Role of Macrophage Lysosomal Enzymes in
the Degradation of Nucleosomes of Apoptotic
Cells
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J Immunol 1999; 163:5346-5352; ;
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References
Chikako Odaka and Toshiaki Mizuochi
Role of Macrophage Lysosomal Enzymes in the Degradation of
Nucleosomes of Apoptotic Cells
Chikako Odaka1 and Toshiaki Mizuochi
P
rogrammed cell death is recognized as the physiological
mechanism by which a large number of unwanted cells are
deleted from the body. Once cells undergo programmed
cell death, their corpses are swiftly engulfed by other cells and
degraded. This engulfment process involves the recognition and
subsequent phagocytosis of cell corpses by engulfing cells. The
process is important for tissue remodeling and for the resolution of
inflammatory responses (1).
The mechanisms by which apoptotic cells can be recognized and
removed have been the subject of intense investigation for the last
few years. An important consequence of the apoptotic process is
cell surface alterations that lead to rapid recognition by phagocytes. A number of surface molecules are involved in the recognition of apoptotic cells by macrophages or dendritic cells, among
which are an uncharacterized lectin inhibited by N-acetylglucosamine (2), the vitronectin receptors (avb3 integrin) (3), which
is thought to cooperate with CD36 in binding to thrombospondin
on the surface of the apoptotic cells (4, 5), a phosphatidyl-L-serine
receptor (6, 7), scavenger receptors (8 –10), and the macrophage
Ag identified by the mAb 61D3 (11), which is identical to CD14
(12). The ABC1 transporter has also been suggested to be involved
in phagocytosis (13). In contrast, little is known about the intracellular processing by which macrophages dispose of apoptotic
cells after engulfment.
The biochemical hallmark of apoptosis is the appearance of a
fragmentation pattern in chromatin, which is indicative of the
DNA cleavage at the linker regions between nucleosomes. The
Department of Bacterial and Blood Products, National Institute of Infectious Diseases, Tokyo, Japan
DNA fragments yield discrete multiples of a 180-bp subunit that is
detected as a “DNA ladder” on agarose gels after isolation of the
DNA from apoptotic cells (14).
The thymus is the organ in which the repertoire of T cells is
selected from a much larger number of immature thymocytes, and
extensive apoptotic cell death occurs in immature thymocyte populations. Although numerous immature thymocytes undergo apoptosis, few dead cells are observed in situ due to rapid engulfment
by phagocytic macrophages in the thymus (15). Recent studies,
using the sensitive TUNEL technique to examine the distribution
of apoptotic thymocytes, demonstrated an increase in the number
of TUNEL-positive cells in the cortex of the thymus within a few
hours after administration of glucocorticoid or anti-CD3 Ab (16 –
18). The clearance of TUNEL-positive apoptotic thymocytes was
found to be conducted by macrophages in thymus. After apoptotic
cells are engulfed by macrophages, the number of TUNEL-positive apoptotic thymocytes gradually reduces and finally becomes
comparable with that of TUNEL-positive cells in untreated thymus
(17, 18). These observations prompted us to predict that the nucleosomal DNA fragments of apoptotic cells might be further degraded when apoptotic cells are phagocytosed by macrophages.
In the present study, we investigated the fate of apoptotic cells
after engulfment by macrophages. In particular, we traced the
DNA fragments of apoptotic cells upon their engulfment by macrophages and observed a disappearance of nucleosomal DNA ladder formation in apoptotic cells. Furthermore, our study demonstrated that the lysosomal enzymes in macrophages were involved
in the degradation of nucleosomes of engulfed apoptotic cells.
These findings will be discussed with reference to the importance
of apoptotic cell scavenger.
Received for publication June 2, 1999. Accepted for publication August 25, 1999.
Materials and Methods
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Cells and reagents
1
Address correspondence and reprint requests to: Dr. Chikako Odaka, Department of
Bacterial and Blood Products, National Institute of Infectious Diseases, 1-23-1,
Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan. E-mail address: [email protected]
Copyright © 1999 by The American Association of Immunologists
IL-2-dependent CTLL-2 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 mM 2-ME, 20 U/ml penicillin, 20 mg/ml streptomycin, and 100 U/ml recombinant mouse IL-2 (19).
Mouse macrophage cell line J774.1 cells were maintained in RPMI 1640
0022-1767/99/$02.00
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Although apoptotic cells are recognized and engulfed by macrophages via a number of membrane receptors, little is known about
the fate of apoptotic cells after the engulfment. We observed in this study that nucleosomal DNA fragments of apoptotic cells
disappeared when they were engulfed by the macrophage cell line J774.1 at 37oC. Pretreatment of J774.1 cells with chloroquine
inhibited intensive DNA degradation, indicating that the cleavage of nucleosomal DNA fragments of apoptotic cells may take place
in the lysosomes of J774.1. When apoptotic cells were exposed to a lysosome-rich fraction derived from J774.1 cells under an acidic
condition, nucleosomal DNA fragments of apoptotic cells were no longer detectable by agarose gel electrophoresis. Additionally,
we found that the lysosome-rich fraction of J774.1 cells contained an acid DNase that is similar to DNase II with respect to its m.w.,
optimal pH, and sensitivity to the inhibitors of DNase II. By exposure of apoptotic cells to the lysosomal-rich fraction, nucleosomal
core histones of apoptotic cells were hydrolyzed along with degradation of nucleosomal DNA fragments. Addition of pepstatin A
to the reaction buffer resulted in accumulation of ;180-bp DNA fragments and inhibition of hydrolysis of nucleosomal core
histones. Leupeptin or CA-074 partially inhibited the degradation of nucleosomal DNA fragments and core histones. These
findings suggest that lysosomal enzymes of macrophages, e.g., DNase II-like acid DNase and cathepsins, are responsible for the
degradation of nucleosomes of apoptotic cells. The Journal of Immunology, 1999, 163: 5346 –5352.
The Journal of Immunology
5347
supplemented with 10% heat-inactivated FCS, 50 mM 2-ME, and the antibiotics. The RPMI 1640 and supplements were purchased from Life
Technologies (Grand Island, NY). Pepstatin A, CA-074, and leupeptin
were obtained from Peptide Institute (Osaka, Japan). All other chemicals
were purchased from Sigma (St. Louis, MO) unless otherwise noted.
was washed away in 40 mM Tris-HCl (pH 7.4), the gel was divided into
three sections. Each gel was incubated in 40 mM Tris-HCl (pH 7.4) or 40
mM sodium acetate buffer (pH 5.2) with or without iodoacetic acid for 20 h
at room temperature. The gels were stained with ethidium bromide (0.5
mg/ml) and illuminated under UV light.
Assay for phagocytosis of apoptotic cells
Detection of histone hydrolysis
One day before assay for phagocytosis, J774.1 cells were seeded at a density of 1 3 106/10 ml in 100-mm2 dishes. CTLL-2 cells were cultured in
the absence of IL-2 for 15 h, with the result that almost all cells showed
apoptosis. These cells were used as apoptotic CTLL-2 in following
experiments.
Apoptotic CTLL-2 cells (1 3 107) were added to macrophage monolayers and then cultured at 37oC for 1 h. After nonphagocytosed apoptotic
cells were removed by extensive washing with RPMI 1640, J774.1 cells
were incubated in RPMI 1640 containing 10% FCS at 37oC or at 4oC for
the indicated period. In some experiments, J774.1 cells were treated with
the indicated concentration of chloroquine. One hour later cells were extensively washed with RPMI 1640 three times and then cultured with apoptotic cells for 1 h. Then, J774.1 cells were washed to eliminate nonphagocytosed apoptotic CTLL-2 cells and incubated in fresh medium for 3 h.
Cells were resuspended in 0.4 N H2SO4 and kept on ice for 30 min. After
centrifugation at 20,000 3 g for 15 min, the supernatant was collected and
then four times the volume of absolute ethanol was added to the samples.
After centrifugation, the pellet was washed with absolute ethanol and allowed to dry at room temperature. The pellets were suspended in 20 ml of
solution containing 0.1 M glycine, 0.2% SDS, and 4 M urea (pH 10).
SDS-PAGE for analysis of histone hydrolysis was performed as described
by Panyin and Chalkely (21) with a slight modification in a 17.5% polyacrylamide gel. The proteins in the gel were stained with Coomassie
brilliant blue.
Detection of DNA cleavage in situ by TUNEL
Isolation and detection of DNA fragments
Cells were resuspended in hypotonic lysis buffer (0.25% Triton X-100, 10
mM Tris-HCl, and 10 mM EDTA, pH 8.0) and centrifuged for 15 min at
20,000 3 g. The supernatant, containing small DNA fragments, was
treated with 100 mg/ml proteinase K and 50 mg/ml RNase A. The DNA
was extracted by phenol/chloroform and precipitated in isopropyl alcohol
containing 0.5 M NaCl. After the sample was centrifuged, the pellet was
washed with 70% ethanol and allowed to dry at room temperature. The
DNA was resuspended in TE buffer (10 mM Tris-HCl and 1 mM EDTA,
pH 8.0), and then electrophoresed on a 2% agarose gel containing ethidium
bromide. Gels were photographed using UV transillumination.
Preparation of the lysosome-rich fraction and apoptotic cell
degradation assay
The lysosome-rich fraction of J774.1 cells was prepared by sequential centrifugations according to the method of de Duve et al. (20) with a slight
modification. In brief, J774.1 cells were washed three times with PBS and
adjusted to a density of 5 3 107 cells/ml in 0.25 N sucrose. The following
fractionation steps were conducted at 4oC. The cell suspension was homogenized and centrifuged at 500 3 g for 12 min to separate crude nuclear
fraction. The supernatant was centrifuged at 5,000 3 g for 10 min and
followed by a final centrifugation of the resultant supernatant at 14,000 3
g for 30 min. The pellet containing lysosome was suspended in 0.01 M
sodium acetate (pH 5.2) and then dialyzed overnight against the same
buffer. The concentration of the lysosome-rich fraction was adjusted to 1
mg/ml. Apoptotic CTLL-2 cells (8 3 105) were incubated with the lysosome-rich fraction (100 mg) in 0.15 M NaCl/5 mM sodium acetate (pH 5.2)
in the absence or presence of the indicated reagents at 37oC. After a 2-h
incubation, the cells were used for isolation of DNA or for extraction of
histones.
Detection of acid DNase activity
Acid DNase was visualized as described by Lacks (21). The lysosome-rich
fraction of J774.1 cells (60 mg) was elecrophoresed on a 12.5% SDSpolyacrylamide gel containing calf thymus DNA at 10 mg/ml. After SDS
Fate of DNA strand breaks in apoptotic cells engulfed by J774.1
cells
In the previous studies, deprivation of IL-2 from IL-2-dependent
CTLL-2 was shown to result in apoptotic cell death (23, 24). In our
study, almost all CTLL-2 cells were found to be dead when cultured in the absence of IL-2 for 15 h, as assessed by trypan blue
dye exclusion (data not shown). When murine macrophage-like
cells J774.1 were exposed to a 10-fold excess of apoptotic CTLL-2
cells and incubated for 1 h at 37oC, ;70% of apoptotic cells were
engulfed by J774.1 cells (data not shown). To examine the processing of apoptotic CTLL-2 cells that were engulfed by J774.1
macrophages, we performed TUNEL staining, which allows detection of DNA strand breaks in the apoptotic CTLL-2 cells (16).
J774.1 cells were coincubated with dead CTLL-2 cells for 1 h at
37oC, and they were extensively washed and fixed. DNA strand
breaks of apoptotic cells in J774.1 macrophages were then evaluated by the TUNEL method. A significant number of DNA breaks
of apoptotic cells was detected in J774.1 cells (Fig. 1a). After
further incubation for 6 h at 37oC or at 4oC, the cells were fixed
and processed for TUNEL staining. In an additional incubation for
6 h at 37oC, the number of TUNEL-positive cells was substantially
decreased in the engulfing cells. (Fig. 1c). The process was inhibited when J774.1 cells were kept at 4oC over a 6-h period (Fig. 1b).
Thus, when J774.1 cells that engulfed apoptotic cells were incubated for 6 h at 37oC, a disappearance of DNA breaks in nuclei of
apoptotic cells was observed.
Degradation of apoptotic DNA fragments in J774.1 cells
DNA fragmentation of apoptotic CTLL-2 cells can be visualized
on a gel as a series of fragments that are multiples of 180 bp (14).
When DNA isolated from apoptotic CTLL-2 cells was subjected to
agarose gel electrophoresis, DNA fragmentation, as demonstrated
by a characteristic “DNA ladder” formation, was observed (data
not shown). After J774.1 cells were coincubated with a 10-fold
excess of apoptotic CTLL-2 cells at 37oC for 1 h, they were extensively washed to eliminate nonphagocytosed apoptotic cells and
incubated in a fresh medium at 37oC for up to 6 h. The cells were
lysed with hypotonic lysis buffer, and the DNA fragments of apoptotic cells engulfed by J774.1 cells were isolated and then subjected to agarose gel electrophoresis.
After J774.1 cells were coincubated with dead CTLL-2 cells for
1 h, nucleosomal DNA ladder formation of apoptotic cells engulfed by J774.1 cells was detectable on the agarose gel (Fig. 2,
lane 2). An additional incubation at 37oC leaded to a disappearance of DNA ladder in a time-dependent manner. In a 2-h incubation, internucleosomal DNA fragments of apoptotic cells were
still visible on the gel (Fig. 2, lane 4), whereas the DNA ladder was
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For TUNEL staining, J774.1 cells were plated on 8-well chamber slides at
1 3 103 cells/well and incubated overnight. Apoptotic CTLL-2 cells (1 3
104 cells/well) were added to the above J774.1 cells and incubated at 37oC
for 1 h. Nonphagocytosed apoptotic cells were removed and then incubated
at 4oC or at 37oC. At the indicated time, the cells were fixed in 4% buffered
formaldehyde and permeabilized with 0.5% saponin/1% BSA in PBS. The
cells were further incubated for 60 min at 37oC in a reaction buffer consisting of 100 mM sodium cacodylate (pH 7.2), 1 mM CoCl2, 10 nM
biotin-16-dUTP (Boehringer Mannheim, Mannheim, Germany), and 100
U/ml TdT (Takara Shuzo, Kyoto, Japan), followed by incubation with avidin-biotin-peroxidase complexes using the Vecstain-ABC kit, (Vector,
Burlingame, CA) for 30 min. Cells were washed and then incubated with
a mixture of 0.06% 3–39-diaminobenzidone tetrahydrochiloride (DAB) and
0.03% H2O2 in 0.1 M Tris-HCl (pH 7.5). Counterstaining was performed
with Meyer’s haematoxylin. Microscopic observations were conducted by
using Microflex UFX-II (Nikon, Tokyo, Japan).
Results
5348
DEGRADATION OF NUCLEOSOMES IN APOPTOTIC CELLS
FIGURE 1. Processing of DNA breaks of apoptotic CTLL-2 cells engulfed by macrophage J774.1 cells. IL-2-dependent CTLL-2 cells cultured in the
absence of IL-2 for 15 h were used as apoptotic CTLL-2 cells. After murine macrophage-like J774.1 cells were exposed to a 10-fold excess of apoptotic
CTLL-2 cells and incubated for 1 h at 37oC, J774.1 cells were extensively washed to eliminate nonphagocytosed apoptotic cells (a) and then incubated for
6 h at 4oC (b) or at 37oC (c). At the end of the incubation period, cells were fixed in 4% buffered formaldehyde and then processed with TUNEL staining
as described in Materials and Methods. All panels were photographed at 3100. Arrowheads indicate apoptotic CTLL-2 cells engulfed by J774.1 cells.
These results are representative of three independent experiments.
fore the exposure of apoptotic CTLL-2 cells. Pretreatment of
J774.1 cells with 50 or 100 mM chloroquine did not affect engulfment of apoptotic cells (data not shown). As shown in Fig. 3, when
apoptotic cells engulfed by untreated J774.1 were incubated for 3 h
at 37oC, nucleosomal DNA fragments were hardly detectable on
the gel (Fig. 3, lane 2). Chloroquine at the concentration of 100
mM was able to inhibit DNA degradation in phagocytosis by macrophages (Fig. 3, lane 3). Chloroquine at 50 mM only slightly
inhibited DNA degradation (Fig. 3, lane 4). Thus, blocking the
acidification of lysosomes/endosomes by chloroquine caused the
inhibitory effect on the processing of DNA cleavage of apoptotic
cells in macrophages. Taken together, these results indicate that
lysosomal enzymes including DNase(s) in macrophages are responsible for degradation of nucleosomal DNA fragments of apoptotic cells.
FIGURE 2. Time-dependent DNA degradation of apoptotic cells engulfed by J774.1 cells. A total of 1 3 107 apoptotic CTLL-2 cells were
added to 1 3 106 macrophage J774.1 monolayers and cultured at 37oC for
1 h. Then, J774.1 cells were extensively washed and incubated in a fresh
medium at 4oC or at 37oC for up to 6 h. At the end of the incubation period,
the fragmented DNA were isolated and then subjected to 2% agarose gel
electrophoresis. Lane 1, J774.1 cells alone; lane 2, apoptotic CTLL-2 cells
engulfed by J774.1 cells. Apoptotic CTLL-2 cells engulfed by J774.1 cells
were incubated for 6 h at 4oC (lane 3), for 2 h at 37oC (lane 4), for 4 h at
37oC (lane 5), or for 6 h at 37oC (lane 6). The size of fragments of HindIII
digest of l-phage shown in the left serves as molecular size markers. The
result is representative of three independent experiments.
FIGURE 3. Chloroquine inhibits DNA degradation of apoptotic cells
engulfed by J774.1 cells. A total of 1 3 107 apoptotic CTLL-2 cells were
added to 1 3 106 J774.1 monolayers and cultured at 37oC for 1 h. Then,
J774.1 cells were extensively washed and incubated in fresh medium at
4oC (lane 1) or at 37oC (lane 2) for 3 h. In cultures with chloroquine,
J774.1 cells were pretreated with 100 mM (lane 3) or 50 mM (lane 4)
chloroquine for 1 h. Cells were extensively washed and cultured with apoptotic cells. One hour later, J774.1 cells were extensively washed and
incubated in a fresh medium for 3 h at 37oC. At the end of the incubation
period, the fragmented DNA was isolated from these cells and subjected to
2% agarose gel electrophoresis. The result is representative of three independent experiments.
Acid DNase activity in the lysosome-rich fraction of
macrophages
We next sought to identify the lysosomal enzymes involved in
DNA degradation of apoptotic cells. To find out whether DNase(s)
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hardly detectable after an additional incubation for 2 h at 37oC
(Fig. 2, lane 5). In a 6-h incubation at 37oC, internucleosomal
DNA fragments of apoptotic cells engulfed by J774.1 cells was no
longer detectable on the gel (Fig. 2, lane 6), which is concordant
with the result obtained by the TUNEL method shown in Fig. 1.
On the other hand, when apoptotic CTLL-2 cells engulfed by
J774.1 cells were incubated for 6 h at 4oC, the DNA ladder remained prominent (Fig. 2, lane 3). These findings suggest that the
degradation of DNA fragments of apoptotic cells occurs in J774.1
cells and that macrophages may contain enzymes capable of digesting the DNA fragments of apoptotic cells. The above process
was significantly inhibited when the experiment was performed at
4oC, i.e., at a temperature that does not permit phagocytosis.
Lysosomes are highly specialized organelles that have a low
internal pH and contain hydrolytic enzymes that have an optimum
acidic pH. Chloroquine is known to raise the pH in lysosomes/
endosomes and to be concentrated inside them (25, 26). To investigate whether lysosomal enzymes of macrophages are involved in
digesting the internucleosomal DNA fragments of apoptotic
CTLL-2 cells, J774.1 cells were pretreated with chloroquine be-
The Journal of Immunology
5349
FIGURE 5. Lysosomal enzymes of J774.1 cells are responsible for degradation of nucleosomal DNA fragments in apoptotic cells. The lysosomerich fraction of J774.1 cells was prepared as described in Materials and
Methods. A total of 8 3 105 apoptotic CTLL-2 cells were incubated at
37oC for 2 h in the absence (lane 1) or the presence of the lysosome-rich
fraction of J774.1 cells (lane 2) with the following reagents: 2 mM ZnSO4
(lane 3), 1 mM iodoacetic acid (lane 4), 0.5 mM leupeptin (lane 5), 0.5 mM
CA-074 (lane 6), 0.5 mM pepstatin A (lane 7), and 0.5 mM leupeptin and
0.5 mM pepstatin A (lane 8). Fragmented DNA was isolated and then
analyzed by electrophoresis on a 2% agarose gel. A 100-bp ladder was run
as a size standard (M). The result is representative of three independent
experiments.
are present in lysosomes, we prepared the lysosome-rich fraction
from J774.1 cells, and then nuclease activity in the isolated lysosome-rich fraction was visualized on a gel with the SDS-PAGE
technique, in which spots with DNase activities appear as dark
bands on fluorescent background (21). As shown in Fig. 4a, DNase
activity in the lysosome-rich fraction was detected at pH 5.2 at
42– 43 kDa, whereas its activity could not be detected at pH 7.4
(Fig. 4b). It was reported that DNase II activity is inhibited by
sulfate ions, zinc ions, or iodoacetic acid (27). Alkylation of one
histidine of DNase II by iodoacetic acid or iodoacetamide inactivates the enzyme (28). When a gel was incubated in the presence
of 1 mM iodoacetic acid under an acidic condition (pH 5.2), the
intense band at the location of ;42– 43 kDa could not be detected
(Fig. 4c). We concluded that the lysosome-rich fraction of J774.1
cells contains a DNase that has an optimum acidic pH and is sensitive to iodoacetic acid.
We further examined whether the lysosome-rich fraction of
J774.1 cells induces the degradation of internucleosomal DNA
fragments in apoptotic cells. After apoptotic CTLL-2 cells were
incubated with or without the lysosome-rich fraction of J774.1
cells for 2 h, DNA was isolated and subjected to agarose gel electrophoresis. Even when the apoptotic cells were incubated in 0.15
M NaCl/5 mM sodium acetate (pH 5.2) for 2 h, the nucleosomal
DNA ladder was observed by agarose gel electrophoresis (Fig. 5,
lane 1). When apoptotic cells were exposed to the lysosome-rich
fraction at pH 5.2, the DNA ladder was no longer detectable on a
agarose gel (Fig. 5, lane 2). No detectable hydrolysis of the DNA
ladder was observed when the incubation was performed at pH 7.4
(data not shown). As described above, DNase II is shown to be
inactivated in the presence of sulfate ions, zinc ions, or iodoacetic
acid (27, 28). Therefore, 2 mM ZnSO4 or 1 mM iodoacetic acid
was included in the reaction buffer. When apoptotic cells were
incubated with the lysosome-rich fraction of J774.1 cells in the
presence of each reagent, degradation of DNA fragments of apoptotic cells was completely inhibited (Fig. 5, lanes 3 and 4). These
results demonstrated that the lysosomal fraction derived from
J774.1 cells was able to degrade nucleosomal DNA fragments of
apoptotic cells into small random-sized fragments that were hardly
visible on a 2% agarose gel. ZnSO4- or iodoacetic acid-sensitive
acid DNase of the lysosome-rich fraction of J774.1 cells may be
responsible for the DNA degradation.
Cathepsins B and D are major lysosomal proteinases, and each
enzyme can contribute to up to 10% of the total lysosomal proteins. So far, a number of studies have discovered several other
cathepsins. To determine whether cathepsins in lysosomes of
J774.1 cells would be involved in the degradation of apoptotic
cells, cathepsin inhibitors were introduced into the reaction buffer
containing apoptotic CTLL-2 cells and the lysosome-rich fraction
of J774.1 cells. Leupeptin strongly inhibits trypsin, plasmin, papain, and cathepsins B and L, but it has little or no inhibitory
activity on cathepsins A and D (29). It inhibits cathepsin L less
effectively than cathepsin B (30). CA-074 exhibits greater inhibitory effects on cathepsin B than leupeptin (31). Pepstatin A is
known as a strong inhibitor of acid proteases, including pepsin,
renin, and cathepsin D (32) and cathepsin E (33). As shown in Fig.
5, lane 7, the addition of pepstatin A induced a significant increase
in the accumulated ;180-bp DNA fragments, i.e., mono-nucleosomal DNA. Leupeptin or CA-074 showed a weak inhibitory effect
on the degradation of nucleosomal DNA fragments of apoptotic
cells (Fig. 5, lanes 5 and 6). Combination of pepstatin A and leupeptin were more effective than pepstatin A alone (Fig. 5, lane 8).
The presence of 1% DMSO in the reaction buffer did not affect the
lysosome-rich fraction-induced DNA degradation (data not
shown). Thus, cathepsins in the lysosome-rich fraction of J774.1
appear to be necessary for the degradation of DNA fragments of
apoptotic cells. Pepstatin A-sensitive cathepsins are likely to be
involved in the degradation of nucleosomal DNA. Moreover, partial inhibition by leupeptin or CA-074 may suggest the involvement of several other lysosomal proteinases in the DNA cleavage.
Proteolysis of nucleosomal core histones in apoptotic cells
exposed to the lysosome-rich fraction of macrophages
As shown in Fig. 5, when apoptotic cells were incubated with the
lysosome-rich fraction of J774.1 cells in the presence of cathepsin
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FIGURE 4. Detection of acid DNase activity in the lysosome-rich fraction of J774.1 cells. The lysosome-rich fraction of J774.1 cells was prepared as described in Materials and Methods. The lysosome-rich fraction
of J774.1 cells was subjected to electrophoresis in a 12.5% SDS-polyacrylamide gel containing calf thymus DNA. After washing out SDS, the gel
was divided into three sections. Each gel was incubated for 20 h at room
temperature in the following solutions; 40 mM sodium acetate (pH 5.2) (a),
40 mM Tris (pH 7.4) (b), and 40 mM sodium acetate (pH 5.2) in the
presence of 1 mM iodoacetic acid (c). Molecular weights at left indicate
migration of prestained standard proteins. The arrowhead indicates active
DNase. These results are representative of three independent experiments.
5350
DEGRADATION OF NUCLEOSOMES IN APOPTOTIC CELLS
histones’ degradation, indicating that nucleosomal core histones of
apoptotic cells may be degraded mainly by pepstatin A-sensitive
lysosomal cathepsins of J774.1 cells (Fig. 6, lane 7). Leupeptin
showed an inhibitory effect (Fig. 6, lane 6), whereas CA-074 had
a weak inhibitory effect on the histone degradation (Fig. 6, lane 5).
Addition of both pepstatin A and leupeptin to the reaction buffer
induced the inhibition of proteolysis of core histones most effectively (Fig. 6, lane 4).
From these results, we concluded that lysosomal proteinases of
J774.1 cells were capable of hydrolyzing nucleosomal core histones. Proteolytic degradation of core histones may be a necessary
step in the cleavage of nucleosomal DNA fragments into small
DNA fragments.
inhibitors, the degradation of mono-nucleosomal DNA fragment
was largely protected. During apoptosis, double-stranded DNA is
cleaved at most accessible internucleosomal linker region, resulting in the generation of mono- and oligo-nucleosomal DNA. DNA
of the nucleosomes is tightly complexed with the core histones
H2A, H2B, H3, and H4 and therefore protected from the cleavage
by endonuclease (34). With respect to these findings, when apoptotic cells are exposed to macrophage lysosomes, proteolytic degradation of core histones may take place simultaneously with the
digestion of nucleosomal DNA fragments into smaller DNA
fragments.
A number of studies have been reported on histone-hydrolyzing
proteinase activities in chromatin isolated from calf thymus (35–
38) or from rat liver (39, 40). On the other hand, it has been
demonstrated that lysosomal cathepsins are capable of hydrolyzing
all types of core histones (41– 43). Therefore, it was hypothesized
that core histones of apoptotic cells might be degraded by lysosomal cathepsins in macrophages. To confirm this, we examined by
in vitro studies using cathepsin inhibitors whether the lysosomerich fraction of J774.1 cells contains histone-hydrolyzing proteinases. After CTLL-2 cells were cultured in the presence or absence
of IL-2 for 16 h, histones of apoptotic CTLL-2 cells were extracted
and thereafter subjected to SDS-PAGE for the detection of histone
subtypes and their hydrolysis. The amount of core histones of apoptotic cells was relatively smaller than that of CTLL-2 cells cultured in the presence of IL-2 (Fig. 6, lane 1 vs lane 2). It may be
due to the release of cellular contents including nucleosomes into
culture medium during apoptosis. However, nucleosomal core histones of apoptotic cells including H2A, H2B, H3, and H4 remained intact during apoptosis (Fig. 6, lane 2). By exposure of
apoptotic CTLL-2 cells to the lysosome-rich fraction of J774.1
cells for 2 h, histones H2A, H2B, H3, and H4, which are bound to
DNA, were degraded almost completely (Fig. 6, lane 3). The proteolytic fragments of these histones were no longer detected because they might be run away from the polyacrylamide gel during
the experimental procedure. Pepstatin A effectively inhibited core
Discussion
In the present study, we observed a rapid disappearance of DNA
strand breaks in apoptotic cells that were phagocytosed by macrophage J774.1 cells, as assessed by TUNEL staining. Furthermore, internucleosomal DNA fragments in apoptotic cells engulfed by J774.1 cells became undetectable when the DNA of
apoptotic cells was analyzed by electrophoresis on an agarose gel.
When the fate of DNA fragments of apoptotic cells after engulfment by resident peritoneal macrophages of BALB/c or C3H/HeJ
mice was examined, we also observed the disappearance of nucleosomal DNA fragments of apoptotic cells (data not shown).
Pretreatment of J774.1 cells with chloroquine inhibited the degradation of DNA fragments in apoptotic cells. This finding implies
that DNA hydrolytic activity of macrophages is mainly localized
in the lysosomes. Furthermore, our study using a cell-free system
indicated that an acid DNase in the lysosomes of J774.1 cells may
be responsible for the degradation of internucleosomal DNA fragments of apoptotic cells.
DNase II hydrolyzes DNA to 39-phosphoryloligonucleotides under acidic conditions and therefore has been designated as an “acid
DNase” (44). de Duke et al. (20), from the results of differential
centrifugation of homogenate of rat liver, demonstrated that DNase
II is a lysosomal enzyme. Furthermore, DNase II was directly isolated from lysosomes in the rat liver (27) and in porcine spleen (45,
46), which provided additional evidence that DNase II is lysosomal. DNase II activity can be detected in various mammalian tissues and species (47). The enzymatic properties of DNase II from
different tissues and animals are found to be very similar, but their
structures and the estimated molecular weights are significantly
diverse. Porcine spleen DNase II is a heterodimeric protein, consisting of a 1:1 complex of an a and b subunit with molecular
masses of 35 kDa and 10 kDa, respectively, whereas DNase II
from other sources consists of a single polypeptide chain with the
following molecular masses: 36 –38 kDa from rat liver (27), 26.5
kDa from bovine liver (48), 45 kDa from human lymphoblasts
(49), and 32 kDa from human urine (50). Although the reasons for
the variability remain unknown, recent studies of molecular cloning of porcine, human, and murine DNase II have explained the
previously reported discrepancies among the molecular weights of
DNase II (51, 52). These sequence analyses indicate that mature
human or murine DNase II is a 344-aa protein, which contains four
potential N-linked glycosylation sites, and that its predicted size is
;42– 44 kDa. The reported a and b subunits of porcine DNase II
are encoded by one cDNA, indicating that the porcine 10-kDa
subunit results from cleavage of a larger precursor protein. In addition, Yasuda et al. (53) indicated that structural organization of
the cDNA encoding human DNase II is similar to those of lysosomal cathepsin families. Thus, our results suggest that a DNase in
the lysosome-rich fraction of J774.1 cells is similar to DNase II
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FIGURE 6. Hydrolysis of core histones by exposure of apoptotic cells
to the lysosome-rich fraction of J774.1 cells. CTLL-2 cells were cultured
in the presence (lane 1) or absence (lane 2) of IL-2 for 15 h. Acid proteins
were extracted from 8 3 105 cells. A total of 8 3 105 apoptotic CTLL-2
cells were incubated at 37oC for 2 h in the presence of the lysosome-rich
fraction of J774.1 cells (lane 3) with the following reagents: 0.5 mM leupeptin and 0.5 mM pepstatin A (lane 4), 0.5 mM CA-074 (lane 5), 0.5 mM
leupeptin (lane 6), and 0.5 mM pepstatin A (lane 7). Acid proteins were
extracted from these cells and subjected to SDS-PAGE. The proteins in the
gel were stained with Coomassie brilliant blue. The molecular masses at
left indicate the migration of standard proteins. Each core histone was run
in the gel and these migrations are shown at right. The result is representative of three independent experiments.
The Journal of Immunology
derived from apoptotic cells, although macrophages are shown to
phagocytose apoptotic cells efficiently. Thus, the capacity of dendritic cells and macrophages to phagocytose apoptotic cells is still
a matter of debate and the difference between the process of engulfment and phagocytosis in these two cell types remains to be
investigated.
Rapid engulfment of apoptotic cells is beneficial for the host
because it prevents the release of potentially toxic and immunogenic intracellular contents from the apoptotic cells into the surrounding tissue (1). Nucleosomes have been found to circulate at
high levels in patients with systemic lupus erythematosus (SLE)
(67). Interestingly, increased rates of apoptosis in lymphoid cells
have been detected both in human and murine lupus (68, 69). It has
been demonstrated that nucleosomes serve as a major immunogen
for pathogenic autoantigen-inducing T cells in both mouse and
human with SLE (70). Therefore, mono- and oligo-nucleosomes
that may be released from poorly engulfed apoptotic cells might
act as an autoantigen in SLE. A reduced phagocytic activity of
SLE patients’ polymorphonuclear leukocytes, monocytes, and
macrophages has been reported (71–73). Moreover, Hermann et al.
(74) recently found that phagocytosis of apoptotic cells is indeed
decreased in SLE patients. Alternatively, it is possible that the
impaired proteolytic hydrolysis of apoptotic cells in macrophages
leads to an decrease of nucleosome degradation, as has been demonstrated by Zurier (75) that sera from SLE patients interfere with
phagocytosis and lysosomal enzyme release from leukocytes. Consequently, a reduction of lysosomal enzymes may cause release of
nucleosomes and serve as an immunogen for the induction of autoreactive lymphocytes.
Additional experiments will define the mechanisms by which
macrophages in both mouse and human with SLE recognize and
engulf apoptotic cells and the intracellular processing by which
their macrophages dispose of apoptotic cells after engulfment.
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with respect to its m.w., optimal pH, and sensitivity to DNase II
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Endonuclease has been proposed to be responsible for the internucleosomal cleavage of the nuclear DNA during apoptosis and
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DNA of the nucleosomes is tightly complexed with the core
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DEGRADATION OF NUCLEOSOMES IN APOPTOTIC CELLS