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Department of Internal Medicine, Division of Pulmonary and Critical Care, and The Heart and Lung Institute, Ohio State University, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-1ß-converting enzyme (ICE)4 (22). Caspase-1 mediates processing of both pro-IL-1ß 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-1ß, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 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 x 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 x 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 x 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.
Caspase inhibitors
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.
Detection of DNA fragmentation
After specific conditions, 4 x 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 x 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 6x loading dye (BlueJuice; Life Technologies) and loaded onto a 1.8% agarose gel in 1x 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 1x 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.
Preparation of lysates and detection of caspase activity
Enzymatic caspase activity measured with amino trifluoromethyl coumarin (AFC). For all AFC preparations, monocytes (3 x 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 x g. Supernatants were mixed with an equal volume of 2x 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).
Cytokine measurements
For IL-1ß 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-1ß (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.
Cell death ELISA
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.
Flow cytometry analysis
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).
Statistical analysis
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1A, 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. 1B). Thus, monocytes cultured in the absence of a
survival stimulus demonstrate spontaneous apoptosis, which can be
prevented by LPS.
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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. 2A). 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. 2B). 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).
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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. 3B).
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.
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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. 4A). This inhibition of
caspase-3 family activity by z-DEVD-FMK correlated with progressive
inhibition of oligonucleosomal DNA cleavage (Fig. 4B).
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). 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.
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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. 6A). However, YVAD-CMK
was physiologically active within monocytes, as was shown by its
ability to block mature IL-1ß 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. 6B). That YVAD-AFC could detect
active caspase-1 was shown by its ready cleavage by recombinant
caspase-1 in control experiments (Fig. 6E). 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. 6B). This activity
remained high at 16 h in agreement with our previous results (Fig. 3
A).
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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-1ß production and requires caspase-1/ICE for processing pro-IL-1ß 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-1ß processing may not be regulated at the level of caspase-1 activation, but by access of pro-IL-1ß 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-
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.
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Acknowledgments |
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Footnotes |
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2 R.J.F. and A.I.D. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Mark D. Wewers, Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Ohio State University, N-325 Means Hall, 1654 Upham Drive, Columbus OH 43210. E-mail address:
4 Abbreviations used in this paper: ICE, IL-1ß-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.
Received for publication November 25, 1998. Accepted for publication May 27, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-1ß prevent programmed cell death (apoptosis) in human peripheral blood monocytes. J. Immunol. 146:1541.[Abstract]
RI and FcRII on monocytes and granulocytes are cytotoxic trigger molecules for tumor cells. J. Immunol. 139:3536.[Abstract]
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