Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate-specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 μg/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3.

Monocytes play a major role in initiating, maintaining, and resolving host inflammatory responses by differentiating into macrophages and dendritic cells and by releasing cell-signaling molecules, including cytokines. In the absence of inflammation, more monocyte precursors develop from the marrow than are needed to replace normal tissue macrophage numbers (1). However, during inflammatory responses, a dramatic up-regulation of monocyte survival and differentiation may be required. Thus, the processes involved in regulating monocyte removal and survival are critical to population control. The importance of monocyte development and differentiation to disease pathogenesis has recently been highlighted by research in animal models in the field of atherogenesis and osteopetrosis (2, 3).

In the absence of an appropriate stimulus, monocytes spontaneously undergo programmed cell death (4, 5, 6, 7). Recently, a family of cysteine-aspartate-specific proteases called caspases has been found to play a major role in programmed cell death. Within this family, a central role has been suggested for caspase-3 (7, 8, 9, 10, 11) and more controversially for caspase-1 (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). Caspase-1 is the prototypical caspase, which was originally identified as IL-1β-converting enzyme (ICE)4 (22). Caspase-1 mediates processing of both pro-IL-1β and pro-IL-18 (23). Additionally, caspase-1 may induce apoptosis, as evidenced by its effect when transfected into fibroblasts and its importance in Fas-mediated apoptosis in caspase-1 knockout animals (12, 13, 14, 15, 16, 17, 18). However, since the discovery of additional ICE-related molecules, other caspases such as caspase-3/CPP32 have been more consistently linked to apoptosis (8).

Generally, caspases exist in cells in an inactive precursor form and require cleavage to generate the active caspase (7). For example, activation of procaspase-3 is tightly regulated by an apoptosis-activating complex, requiring proteolytic removal of an amino-terminal prodomain to produce the active caspase (24, 25, 26, 27). Once activated, caspase-3 performs a number of executioner functions, including the activation of a latent cytosolic endonuclease, caspase-activated deoxyribonuclease (CAD). CAD normally exists intracellularly in an inactive form bound to I-CAD. Caspase-3 cleaves I-CAD, resulting in the release of CAD (28, 29, 30). CAD cleaves DNA into oligonucleosomal fragments that are released into the cytosol. The presence of these cytosolic fragments are landmarks for apoptotic cell death (31, 32).

Due to the importance of caspases in determining either programmed cell death or cytokine activation, we sought to study which caspases are important in human monocyte death and survival. Although previous investigators have shown caspase activity in monocytic cell lines, this is the first study to address the issue in peripheral human monocytes. Our results identify an important role for caspase-3 activity in spontaneous monocyte apoptosis, which is prevented by endotoxin. Interestingly, we fail to identify a role for caspase-1 activation in monocyte death and unexpectedly after endotoxin stimulation. Although LPS protects monocytes from apoptosis and is associated with the processing of pro-IL-1β, no change in baseline caspase-1 activity is detectable. Conversely, LPS prevents the activation of caspase-3, which is activated spontaneously in fresh blood monocytes.

Human monocytes were purified by clumping, as previously described by Graziano and Fanger (33). This method was chosen to limit potential confounding factors involved in other methods of purification, such as adherence or LPS contamination, which may activate cells. Briefly, fresh human monocytes were obtained from normal donors and diluted 1/1 with sterile saline solution. The solution was subsequently centrifuged through a Histopaque-1077 gradient column (Sigma, St. Louis, MO) at 600 × g for 20 min at 4°C. The mononuclear layer was removed, washed, and spun twice in RPMI 1640 (Life Technologies, Grand Island, NY), and the cells were counted. The cells were resuspended in RPMI 1640/10% FBS (HyClone, Logan, UT) at a concentration of 5 × 107 cells/ml. Cells were rotated at 70 rpm on a horizontal rotor for 1 h at 4°C to induce clumping and then sedimented by gravity for 20 min through FBS at 4°C. The sedimented cells were subsequently washed twice in RPMI 1640 and resuspended in RPMI at a final concentration of 1 × 106 cells/ml. The population of monocytes obtained was on average 70–80% pure. In all experiments, monocytes were incubated at a concentration of 1 × 106 cells/ml in serum-free RPMI 1640 at 37°C in 5% CO2. In selected experiments, LPS (LPS Westphal preparation, Escherichia coli 0127:B8; Difco, Detroit, MI) was incubated with monocytes at 1 ng/ml or 1 μg/ml, as indicated.

The generalized caspase inhibitor z-VAD-FMK (Enzyme Systems Products, Livermore, CA); YVAD-CMK, an ICE/caspase-1 family inhibitor (Calbiochem, San Diego, CA); and z-DEVD-FMK, a caspase-3 family inhibitor (Enzyme Systems Products) were utilized. These inhibitors at 1, 10, and 100 μM in DMSO (Sigma) were added to samples of fresh monocytes and incubated overnight in polypropylene tubes at 37°C in a 5% CO2 environment. A DMSO control (0.1% v/v) was also included as a control for the highest concentration of inhibitors.

After specific conditions, 4 × 106 monocytes were harvested by centrifugation. The supernatant was removed and monocytes were resuspended in 100 μl of hypotonic lysis buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.9, 10 mM EDTA, 50 μg/ml RNase A) at room temperature for 10 min. Samples were then centrifuged at 16,000 × g, and the supernatant was placed on a DNA Miniprep system (Wizard Plus series SV 9600; Promega, Madison, WI). After washing with 750 μl and 250 μl of 70% ethanol, the DNA was eluted with 100 μl of water at 65°C and concentrated to the desired volume. Samples were mixed with 6× loading dye (BlueJuice; Life Technologies) and loaded onto a 1.8% agarose gel in 1× TAE buffer (40 mM Tris base, 2 mM EDTA, 20 mM glacial acetic acid). The gel was subsequently stained with a 1/10,000 dilution of Syber Green (Molecular Probes, Eugene, OR) in 1× TAE buffer for 30 min to 1 h. The DNA ladders were imaged using a gel imaging system (Bio-Rad, Hercules, CA). A 123-bp DNA marker (Life Technologies) was included.

Enzymatic caspase activity measured with amino trifluoromethyl coumarin (AFC).

For all AFC preparations, monocytes (3 × 106 cells) were collected by centrifugation and washed with KPM buffer (50 mM KCl, 50 mM PIPES, 10 mM EGTA, 1.92 mM MgCl2, pH 7.0, 1 mM DTT, 0.1 mM PMSF, 10 μg/ml of cytochalasin B, and 2 μg/ml of protease inhibitors: chymostatin, pepstatin, leupeptin, antipain). Cells were snap frozen in liquid nitrogen and lysed by four cycles of freeze thawing. The presence of active caspases was determined by AFC assay using a specific fluoro-substrate, as previously described (34). Lysates were incubated with DEVD-AFC in a cyto-buffer (10% glycerol, 50 mM PIPES, pH 7, 1 mM EDTA) containing 1 mM DTT and 20 μM DEVD-AFC (Enzyme Systems Products). Extracts were also incubated with YVAD-AFC (Enzyme Systems Products) in a YVAD cyto-buffer (10% sucrose, 100 mM HEPES, pH 7.5, 0.1% CHAPS, 10 mM DTT). Specifically, 20 μM YVAD-AFC was added to lysates and incubated for 45 min at room temperature before measurement. Standard recombinant caspase-1 was a gift from Nancy Thornberry (Merck Research Laboratory, Rahway, NJ). In both instances, release of free AFC was determined using a Cytofluor 4000 fluorometer (Perseptive Diagnostics, Framingham, MA; filters: excitation, 400 nm; emission, 505 nm).

Complexation to biotinylated caspase substrate.

Alternatively, active caspases were detected by affinity label, as described by Faleiro et al. (10). Briefly, monocyte pellets were resuspended in KPM buffer and lysed as previously described, but in the presence of 8 μl of 20 μM affinity label solution (biotin-DEVD-aomk or biotin-YVAD-cmk; Biosyn, Belfast, Ireland). Stocks of biotinylated substrates were diluted to 20 μM into MDB buffer (50 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 1 mM DTT, pH 7). Lysates were incubated for 15 min at 37°C and centrifuged for 20 min at 15,000 × g. Supernatants were mixed with an equal volume of 2× SDS-PAGE buffer (Bio-Rad). Proteins were separated in 15% acrylamide gels by electrophoresis. Gels were transferred to PVDF-PSQ (Millipore, Bedford, MA) for 1 h at 200 mAmp. Membranes were incubated for 20 min with avidin-Neutralite (Molecular Probes) at 1 μg/ml in PT buffer (20 mM Tris, 150 mM NaCl, 0.05% Tween-20) containing 1% BSA (PT-BSA buffer). Membranes were washed in PT buffer and then incubated in biotinylated HRP (Molecular Probes) at 25 ng/ml in PT-BSA buffer. Proteins were visualized by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL).

Antigenic detection of caspase cleavage products.

mAbs that specifically recognize caspase-3 were obtained against purified full-length recombinant protein (gift from Y. Lazebnik, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Membranes were blocked at 4°C overnight in PT buffer containing 3% milk and 2% BSA (PT-M). Membranes were incubated with anti-caspase 3 Ab (1:1000) for 1 h at room temperature in PT-M buffer with 0.05% Tween-20 (PT-MT). After five washes in PT buffer containing 0.05% Tween-20 (PT-T), membranes were incubated with anti-mouse HRP (1:5000; Amersham) for 1 h in PT-MT buffer. After washing in PT-T buffer, proteins were visualized by ECL (Amersham).

For IL-1β and IL-8 quantification, LPS (Sigma) at a concentration of 100 ng/ml was added to aliquots of monocyte samples with various concentrations of the caspase inhibitor, YVAD-CMK. Cells were incubated overnight at 37°C in a 5% CO2 environment. Samples were centrifuged at 1200 rpm for 5 min, and the supernatants were removed to a fresh tube. Supernatants were assayed by enzyme-linked immunoassay for both IL-1β (35) and IL-8 (R &D Systems, Minneapolis, MN) production to assess the specificity of the caspase-1 inhibitor, YVAD-CMK. Samples were read at 450 nm on an automated plate reader (Dynatech MR 600, Chantilly, VA). Results were expressed as percentage of cytokine release in comparison with control cells.

A quantitative enzyme-linked immunoassay (Boehringer Mannheim, Indianapolis, IN) that detects DNA fragments was used following the manufacturer’s recommendations. This detects mono- and oligonucleosomal DNA using the cytoplasmic fractions of cell lysates. Briefly, anti-histone Ab is coated onto a microtiter plate. After a washing step, the wells are incubated with 200 μl of incubation buffer for 30 min. The wells are again washed and incubated with 100 μl of sample for 90 min at room temperature. Following another wash step, the wells are then washed and incubated with 100 μl of anti-DNA peroxidase for an additional 90 min. Addition of substrate solution produces a color change after 10–20 min. This color change is compared with a blank well with substrate added. The plate was read at 405 nm on an automated plate reader (Dynatech MR 600). The assay can detect apoptotic DNA from as little as 50 cells/well.

Utilizing an apoptosis detection kit (R&D Systems, Minneapolis, MN), staining of monocytes with both annexin V and propidium iodide was done as recommended by the manufacturers to quantitatively determine the percentage of cells undergoing apoptosis. Briefly, cells were washed with PBS and resuspended in the binding buffer provided. Fluorescein-conjugated annexin V and propidium iodide were incubated for 20 min with monocytes cultured for defined time periods (2–16 h). The monocyte population was selected by gating on CD45/CD14-positive cells (Becton Dickinson Immunocytometry Systems, San Jose, CA) and analyzed on a flow cytometer (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ).

All data were expressed as mean ± SEM. Paired t tests were used for single comparisons (Microsoft Excel; Microsoft, Redmond, WA). For comparisons that involved multiple variables and observations, two- and three-way ANOVA (JMP; SAS Institute, Cary, NC) was used. Having passed statistical significance by ANOVA, individual comparisons were made using the contrast method. Statistical significance was defined as a p value <0.05.

Initial studies focused on developing an in vivo system that allowed the study of monocyte apoptosis. When cultured overnight (16 h) in serum-free conditions in the absence of endotoxin, monocytes showed evidence of apoptosis as determined by annexin V staining (Table I). We also evaluated monocyte death both by cell death ELISA and oligonucleosomal DNA cleavage. As illustrated in Fig. 1,A, a greater than 2-fold increase in mono- and oligonucleosomal DNA was seen in monocytes cultured in LPS-free media in contrast to LPS-treated cells (1 ng/ml). In addition, monocytes cultured for 16 h without LPS showed evidence of apoptotic cell death, as indicated by DNA ladder formation, whereas fresh cells (time zero) and LPS (1 μg/ml)-treated cells did not (Fig. 1 B). Thus, monocytes cultured in the absence of a survival stimulus demonstrate spontaneous apoptosis, which can be prevented by LPS.

Table I.

Effect of time in culture on percent of monocytes undergoing spontaneous apoptosisa

Annexin V Positive Cells (% of total monocytes)
0 h2 h4 h6 h8 h16 h
Donor 1 28.3 35.0 26.7 31.9 40.7 78.4 
Donor 2 19.0 23.3 36.8 26.6 19.5 61.3 
Donor 3 25.0 24.2 24.9 23.6 26.8 69.8 
Mean± SEM (%) 25± 1.1 24± 2.1 25± 2.6 24± 2.1 27± 2.3 70± 1.0 
Annexin V Positive Cells (% of total monocytes)
0 h2 h4 h6 h8 h16 h
Donor 1 28.3 35.0 26.7 31.9 40.7 78.4 
Donor 2 19.0 23.3 36.8 26.6 19.5 61.3 
Donor 3 25.0 24.2 24.9 23.6 26.8 69.8 
Mean± SEM (%) 25± 1.1 24± 2.1 25± 2.6 24± 2.1 27± 2.3 70± 1.0 
a

Annexin staining of monocytes at various time points. Cells were fixed and labeled with annexin V and propidium iodide. The monocyte population was selected by gating on CD45/CD14 positive cells and analyzed on a flow cytometer (FACSCalibur) for annexin V staining.

FIGURE 1.

Monocyte apoptosis occurs in the absence of a survival stimulus. A, Cell death ELISA. Mono- and oligonucleosomal DNA fragment generation, as detected in cell lysates by ELISA for unstimulated and LPS stimulated (1 ng/ml) monocyte cultures after 16 h. Results are expressed as the mean ± SEM from nine experiments. The asterisk denotes two-tailed Student’s t test, p < 0.02. B, DNA laddering in monocytes. Cytosolic DNA (4 × 106 cells/lane) was purified from monocytes cultured in the presence or absence of LPS (1 μg/ml) and analyzed by agarose gel electrophoresis.

FIGURE 1.

Monocyte apoptosis occurs in the absence of a survival stimulus. A, Cell death ELISA. Mono- and oligonucleosomal DNA fragment generation, as detected in cell lysates by ELISA for unstimulated and LPS stimulated (1 ng/ml) monocyte cultures after 16 h. Results are expressed as the mean ± SEM from nine experiments. The asterisk denotes two-tailed Student’s t test, p < 0.02. B, DNA laddering in monocytes. Cytosolic DNA (4 × 106 cells/lane) was purified from monocytes cultured in the presence or absence of LPS (1 μg/ml) and analyzed by agarose gel electrophoresis.

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To investigate a potential role played by caspases in monocyte apoptosis, we measured the presence of active caspases by a sensitive Western blot technique that labels active caspase with the biotinylated probe DEVD-biotin. This probe labels caspase-3-like molecules (36). Furthermore, we utilized z-VAD-FMK, a generalized irreversible caspase inhibitor that binds to the active site of caspases and blocks their biological activity. Using the DEVD-biotin system, monocytes cultured overnight (DMSO control) demonstrated marked activation of caspase-3-like activity when compared with fresh monocytes (Fig. 2,A). As expected, increasing doses of z-VAD-FMK prevented detection of active caspase-3 family proteases. Furthermore, consistent with a functional effect of z-VAD-FMK, a progressive dose-dependent decrease in DNA ladder formation was seen with increasing concentrations of z-VAD-FMK (Fig. 2 B). Therefore, activation of the apoptotic pathway in monocytes, as indicated by DNA ladder formation and caspase activation, can be prevented by a general caspase inhibitor. The suppression by the caspase inhibitor was specific, as a nonblocking peptide FMK substrate (z-Phe-Ala-FMK) did not prevent apoptosis (not shown).

FIGURE 2.

Progressive inhibition of monocyte apoptosis by a generalized caspase inhibitor. A, Monocytes were cultured with increasing concentrations of z-VAD-FMK for 16 h. Monocyte lysates were labeled with biotinylated DEVD, and protein was separated by SDS-PAGE (1.5 × 106 cells/lane). The presence of caspase-3-like activity was determined by Western blotting using avidin-biotin labeling. This is representative of two identical experiments. B, Effects of z-VAD-FMK on monocyte oligonucleosomal DNA ladder formation. Monocytes were cultured as in A, and cytosolic DNA was analyzed by agarose gel electrophoresis. Lane 1, fresh monocytes; lanes 2–4, z-VAD-FMK at 1, 10, and 100 μM; and lane 5, DMSO control for z-VAD-FMK diluent. The gel shown is representative of four separate experiments.

FIGURE 2.

Progressive inhibition of monocyte apoptosis by a generalized caspase inhibitor. A, Monocytes were cultured with increasing concentrations of z-VAD-FMK for 16 h. Monocyte lysates were labeled with biotinylated DEVD, and protein was separated by SDS-PAGE (1.5 × 106 cells/lane). The presence of caspase-3-like activity was determined by Western blotting using avidin-biotin labeling. This is representative of two identical experiments. B, Effects of z-VAD-FMK on monocyte oligonucleosomal DNA ladder formation. Monocytes were cultured as in A, and cytosolic DNA was analyzed by agarose gel electrophoresis. Lane 1, fresh monocytes; lanes 2–4, z-VAD-FMK at 1, 10, and 100 μM; and lane 5, DMSO control for z-VAD-FMK diluent. The gel shown is representative of four separate experiments.

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To determine the kinetics of caspase activation during monocyte apoptosis, cells were incubated for different time periods before lysates were prepared with biotinylated DEVD. Evidence for caspase-3 family activation was seen as early as 4 h in culture (Fig. 3,A). This caspase-3-like activity correlated with subsequent oligonucleosomal DNA fragmentation. DNA ladders were detected as early as 8 h with a progressively more defined signal up to 16 h in culture (Fig. 3 B). Thus, caspase-3-like activity is detectable before DNA laddering, which is consistent with the known role of activated caspase-3 in generating oligonucleosomal DNA fragments.

FIGURE 3.

Kinetics of monocyte caspase activation and DNA laddering. A, Lysates from monocytes cultured for different time periods were labeled with biotinylated DEVD-aomk. Proteins were separated by SDS-PAGE and detected by immunoblot (1.5 × 106 cells/lane). The blot shown is representative of two identical experiments. B, Monocyte oligonucleosomal DNA ladder formation was determined at similar time points as in A, utilizing agarose gel electrophoresis of cytosolic DNA.

FIGURE 3.

Kinetics of monocyte caspase activation and DNA laddering. A, Lysates from monocytes cultured for different time periods were labeled with biotinylated DEVD-aomk. Proteins were separated by SDS-PAGE and detected by immunoblot (1.5 × 106 cells/lane). The blot shown is representative of two identical experiments. B, Monocyte oligonucleosomal DNA ladder formation was determined at similar time points as in A, utilizing agarose gel electrophoresis of cytosolic DNA.

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To further define whether caspase-3 family proteases become activated in monocyte apoptosis, we utilized an irreversible inhibitor of caspase-3 family caspases, z-DEVD-FMK. Monocytes were cocultured for 16 h with incremental doses of z-DEVD-FMK. After lysing the cells, active caspases present in the lysates were detected with biotinylated DEVD, as previously described. A dose-response inhibition of active caspase-3 family proteases was seen (Fig. 4,A). This inhibition of caspase-3 family activity by z-DEVD-FMK correlated with progressive inhibition of oligonucleosomal DNA cleavage (Fig. 4 B).

FIGURE 4.

Inhibition of monocyte apoptosis by a caspase-3 inhibitor. Monocytes were cultured for 16 h in the presence of increasing concentrations of z-DEVD-FMK (0, 1, 10, and 100 μM) or DMSO control. A, Monocyte lysates were labeled with biotinylated DEVD-aomk, separated by SDS-PAGE (1.5 × 106 cells/lane), and transferred to PVDF membrane, and biotinylated caspases were detected by Western blotting. The blot is representative of four different experiments. B, DNA ladder formation. Monocytes from A were also analyzed for DNA ladder formation by agarose gel chromatography of cytosolic DNA. Shown is the effect of increasing concentrations of z-DEVD-FMK (1, 10, and 100 μM).

FIGURE 4.

Inhibition of monocyte apoptosis by a caspase-3 inhibitor. Monocytes were cultured for 16 h in the presence of increasing concentrations of z-DEVD-FMK (0, 1, 10, and 100 μM) or DMSO control. A, Monocyte lysates were labeled with biotinylated DEVD-aomk, separated by SDS-PAGE (1.5 × 106 cells/lane), and transferred to PVDF membrane, and biotinylated caspases were detected by Western blotting. The blot is representative of four different experiments. B, DNA ladder formation. Monocytes from A were also analyzed for DNA ladder formation by agarose gel chromatography of cytosolic DNA. Shown is the effect of increasing concentrations of z-DEVD-FMK (1, 10, and 100 μM).

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To specifically delineate which of the DEVD-dependent caspases are involved in monocyte apoptosis, an anti-caspase-3/CPP-32 Ab was utilized for Western blotting. The Ab recognizes both the precursor form and the processed forms of caspase-3 (8, 37, 38). With progressive time in culture, a decrease in procaspase-3 is seen with a concomitant increase in the processed fragments (Fig. 5). These data suggest that caspase-3 itself is responsible for the z-DEVD-inhibitable activity, consistent with a central role for caspase-3 in monocyte apoptosis.

FIGURE 5.

Caspase-3 is involved in monocyte apoptosis. Lysates from monocytes incubated for different time periods in endotoxin-free RPMI 1640 were separated by SDS-PAGE (3 × 106 cells/lane), blotted onto PVDF membrane, and probed with anti-caspase-3-specific Ab. Proteins were visualized with ECL.

FIGURE 5.

Caspase-3 is involved in monocyte apoptosis. Lysates from monocytes incubated for different time periods in endotoxin-free RPMI 1640 were separated by SDS-PAGE (3 × 106 cells/lane), blotted onto PVDF membrane, and probed with anti-caspase-3-specific Ab. Proteins were visualized with ECL.

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To determine whether caspase-1 family proteases are necessary for monocyte apoptosis, cells were cultured for 16 h with increasing concentrations of YVAD-CMK (caspase-1-like inhibitor). In contrast to z-VAD-FMK and z-DEVD-FMK, monocytes cultured in the presence of YVAD-CMK showed no protection from apoptosis, as indicated by the presence of DNA laddering at all dose concentrations that were used (Fig. 6,A). However, YVAD-CMK was physiologically active within monocytes, as was shown by its ability to block mature IL-1β release in LPS-treated monocytes in a dose-dependent manner while not affecting IL-8 release (Fig. 6, C and D). As further evidence that the caspase-1 family does not play a role in this model of monocyte apoptosis, we determined caspase-1 activity in monocyte cultures at various time points utilizing a fluorogenic substrate. This peptide-tagged fluorogenic substrate is recognized by the catalytic site of active caspase-1 family proteases resulting in fluorescence. YVAD-AFC added to lysates showed no change in activity after 16 h in culture compared with baseline (Fig. 6,B). That YVAD-AFC could detect active caspase-1 was shown by its ready cleavage by recombinant caspase-1 in control experiments (Fig. 6,E). In contrast, when DEVD-AFC (fluorogenic substrate for active caspase-3 family) was added to cell lysates, a 6-fold increase in activity was seen in cells cultured for longer than 4 h (Fig. 6,B). This activity remained high at 16 h in agreement with our previous results (Fig. 3 A).

FIGURE 6.

Role of caspase-1 in monocyte apoptosis. A, Effect of YVAD-CMK on monocyte apotosis. Monocytes were incubated with increasing concentrations of YVAD-CMK (0, 1, 10, and 100 μM or DMSO control). Samples were analyzed after 16 h in culture for DNA ladder formation, as described in Fig. 1. This is representative of four identical experiments. B, Monocyte caspase activity over time in culture. Monocytes were incubated for various time periods. Lysates were analyzed for the presence of caspase-1 and caspase-3 activity using fluorogenic substrates, YVAD-AFC (open symbols) and DEVD-AFC (filled symbols), respectively. Two separate donors were studied (donor 1, circle; donor 2, square). C, Effect of YVAD-CMK (caspase-1 inhibitor) on monocyte IL-1β release (n = 5). Monocytes were incubated overnight with the inhibitor in the presence of LPS (100 ng/ml). Results are expressed as percentage of control. ANOVA results for IL-1β release in the presence of YVAD-CMK revealed a p value <0.001. Individual comparisons were statistically different from baseline (p value <0.001, denoted by asterisks). D, Effect of YVAD-CMK on IL-8 production (n = 5). Monocytes were cultured as in C, and IL-8 release was determined by ELISA, with results being expressed as percentage of control. ANOVA results for IL-8 release in the presence of YVAD-CMK did not reach statistical significance (p = 0.96). E, Detection of purified ICE activity with YVAD-AFC. Cleavage of YVAD-AFC substrate with various concentrations of recombinant caspase-1 is shown.

FIGURE 6.

Role of caspase-1 in monocyte apoptosis. A, Effect of YVAD-CMK on monocyte apotosis. Monocytes were incubated with increasing concentrations of YVAD-CMK (0, 1, 10, and 100 μM or DMSO control). Samples were analyzed after 16 h in culture for DNA ladder formation, as described in Fig. 1. This is representative of four identical experiments. B, Monocyte caspase activity over time in culture. Monocytes were incubated for various time periods. Lysates were analyzed for the presence of caspase-1 and caspase-3 activity using fluorogenic substrates, YVAD-AFC (open symbols) and DEVD-AFC (filled symbols), respectively. Two separate donors were studied (donor 1, circle; donor 2, square). C, Effect of YVAD-CMK (caspase-1 inhibitor) on monocyte IL-1β release (n = 5). Monocytes were incubated overnight with the inhibitor in the presence of LPS (100 ng/ml). Results are expressed as percentage of control. ANOVA results for IL-1β release in the presence of YVAD-CMK revealed a p value <0.001. Individual comparisons were statistically different from baseline (p value <0.001, denoted by asterisks). D, Effect of YVAD-CMK on IL-8 production (n = 5). Monocytes were cultured as in C, and IL-8 release was determined by ELISA, with results being expressed as percentage of control. ANOVA results for IL-8 release in the presence of YVAD-CMK did not reach statistical significance (p = 0.96). E, Detection of purified ICE activity with YVAD-AFC. Cleavage of YVAD-AFC substrate with various concentrations of recombinant caspase-1 is shown.

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As shown previously (Fig. 1), LPS protected monocytes from apoptosis. To determine whether LPS had effects on caspase activity, monocytes were incubated in the presence or absence of LPS for 0 and 16 h. Lysates were analyzed with both YVAD-AFC and DEVD-AFC. Caspase-1-like activity did not change in the presence or absence of LPS, from time 0 to 16 h in culture. In contrast, caspase-3-like activity in non-LPS-treated cells increased more than 4-fold from time 0 to 16 h in culture. In LPS-treated cells, no significant change was seen in caspase-3-like activity during this time period (Fig. 7). These data suggest that LPS protected monocytes from undergoing apoptosis at least partially by regulating caspase-3-like activity.

FIGURE 7.

Effect of LPS on monocyte apoptosis. Monocytes were incubated with or without endotoxin (LPS, 1 μg/ml). Lysates were prepared from monocytes harvested fresh or after 16 h in culture. The presence of caspase-1- and caspase-3-like activity was determined using fluorogenic substrates YVAD-AFC (open bars) and DEVD-AFC (filled bars), respectively. Results are expressed as the mean ± SEM from three separate experiments. When analyzed by both two- and three-way analysis of variance for the effect of LPS, time, and caspase type, the p value was <0.015. For individual comparisons, DEVD-AFC activity was significantly increased at 16 h vs 0 h (p < 0.001). Comparisons are denoted by an asterisk. There was no such increase at 16 h with LPS for DEVD-AFC (p = 0.33, denoted by ¶), nor for YVAD-AFC comparisons with time and LPS variables.

FIGURE 7.

Effect of LPS on monocyte apoptosis. Monocytes were incubated with or without endotoxin (LPS, 1 μg/ml). Lysates were prepared from monocytes harvested fresh or after 16 h in culture. The presence of caspase-1- and caspase-3-like activity was determined using fluorogenic substrates YVAD-AFC (open bars) and DEVD-AFC (filled bars), respectively. Results are expressed as the mean ± SEM from three separate experiments. When analyzed by both two- and three-way analysis of variance for the effect of LPS, time, and caspase type, the p value was <0.015. For individual comparisons, DEVD-AFC activity was significantly increased at 16 h vs 0 h (p < 0.001). Comparisons are denoted by an asterisk. There was no such increase at 16 h with LPS for DEVD-AFC (p = 0.33, denoted by ¶), nor for YVAD-AFC comparisons with time and LPS variables.

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Apoptosis is a cell suicide program that has been conserved through evolution. It results in the cell death through a tightly regulated process resulting in the removal of damaged or unwanted tissue without eliciting an inflammatory response. The processes that elicit activation of the apoptotic program are diverse. The present study asks the question of what role caspases play in monocytes undergoing spontaneous apoptosis in vitro. It further defines the specific roles of caspase-3 and caspase-1 in monocyte apoptosis and in LPS activation.

In agreement with previous observations, we found that monocytes can be prevented from undergoing apoptosis with the addition of endotoxin to cell culture (4, 5). In the absence of a survival stimulus, monocytes undergo programmed cell death. Consistent with previous studies, it is evident that apoptotic monocytes show characteristic changes on electron microscopy and demonstrate oligonucleosomal DNA fragments on DNA gel electrophoresis (4, 5, 32). To demonstrate the role played by caspases, we utilized a biotinylated caspase-3 substrate. As shown in Fig. 3, caspase activity precedes DNA laddering, which is consistent with reports that caspase activation is required for DNA fragmentation (28). As has been previously reported in monocytic tumor cell lines, we show that monocyte apoptosis can be prevented by inhibition of caspase activity with a generalized inhibitor, z-VAD-FMK (39). Subsequently, we demonstrate inhibition of monocyte apoptotic cell death with a caspase-3 family inhibitor, z-DEVD-FMK, but not with a caspase-1 family inhibitor, YVAD-CMK. Fluorogenic substrates also show increased caspase-3-like activity with time, but no change in caspase-1-like activity. As further proof of the specific role of caspase-3 in apoptosis, we found progressive activation of caspase-3 with time in culture by using an anti-caspase-3 Ab. This is consistent with previous work in hemopoietic stem cell lines and other cell lines, which link caspase-3 with apoptosis (9, 10, 40, 41). These findings, however, question the role of caspase-1/ICE in monocyte apoptosis. The lack of caspase-1 activity in monocyte apoptosis contrasts with previous studies in cell lines and knockout models, which support its role in apoptosis (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). Because the closest link between apoptosis and caspase-1 involves the Fas system, it is conceivable that spontaneous human monocyte apoptosis is caspase-1 independent, while Fas-mediated apoptosis may use a caspase-1 pathway. This hypothesis needs to be confirmed, but finds support from studies in ICE knockout mice that demonstrate resistance to Fas-mediated apoptosis, but remain sensitive to apoptosis induced by other stimuli (13).

It had previously been believed that removal of cytokine stimulation from a hemopoietic cell caused cessation of a survival-signaling pathway, resulting in death of the cell. By comparing DEVD-AFC activity in cell cultures at time 0 and after 16 h, in the presence or absence of LPS (survival stimulus), we demonstrated that in the absence of LPS (i.e., loss of a survival signal), a dramatic increase in active caspase-3 family activity occurred, resulting in apoptotic cell death. Thus, LPS appears to prevent apoptosis by inhibition of activation of the caspase death cascade. It is particularly noteworthy that we did not detect any significant change in caspase-1 activity with LPS. This was unexpected considering that LPS stimulates mature IL-1β production and requires caspase-1/ICE for processing pro-IL-1β to its active form. Nevertheless, we and others have failed to detect caspase-1/ICE activity in human monocytes, macrophages, and THP-1 cell lines (42, 43). The lack of caspase-1 activity may be due to insufficient sensitivity. However, our data do show low, but detectable activity, even at baseline, using the YVAD-AFC substrate. Singer et al. (44) has previously shown evidence of caspase-1 activity in monocytes of both LPS-treated and control monocytes by immunoelectron microscopy. Taken together, these findings suggest that caspase-1 activity may exist constitutively in a specialized compartment. Thus, pro-IL-1β processing may not be regulated at the level of caspase-1 activation, but by access of pro-IL-1β to the active caspase-1/ICE compartment. The regulation of caspase-1 remains an area of active investigation.

The caspase-1 and caspase-3 families may have roles other than in the apoptotic program. For example, in THP-1 monocytic cells, caspase-1 cleaves pro-IL-18 to a biologically active mature IL-18 (23). In contrast, caspase-3 was found to cleave precursor and mature IL-18 to biologically inactive units. IL-18 has numerous immunologic functions, which include enhancing NK cell cytotoxicity and stimulating IFN-γ and GM-CSF production by monocytes, both of which can act as a survival signal. In this situation, it would appear that caspase-3 may down-regulate the inflammatory response. Additionally, another proinflammatory cytokine, IL-16, is also cleaved by caspase-3 in both COS cells and lymphocytes to its mature active form. We suggest that caspase activation in lymphocytes may result in activation of pathways other than apoptosis (45). To date, in monocytes a role for caspase-3 other than in apoptosis has not been defined.

Although our findings point to a critical role for caspase-3 in monocyte apoptosis, it does not rule out the possibility that other caspase-3 family proteases also play a crucial role in monocyte apoptosis. For example, caspase-6 and caspase-7 also may be inhibited by z-DEVD-FMK. Nevertheless, our results do show evidence that caspase-3 itself is cleaved in the process of monocyte apoptosis, which suggests it is responsible for the DEVD-AFC activity (Fig. 5).

In conclusion, in the absence of an inflammatory stimulation, monocytes undergo spontaneous apoptosis characterized by activation of caspase-3 and oligonucleosomal DNA ladder formation. This apoptosis program is prevented by the addition of LPS or specific inhibitors of caspase-3.

We thank Thomas L. Clanton, Clay B. Marsh, James E Gadek, and Veela Mehta for their comments and support.

1

This work is supported by National Institutes of Health Grants HL40871 and HL53229. R.J.F. is a Glaxo-Wellcome Pulmonary Fellowship recipient for 1998.

4

Abbreviations used in this paper: ICE, IL-1β-converting enzyme; AFC, amino trifluoromethyl coumarin; CAD, caspase-activated deoxyribonuclease; PVDF, polyvinylidene difluoride; DEVD, Asp-Glu-Val-Asp; YVAD, Tyr-Val-Ala-Asp; CMK, chloromethyl ketone; FMK, fluoromethyl ketone; aomk, acyloxymethyl ketone.

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