrast, the inhibitor had no significant effect on spontaneous apoptosis after 2 h of culture. This was also true upon measurement of spontaneous apoptosis after 6 h and 22 h (data not shown), confirming that TNF-{alpha}-induced apoptosis but not spontaneous apoptosis critically depended on activation of caspase-8. Taken together, these findings suggest that TNF-{alpha} exerts its effect, not simply by amplifying an intracellular signaling pathway, which underlies spontaneous apoptosis. Thus, different molecular requirements seem to underlie spontaneous and TNF-{alpha}-induced apoptosis with respect to the activation of caspases.



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  		Figure 3. Apoptosis measured by the loss of DNA content. PMN were treated for 1 h with 100 µM inhibitor Z-AEVD-FMK (+AEVD) or vehicle (–AEVD). After treatment with the inhibitor, PMN were stimulated for 2 h with TNF-{alpha} (300 U/ml) or were left unstimulated to measure spontaneous apoptosis (unstimulated). Numbers indicate apoptotic cells in percent of total cell number. Mean ± SD, n = 8. ***, P< 0.05; n.s., not significant.

A role for caspase-10 in spontaneous apoptosis
To elucidate the intracellular pathway that may be responsible for spontaneous apoptosis, apoptosis was measured in the presence of the Z-IETD-FMK inhibitor, which is known to inhibit not only caspase-8 but also caspase-10 [27 ]. By detecting the loss of DNA content, we measured spontaneous and TNF-{alpha}-induced apoptosis in the presence of this inhibitor after 2 h of culture, but no significant reduction of spontaneous apoptosis was detectable (data not shown), which may be a result of the fact that the percentage of cells that underwent apoptosis spontaneously was very low at this time point. However, the inhibitor significantly reduced spontaneous apoptosis from 13.9% in the untreated control to 7.6% after 6 h of culture (Fig. 4 ). As spontaneous apoptosis was not affected in the presence of the potent caspase-8 inhibitor Z-AEVD-FMK as shown above, the down-regulation of spontaneous apoptosis in the presence of the Z-IETD-FMK inhibitor suggests that spontaneous apoptosis of human PMN involves the activation of caspase-10.



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  		Figure 4. Evidence for an involvement of caspase-10 in spontaneous apoptosis as measured by the loss of DNA content. PMN were treated for 1 h with 100 µM inhibitor Z-IETD-FMK (+IETD) or vehicle (–IETD). After treatment with the inhibitor, PMN were stimulated for 6 h with TNF-{alpha} (300 U/ml) or left unstimulated to measure spontaneous apoptosis (unstimulated). Numbers indicate apoptotic cells in percent of total cell number. Mean ± SD, n = 7. ***, P < 0.05; n.s., not significant.

To confirm this finding, apoptosis was measured in the presence of the Z-IETD-FMK inhibitor by studying the morphology of the nuclei of PMN at 4 h after the onset of culture (Fig. 5A ). The quantitative analysis revealed that the inhibitor significantly reduced spontaneous apoptosis from 24.8% in the untreated control to 8.1% (Fig. 5B) . Moreover, the TNF-{alpha}-induced apoptosis was reduced from 42.7% to 22.7% in the presence of the inhibitor, which was probably a result of the fact that the Z-IETD-FMK inhibitor also affects caspase-8. However, the observed inhibition of spontaneous apoptosis seems to be a result of the inhibition of caspase-10, suggesting that caspase-10 is expressed in human PMN and may have a functional impact on spontaneous PMN apoptosis.



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  		Figure 5. Activation of caspase-10 is critical for spontaneous apoptosis of human PMN, which were treated with 100 µM caspase inhibitor Z-IETD-FMK (+IETD) or vehicle (–IETD) for 1 h and were stimulated for 4 h with TNF-{alpha} (300 U/ml) or left unstimulated to measure spontaneous apoptosis (unstimulated). (A) Fluorescence microscopy of acridine orange-stained PMN. Arrows indicate apoptotic PMN. (B) Data represent apoptotic PMN with condensed nuclei in percent of total cell number. Mean ± SD, n = 3. ***, P < 0.05, versus unstimulated control; n.s., not significant.

To verify the putative role of caspase-10 in spontaneous PMN apoptosis, we studied its expression at the mRNA level using the RT-PCR technique by the use of primers that allow the detection of the different caspase-10 isoforms (Fig. 6 ). For control, we measured the expression of caspase-10 isoforms in Jurkat cells and in HL-60 cells and found a high expression of caspase-10/b and/or -10/d as well as a high expression of caspase-10/a in both cell types, whereas the expression of caspase-10/c was rather moderate. In human PMN, we were able to detect a high expression of caspase-10/b and/or -10/d and a low expression of caspase-10/a and -10/c.



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  		Figure 6. Expression of different caspase-10 isoforms in human PMN. Analysis of mRNA expression of the different caspase-10 isoforms by RT-PCR. Total RNA was obtained from PMN freshly isolated from the circulation of HL-60 cells and Jurkat cells. The PCR products on a 2% agarose gel are shown. The result is representative of three independent experiments. M, marker.

To study the involvement of the different caspase-10 isoforms in spontaneous apoptosis of human PMN, the activation of the isoforms was investigated by detecting their proteolytic cleavage using the Western blotting technique. As shown in Figure 7A , PMN freshly isolated from the circulation showed a high expression of caspase-10/d and -10/b as well as a moderate expression of caspase-10/a and -10/c. When PMN were aged in culture, we observed a down-regulation of caspase-10/b in PMN, which underwent spontaneous apoptosis at 22 h after the onset of culture. This effect was accompanied by the appearance of a 47-kDa cleavage product, which has previously been reported to occur during activation of caspase-10/b [28 ]. In contrast, no down-regulation of other caspase-10 isoforms was observed, suggesting that spontaneous apoptosis is associated with a differential activation of caspase-10/b. However, no down-regulation of any of the caspase-10 isoforms was observed in the presence of TNF-{alpha}, suggesting that caspase-10 is not involved in TNF-{alpha}-induced apoptosis. Thus, activation of caspase-10/b seemed to be critical for spontaneous apoptosis but not for TNF-{alpha}-mediated apoptosis of human PMN. In contrast, TNF-{alpha} induced the complete proteolytic cleavage of caspase-8 within 2 after stimulation, confirming the important role of caspase-8 in TNF-{alpha}-mediated apoptosis (Fig. 7B) . When compared with the effect of TNF-{alpha}, spontaneous apoptosis was only associated with a slight processing of caspase-8 at early time points (2 h and 6 h), which may confirm the differential involvement of caspases in spontaneous and TNF-{alpha}-induced apoptosis.



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  		Figure 7. Caspase-10/b is proteolytically cleaved upon spontaneous apoptosis but not on TNF-{alpha}-mediated apoptosis of human PMN, which were incubated for 0, 2, 6, or 22 h in culture without further stimulation for induction of spontaneous apoptosis (unstimulated) or were treated with TNF-{alpha} (300 U/ml). (A) Western blot for caspase-10. (B) Western blot for caspase-8. The results are representative of three independent experiments.

To further confirm the involvement of caspase-10 in spontaneous apoptosis, the enzymatic activity of caspase-10 was studied using a fluorogenic IETD substrate (Fig. 8 ). First of all, we measured caspase-10 activity in whole cell lysates of PMN. When compared with PMN, freshly isolated from the circulation (0 h), we found a significant increase of caspase-10 activity in PMN, which were aged for 6 h in culture. This was also true when the enzymatic activity was measured in the immunoprecipitates of caspase-10, demonstrating that the cleavage of the IETD substrate was a result of the specific action of caspase-10. Moreover, the measurement of the enzymatic activity in the immunodepletates showed a significant decrease of caspase-10 activity when compared with the whole cell lysates at 6 h of culture, demonstrating that the observed effect is specific for caspase-10. Taken together, this finding shows that caspase-10 was activated during spontaneous apoptosis of PMN.



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  		Figure 8. Increase of caspase-10 activity upon spontaneous apoptosis of human PMN, which were lysed immediately after isolation (0 h) or after incubation for 6 h in culture without further stimulation for induction of spontaneous apoptosis. Caspase-10 activity was measured in the whole cell lysates, in the immunoprecipitates of caspase-10 (precipitate), and the whole cell lysates, which were depleted for caspase-10 (depletate) using the fluorogenic Z-IETD-AFC substrate. Data represent mean fluorescence intensity ± SD, n = 3. ***, P < 0.05; n.s., not significant.


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DISCUSSION
 
In the present study, we used a model in which we were able to measure spontaneous and TNF-{alpha}-induced apoptosis of human PMN, which were freshly isolated from the circulation and aged in culture using three independent methods, i.e., detection of the nuclear morphology, measurement of the internucleosomal DNA fragmentation (DNA laddering), and analysis of the intracellular DNA content. TNF-{alpha} was found to substantially induce PMN apoptosis at early time points (2 h and 6 h), a finding that is in accordance with the literature [4 , 26 ]. At later time points (~20 h), TNF-{alpha} has been reported earlier to show a relative ineffectiveness to induce or accelerate spontaneous apoptosis, which may be a result of the fact that the majority of PMN already died spontaneously at this time point [4 ]. In the present study, the mechanisms underlying the initiation of spontaneous and TNF-{alpha}-induced apoptosis were studied at early time points (2–6 h). The experiments with the caspase-8 inhibitor Z-AEVD-FMK, which blocked TNF-{alpha}-mediated apoptosis of PMN but not spontaneous apoptosis, revealed that the increased susceptibility of PMN to undergo apoptosis in the presence of TNF-{alpha} was not simply a result of enhancement of spontaneous apoptosis. As the Z-AEVD-FMK inhibitor was not able to block spontaneous apoptosis significantly, this finding supported the idea that spontaneous and TNF-{alpha}-mediated apoptosis of PMN result from different molecular pathways. Whereas TNF-{alpha}-mediated apoptosis was found to depend on the activation of caspase-8, which is in accordance with the literature [26 ], caspase-8 seemed not to play a predominant role for the induction of spontaneous apoptosis. This prompted us to study the putative expression of caspase-10 in human PMN, which is similar to caspase-8, known to act as an initiator caspase located upstream in apoptotic signaling pathways [22 ]. Four different splice variants of caspase-10 have been identified, designated as caspase-10/a [Fas-associated death domain (FADD) protein interleukin-1ß-converting enzyme-2], caspase-10/b (mammalian Ced-3 homologue-4), caspase-10/c, and caspase-10/d with molecular masses of 55 kDa, 59 kDa, 40 kDa, and 66 kDa, respectively [25 , 29 , 30 ]. Caspase-10/a has been shown to be recruited to Fas and TNF-receptor 1 (p55) in a FADD-dependent manner [30 ], and caspase-10/b was found to activate caspase-3 and -7 [25 ]. Kobayashi et al. [31 ] detected the expression of caspase-10 in human PMN by the oligonucleotide microarray technique. We identified different isoforms of caspase-10 in human PMN at the mRNA and at the protein level. Moreover, we observed the proteolytic cleavage of caspase-10/b and the appearance of its specific 47-kDa cleavage product upon spontaneous apoptosis. In contrast, the expression of caspase-10/a, -10/c, and -10/d was found to be unaffected upon spontaneous apoptosis. It is interesting that no down-regulation of any of the caspase-10 isoforms was detectable in the presence of TNF-{alpha}. This may show that stimulation of PMN with TNF-{alpha} not only promotes the activation of caspase-8 but also seems to prevent the activation of caspase-10, which occurs during spontaneous apoptosis. Thus, a molecular switch may determine whether PMN die spontaneously or as a result of cytokine-mediated apoptosis. Without further stimulation, PMN die spontaneously upon activation of caspase-10/b, whereas the presence of TNF-{alpha} interrupts this pathway and favors the activation of caspase-8, which triggers cytokine-mediated apoptosis. The finding that TNF-{alpha} induced apoptosis of human PMN at early time points is in accordance with the literature [26 , 32 ]. Maianski et al. [32 ] demonstrated that TNF-{alpha} induced the full activation of caspase-8, which resulted in the appearance of the active 18-kDa subunit of caspase-8 within 6 h after stimulation. In contrast, spontaneous apoptosis was only associated with the appearance of the large 43/41-kDa fragment of caspase-8, whereas the 18-kDa subunit was not detectable within 6 h of culture, confirming that spontaneous and TNF-{alpha}-induced apoptosis may result from different molecular mechanisms. In addition to this caspase-dependent cell death, Maianski et al. [32 ] reported that TNF-{alpha} induces a caspase-independent cell death in human neutrophils, but mechanisms underlying this death pathway are unknown.

As caspase-8 and -10 show similar substrate specificities, the use of caspase inhibitors bears strong limitations. Garcia-Calvo et al. [27 ] tested the effect of different petide-based and macromolecular inhibitors of the caspase family. In peptide-binding studies, they showed a similar dissociation constant (Ki) of caspase-8 for Z-AEVD-FMK (1.6 nM) and Z-IETD-FMK (1.05 nM), suggesting a similar inhibitory effect of the two substances on caspase-8. In contrast, the Ki rates of caspase-10 for Z-AEVD-FMK (320 nM) and Z-IEDT-FMK (27 nM) showed an ~12-fold higher specificity of Z-IETD-FMK, indicating that the Z-AEVD-FMK inhibitor was less effective in inhibiting caspase-10 when compared with the Z-IETD-FMK inhibitor. Using these two inhibitors, we found striking differences upon treatment of human PMN: Whereas TNF-{alpha}-mediated apoptosis was almost completely blocked in the presence of the Z-AEVD-FMK inhibitor, it had no effect on spontaneous apoptosis, which was measured by different methods including DNA laddering. In contrast, the Z-IETD-FMK inhibitor blocked cytokine-mediated apoptosis as well as spontaneous apoptosis. Thus, the inhibitor studies support the view that spontaneous but not TNF-{alpha}-mediated apoptosis depends on the activation of caspase-10. However, it remains the unresolved question whether extracellular signals are required to activate caspase-10 and the so-called "spontaneous apoptosis" of PMN. Although no stimuli were added when PMN were aged in culture, it cannot be ruled out that autocrine signals, which may be generated by PMN upon aging, may induce spontaneous apoptosis.

Altogether, our study shows that the capability of TNF-{alpha} to promote apoptosis of PMN is not simply a result of an acceleration of spontaneous apoptosis. Whereas caspase-8 was found to be critical for the induction of PMN apoptosis by TNF-{alpha}, spontaneous apoptosis was associated with a specific down-regulation of the caspase-10/b proenzyme and a specific induction of the enzymatic activity of caspase-10. Moreover, the proteolytic cleavage of caspase-10/b was prevented in the presence of TNF-{alpha}, suggesting a molecular switch between caspase-10 and -8 in the upstream death-signaling pathway that determines whether the cells die spontaneously by apoptosis or upon stimulation by inflammatory mediators such as TNF-{alpha}. As apoptosis of PMN is not only involved in the maintenance of PMN homeostasis in the circulation under normal, noninflammatory conditions [33 ] but is also critical for the elimination of PMN in the tissue during the final resolution of inflammation [11 ], these divergent molecular mechanisms may have an impact for the regulation of PMN apoptosis in diverse (patho-) physiological settings and may therefore bear implications for novel therapeutic concepts.


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ACKNOWLEDGEMENTS
 
Deutsche Forschungsgemeinschaft (SFB 366/C3) supported this study. The expert technical assistance of Ms. A. Günther is acknowledged.

Received July 9, 2003; revised December 17, 2003; accepted December 24, 2003.


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