Nitric Oxide Protects Macrophages from
Hydrogen Peroxide-Induced Apoptosis by
Inducing the Formation of Catalase
This information is current as
of June 15, 2017.
Yasuhiro Yoshioka, Tatsuya Kitao, Takashi Kishino, Akiko
Yamamuro and Sadaaki Maeda
J Immunol 2006; 176:4675-4681; ;
doi: 10.4049/jimmunol.176.8.4675
http://www.jimmunol.org/content/176/8/4675
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References
The Journal of Immunology
Nitric Oxide Protects Macrophages from Hydrogen
Peroxide-Induced Apoptosis by Inducing the Formation
of Catalase1
Yasuhiro Yoshioka,2 Tatsuya Kitao, Takashi Kishino, Akiko Yamamuro, and Sadaaki Maeda
M
acrophages play a significant role in innate immunity
and inflammation. When activated by pathogens and
proinflammatory cytokines, they produce large
amounts of NO, superoxide anion, and H2O2. These reactive oxygen species (ROS)3 exert strong cytotoxicity against microorganisms and many cells, including the macrophages themselves (1– 4).
H2O2 is considered to be one of the most toxic ROS. Although
H2O2 itself is not highly reactive, it can readily diffuse across
cellular membrane and is converted into highly reactive hydroxyl
radicals through the Fenton reaction in the presence of metal ions
(5). The hydroxyl radical is able to cause the degradation of most
biological macromolecules, e.g., peroxidation of lipids, oxidation
of protein thiols, DNA base damage, and strand breakage of nucleic acids (6). Mitochondria are a main target for damage by
H2O2. H2O2 is known to induce the mitochondrial permeability
transition and to disrupt the mitochondrial membrane potential.
Such conditions can cause the release of cytochrome c from the
mitochondria to the cytosol (7). The release of cytochrome c constitutes an important step in the activation of a specific subgroup,
CPP32, of caspases, and thus promotes apoptosis (8).
The predominant enzymatic defense systems against H2O2 are
the glutathione redox cycle and catalase. Glutathione peroxidase
oxidizes glutathione to glutathione disulfide and reduces H2O2 to
H2O. Glutathione reductase regenerates glutathione from oxidized
Department of Pharmacotherapeutics, Faculty of Pharmaceutical Sciences, Setsunan
University, Hirakata, Osaka, Japan
glutathione disulfide, using reducing equivalents from NADPH
(9). Catalase protects cells from the accumulation of H2O2 by converting it to H2O and O2 (10). It has been demonstrated that both
the glutathione redox cycle and catalase play an important role in
detoxification of extracellular H2O2 in macrophages (11).
NO is produced by a family of NO synthases composed of 3
isoforms, which are the constitutive endothelial and neuronal isoforms and the inducible isoform (12). Recent reports indicate that
NO displays both pro- and antiapoptotic properties. Endogenously
generated or exogenously supplied NO induces apoptosis in thymocytes (13), hepatocytes (14), macrophages (15), and several
other cells (16 –18). In contrast, NO at a low nontoxic concentration induces resistance to TNF-␣-induced hepatotoxicity (19) and
inhibits Fas-induced apoptosis in B lymphocytes (20). We also
reported recently that NO at a low concentration protected macrophages from NO-induced apoptosis (21). In addition, it has been
reported that NO enhances the cytotoxic activity of H2O2 in lymphoma cells (22), hepatoma cells (23), and several other cells (24 –
26). In contrast, NO inhibits H2O2-induced apoptosis in endothelial cells (27), cardiomyoblasts (28), and several other types of
cells (29 –31). However, the effect of NO on H2O2 toxicity in
macrophages has been poorly investigated.
In the present study, we investigated the effect of NO against
H2O2 toxicity in murine macrophage-like RAW264 cells. We
found that NO at a low concentration protected RAW264 cells
from H2O2-induced apoptosis by inducing the synthesis of
catalase.
Received for publication October 5, 2005. Accepted for publication February 6, 2006.
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.
Materials and Methods
Abs and chemicals
1
This work was supported by grants from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan.
2
Address correspondence and reprint requests to Dr. Yasuhiro Yoshioka, Department
of Pharmacotherapeutics, Faculty of Pharmaceutical Sciences, Setsunan University,
Hirakata, Osaka 573-0101, Japan. E-mail address: [email protected]
3
Abbreviations used in this paper: ROS, reactive oxygen species; SNP, sodium nitroprusside; NOC18, 1-hydroxy-2-oxo-3,3-bis-(2-aminoethyl)-1-triazene; BSO, Lbuthionine-(S,R)-sulfoximine; CHX, cycloheximide; 3-AT, 3-amino-1,2,4-triazole;
DCF-DA, 2⬘,7⬘-dichlorofluorescein diacetate; DBcGMP, dibutyryl-cGMP; DIG,
digoxigenin.
Copyright © 2006 by The American Association of Immunologists, Inc.
Anti-catalase polyclonal Ab was obtained from Calbiochem. Anticaspase-3 polyclonal Ab was purchased from Cell Signaling Technology,
and anti-caspase-9 mAb was obtained from Medical & Biological Laboratories. The fluorescent DNA-binding dye Hoechst 33342, the glutathione
synthesis inhibitor L-buthionine-(S,R)-sulfoximine (BSO), the catalase inhibitor 3-amino-1,2,4-triazole (3-AT), the protein synthesis inhibitor cycloheximide (CHX), and the cell-permeable cGMP analog dibutyryl-cGMP
(DBcGMP) were obtained from Sigma-Aldrich. All other chemicals were
purchased from Wako Pure Chemical.
0022-1767/06/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
We investigated the cytoprotective effect of NO on H2O2-induced cell death in mouse macrophage-like cell line RAW264. H2O2treated cells showed apoptotic features, such as activation of caspase-9 and caspase-3, nuclear fragmentation, and DNA fragmentation. These apoptotic features were significantly inhibited by pretreatment for 24 h with NO donors, sodium nitroprusside and
1-hydroxy-2-oxo-3,3-bis-(2-aminoethyl)-1-triazene, at a low nontoxic concentration. The cytoprotective effect of NO was abrogated
by the catalase inhibitor 3-amino-1,2,4-triazole but was not affected by a glutathione synthesis inhibitor, L-buthionine-(S,R)sulfoximine. NO donors increased the level of catalase and its activity in a concentration-dependent manner. Cycloheximide, a
protein synthesis inhibitor, inhibited both the NO-induced increase in the catalase level and the cytoprotective effect of NO. These
results indicate that NO at a low concentration protects macrophages from H2O2-induced apoptosis by inducing the production
of catalase. The Journal of Immunology, 2006, 176: 4675– 4681.
4676
NO PROTECTS MACROPHAGES FROM H2O2-INDUCED APOPTOSIS
Cell culture and treatment with drugs
Isolation of RNA and Northern blot analysis
Clonal murine macrophage-like RAW264 cells were maintained in DMEM
supplemented with 10% heat-inactivated FCS, as described previously (21,
32). The cells were plated at a density of 2 ⫻ 104/well in 96-well tissue
culture plates for viability experiments, 1 ⫻ 106 in 60-mm-diameter dishes
for DNA degradation analysis, or 2 ⫻ 106 in 100-mm-diameter dishes for
Western blotting. BSO or CHX was added 1 h before exposure to NO
donors, and 3-AT was added 1 h before exposure to 1 mM H2O2.
Cells were lysed with denaturing solution containing 4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol. After having been transferred to a polypropylene tube, total
RNA was extracted by the sequential addition of 2 M sodium acetate (pH
4.0), water-saturated phenol, and chloroform-isoamylalcohol (49:1), followed by centrifugation at 1800 ⫻ g for 40 min at 4°C and then precipitated with isopropanol and ethanol. Total RNA (10 ␮g/lane) was separated
by electrophoresis on 1% agarose-formaldehyde gels and transferred onto
a Hybond N⫹ membrane (Amersham Biosciences). A 720-bp digoxigenin
(DIG)-labeled DNA probe was generated with a DIG-High Prime kit
(Roche Applied Science) using two primers, 5⬘-GCTGAAGTTGAACA
GATG-3⬘ and 5⬘-GTCATCAGCGTGAGTCTG-3⬘, and the full-length
cDNA of catalase (Invitrogen Life Technologies) as a template. Northern
hybridization was performed in DIG Easy Hyb (Roche Applied Science) at
42°C for 12 h. The membrane was washed with 2 ⫻ SSC at room temperature for 20 min and with 2 ⫻ SSC containing 3% SDS at 42°C for 45
min. It was then incubated with alkaline phosphatase-conjugated anti-DIG
Ab (Roche Applied Science), and the reaction product was detected by
using a CDP-Star chemiluminescence detection system (Roche Applied
Science). The equality and integrity of total RNA were examined by staining the RNA on the membrane with ethidium bromide.
Assessment of cell viability and apoptosis
Cell viability was determined by the colorimetric MTT assay described
previously (33). Apoptotic chromosomal condensation and fragmentation
were determined by using the chromatin dye Hoechst 33342. Cells were
stained with Hoechst 33342, and apoptotic nuclei were counted under a
fluorescence microscope as described earlier (21).
DNA agarose gel electrophoresis
Western blot analysis
Depending on the experiment, 2–30 ␮g of the cell lysate was blotted onto
polyvinylidene difluoride membranes after separation of the protein by
SDS-PAGE. The blots were then blocked for 1 h in TBS containing 0.1%
Tween 20 and 5% nonfat dry milk and incubated for 1–3 h at room temperature with the primary Ab (1/2000 dilution of Ab against catalase; and
1/1000 dilution of Abs against caspase-3 and caspase-9). Next, the membrane was rinsed five times with TBS/0.1% Tween 20 and incubated for 1 h
with HRP-conjugated anti-mouse or anti-rabbit IgG polyclonal Abs (Cappel). Signals were detected by the ECL method.
Measurement of ROS levels
Cells were incubated with 100 ␮M 2⬘,7⬘-dichlorofluorescein diacetate
(DCF-DA; Molecular Probes) in DMEM for 30 min to assess H2O2-mediated oxidation of it to the fluorescent compound DCF. Then the cells
were treated with 1 mM H2O2 for 30 min and subsequently washed with
PBS and lysed with 1% Triton X-100/PBS. The fluorescence of cell lysates
was measured by using a fluorometer (excitation at 390 nm, emission at
460 nm; MTP-100F; Corona Electric).
Measurement of glutathione levels
Intracellular glutathione content was determined by the Griffith (34) modification of Tietze’s enzymatic procedure (35). Briefly, cells (1 ⫻ 106) were
resuspended in 100 ␮l of hydrochloric acid (10 mM) and lysed by freezingthawing. After centrifugation for 5 min at 10,000 ⫻ g, the supernatant
solution was deproteinized with 5-sulfosalicylic acid (10%; 50 ␮l). The
acid-precipitated proteins were pelleted by centrifugation at 4°C for 5 min
at 10,000 ⫻ g. For determination of the glutathione content, aliquots of the
acid-soluble supernatant were mixed with a solution of 114 mM sodium
phosphate (pH 7.5), 5.0 mM EDTA, 0.19 mM NADPH, and 0.6 mM 5,5dithiobis(2-nitrobenzoic acid) in a total volume of 1 ml. Upon addition of
glutathione reductase, the increase in absorption at 412 nm was monitored
and used to determine the amount of glutathione in the sample by comparison to a reference curve generated with known amounts of glutathione
standard. Protein content was determined by using a BCA protein assay kit
(Pierce).
Measurement of catalase activity
Catalase activity was measured according to the method described by Aebi
(36). Briefly, cells were lysed with a buffer consisting of 50 mM sodium
phosphate (pH 7.5) and 0.5% Triton X-100. Catalase activity was measured
at 30°C by the consumption of H2O2 at 240 nm in a spectrophotometer
(BioSpec-mini; Shimadzu). The reaction was performed in a solution of 10
mM H2O2 in 50 mM Tris-HCl (pH 8.0) containing 20 ␮l of cell lysate in
a final volume of 1 ml. One unit was defined as the amount of enzyme that
decomposed 1.0 mmol of H2O2 per minute at 30°C. The activity was reported as units per mg of protein, with the protein concentration determined
by using a BCA protein assay kit.
Statistical evaluation
The results were expressed as means ⫾ SE obtained from three to four
independent experiments. One-way ANOVA was used to test for differences between group means. When appropriate, post hoc multiple comparisons were performed to test for differences between experimental
groups (Scheffe’s test or Dunnett’s test). When the p value was ⬍0.05, the
difference was considered to be significant.
Results
Pretreatment with NO donors prevented H2O2-induced apoptosis
We estimated H2O2-induced damage to RAW264 cells by using
the MTT assay. Treatment of the cells with H2O2 for 24 h caused
loss of viability in a concentration-dependent manner (Fig. 1A).
We next investigated the cytoprotective effect of 2 NO donors,
sodium nitroprusside (SNP) and 1-hydroxy-2-oxo-3,3-bis-(2-aminoethyl)-1-triazene (NOC18) on the H2O2 toxicity. It has been
reported that SNP releases NO after reductive metabolism and that
NOC18 releases NO with a half-life of ⬃21 h in aqueous solution
(37, 38). SNP or NOC18 at a concentration ⬎1 mM induced cell
death (data not shown). Pretreatment with SNP or NOC18 at low
concentrations (30 –300 ␮M) for 24 h protected the cells from
H2O2-induced death (Fig. 1B). In addition, pretreatment of the
cells with 100 ␮M SNP or 100 ␮M NOC18 attenuated H2O2induced apoptosis and DNA fragmentation (Fig. 2, A and B). Furthermore, the activation of caspase-9 and caspase-3 by H2O2 was
suppressed by pretreatment with these reagents (Fig. 2C). These
results indicate that NO protects macrophages from H2O2-induced
apoptosis.
Involvement of catalase in cytoprotection by NO
To further elucidate of the mechanism underlying the cytoprotective effect of NO, we analyzed intracellular ROS levels after H2O2
treatment by using the DCF-DA method. Treatment with 1 mM
H2O2 for 30 min significantly increased the intracellular ROS level
(Fig. 3A). Pretreatment with 100 ␮M SNP or 100 ␮M NOC18 for
24 h reduced the degree of increase in the ROS level. This result
suggests that the cytoprotection by NO was mediated by enhancing
intracellular H2O2-decomposing systems. It has been reported that
such systems, so-called antioxidant defense systems, mainly depend on the glutathione redox cycle and catalase (11). Next, we
examined the effects of BSO and 3-AT on the cytoprotection by
NO. Intracellular glutathione contents were completely depleted
by 3 mM BSO and were not changed by the treatment with 100
␮M SNP or 100 ␮M NOC18 (Table I). The cytoprotective effects
of SNP and NOC18 were not affected by 3 mM BSO (Fig. 3B). In
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For DNA laddering analysis, cells were lysed in 200 ␮l of lysis buffer
consisting of 50 mM Tris-HCl (pH 7.5), 20 mM EDTA, and 1% Nonidet
P-40. After lysis, the DNA fragments were separated from the intact chromatin by centrifugation for 5 min at 1600 ⫻ g. The supernatant was successively treated with 0.5 mg/ml RNase A for 2 h at 56°C and 0.5 mg/ml
proteinase K for 4 h at 37°C. The DNA samples were ethanol precipitated
and then loaded onto 1.0% agarose gels and separated by electrophoresis in
1 ⫻ Tris/borate/EDTA running buffer for 40 min at 100 V. The DNA band
pattern was visualized by ethidium bromide staining.
The Journal of Immunology
FIGURE 3. Inhibition of catalase blocked cytoprotection by NO. A,
RAW264 cells were treated with 100 ␮M SNP or 100 ␮M NOC18 for 24 h.
Then they were incubated with DCF-DA for 30 min and subsequently
incubated with 1 mM H2O2 for 30 min. The cells were washed with PBS
and lysed with 1% Triton X-100/PBS. Fluorescence of oxidized DCF was
measured by fluorometry. (B and C) RAW264 cells were treated with 100
␮M SNP or 100 ␮M NOC18 for 24 h in the absence or presence of 3 mM
BSO. Then the cells were treated with 1 mM H2O2 in the absence or
presence of 30 mM 3-AT for 24 h. Cell viability was determined by
using the colorimetric MTT assay. Results show the mean ⫾ SE obtained from three or four independent experiments. ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ,
p ⬍ 0.001, significantly different from values without SNP or NOC18
pretreatment.
contrast, the cytoprotective effects of SNP and NOC18 were abolished by 30 mM 3-AT (Fig. 3C). These results suggest that the
cytoprotective effect of NO was mediated by the activation of
catalase.
NO increased the level of catalase and its activity
When we examined the effect of NO on the catalase activity in
RAW264 cells, we found that treatment of the cells with SNP or
NOC18 for 24 h increased their catalase activity in a concentration-dependent manner (Fig. 4A). Next, we examined the effect of NO on the protein level of catalase. In agreement with
FIGURE 2. Pretreatment with NO donors attenuated H2O2-induced apoptosis in macrophages. RAW264 cells were treated with 100 ␮M SNP or
100 ␮M NOC18 for 24 h. Then the cells were treated with 1 mM H2O2 for
the indicated periods (A) or for 6 h (B). Analysis of apoptotic nuclear
fragmentation (A) and DNA degradation (B) was performed as described in
Materials and Methods. C, Cleavage of caspase-9 and caspase-3 was determined by Western blotting. Results show the mean ⫾ SE obtained from
four independent experiments (A) and a representative of three independent
experiments (B and C). ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01, significantly different
from values without SNP or NOC18 pretreatment.
Table I. Effect of BSO on the level of intracellular glutathionea
Control
SNP
NOC18
None
BSO
9.43 ⫾ 0.96
10.22 ⫾ 1.52
9.80 ⫾ 1.28
0.20 ⫾ 0.08
0.26 ⫾ 0.15
0.16 ⫾ 0.10
a
RAW264 cells were incubated with 100 ␮M SNP or 100 ␮M NOC18 in the
presence or absence of 3 mM BSO for 24 h. Intracellular glutathione levels were
determined as described in Materials and Methods. Data are presented as mean values
(nmol/1 ⫻ 106 cells) ⫾ SE from three independent experiments.
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FIGURE 1. Pretreatment with NO donors protected macrophages from
H2O2-induced cell death. A, RAW264 cells were incubated with various
concentrations of H2O2 for 24 h. Cell viability was then determined by
conducting the colorimetric MTT assay. B, Cells were treated with SNP or
NOC18 at the indicated concentrations for 24 h. Then they were incubated
with 1 mM H2O2 for 24 h. The cell viability was determined by using the
MTT assay. Results show the mean ⫾ SE obtained from three or four
independent experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01, significantly different
from values without SNP or NOC18 pretreatment.
4677
4678
NO PROTECTS MACROPHAGES FROM H2O2-INDUCED APOPTOSIS
FIGURE 5. Effect of H2O2 on the levels of catalase protein. A,
RAW264 cells were treated with 1 mM H2O2 for the indicated periods. The
levels of catalase protein were determined by Western blotting. B,
RAW264 cells were treated with SNP or NOC18 at the indicated concentrations for 24 h. Then they were treated with 1 mM H2O2 for 12 h. The
levels of catalase protein were determined by Western blotting. Results
show representative blots from three independent experiments.
It has been demonstrated that many of the physiological actions of
NO are mediated by an increase in the cGMP concentration via the
FIGURE 4. Effect of SNP or NOC18 on the levels of catalase and its
activity. RAW264 cells were treated with SNP or NOC18 at the indicated
concentrations for 24 h. A, The catalase activity was measured by the
consumption of H2O2 at 240 nm in a spectrophotometer as described in
Materials and Methods. B and C, The levels of catalase protein and mRNA
were determined by Western blotting and Northern blotting, respectively.
Results show the mean ⫾ SE obtained from four independent experiments
(A) and a representative blot of four independent experiments (B and C).
ⴱⴱ, p ⬍ 0.01, significantly different from the control.
the increase in the enzyme activity, treatment of the cells with
SNP or NOC18 for 24 h increased their catalase levels in a
concentration-dependent manner (Fig. 4B). The catalase mRNA
level also was increased concentration-dependently by treatment with SNP or NOC18 (Fig. 4C). Treatment with 1 mM
H2O2 did not affect the level of catalase protein, and the increase in the amount of catalase protein caused by SNP or
NOC18 was also unaffected by treatment with 1 mM H2O2 for
12 h (Fig. 5). Next, we examined the effect of CHX on both
NO-induced cytoprotection and catalase induction. Both the induction of catalase and the increase in catalase activity by treatment with 100 ␮M SNP or 100 ␮M NOC18 for 24 h were
inhibited by 300 nM CHX (Fig. 6, A and B). CHX also negated
the cytoprotective effects of SNP and NOC18 (Fig. 6C). These
results suggest that NO protected the macrophages from H2O2
toxicity by inducing the synthesis of catalase.
FIGURE 6. Effect of CHX on the cytoprotection and the induction of
catalase by NO. RAW264 cells were treated with 100 ␮M SNP or 100 ␮M
NOC18 for 24 h in the absence or presence of 300 nM CHX. A, The levels
of catalase protein were determined by Western blotting. B, The catalase
activity was measured. C, RAW264 cells were treated with 100 ␮M SNP
or 100 ␮M NOC18 for 24 h in the absence or presence of 300 nM CHX.
Then the cells were incubated with 1 mM H2O2 for 24 h. Cell viability was
determined by using the colorimetric MTT assay. Results show a representative blot of four independent experiments (A) and the mean ⫾ SE
obtained from three independent experiments (B and C). ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ,
p ⬍ 0.001, significantly different from values without SNP or NOC18
pretreatment.
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NO-cGMP signaling pathway was not involved in the
cytoprotection or induction of catalase by NO
The Journal of Immunology
activation soluble guanylate cyclase (39). Therefore, we also examined the effect of DBcGMP on the levels of catalase and H2O2induced cell death in RAW264 cells. Pretreatment with DBcGMP
for 24 h did not affect the level of catalase nor did it protect
RAW264 cells from H2O2-induced cell death (Fig. 7). These results indicate that the cytoprotection and the induction of catalase
by NO were not mediated by the NO-cGMP signaling pathway.
Discussion
The major findings of the present study are that NO protected
RAW264 cells from H2O2-induced apoptosis, and that the cytoprotective effect of NO was mediated by the induction of catalase.
Our present study provides the first evidence that NO increases the
protein level of catalase and its activity.
Catalase and the glutathione redox cycle play a central role in
the detoxification of H2O2 in many types of cells (40 – 43). It has
been demonstrated that alveolar macrophages are protected against
exogenous oxidants by catalase and the glutathione redox cycle
(11). Consistent with these reports, in the present study, the catalase inhibitor 3-AT and the glutathione synthesis inhibitor BSO
facilitated H2O2-induced cell death in RAW264 cells. However,
the cytoprotective effect of NO against H2O2 toxicity was lost in
the presence of 3-AT but not in that of BSO (Fig. 3, B and C),
indicating that the cytoprotective effect of NO was mediated by
catalase, not by the glutathione redox cycle. In agreement with
these findings, NO increased the catalase activity but affected neither the glutathione content nor glutathione peroxidase activity
(Fig. 4A, Table I, and data not shown). It has been reported that
3-AT also inhibits heme-containing peroxidases such as myeloperoxidase and lactoperoxidase (44, 45). Among these enzymes,
myeloperoxidase, which is expressed in macrophages, has the ability to reduce the amount of H2O2 (46, 47). However, it was reported earlier that myeloperoxidase accelerates H2O2-induced cell
death in a human leukemia cell line HL-60 and a melanoma cell
line B-16 (48, 49). Although we cannot exclude the possibility that
myeloperoxidase contributed to the cytoprotection by NO, these
reports would suggest that myeloperoxidase did not participate in
the cytoprotective effect of NO on H2O2-induced cell death in
RAW264 cells.
It has been reported that the level of catalase protein is decreased in the kidneys of animals with macrophages that produced
high levels of NO (50). In contrast, our results demonstrated that
NO increased the level of catalase in RAW264 cells. This difference may be attributed to the cell type-specific effect of NO on the
level of catalase. In this context, we observed that NO increased
the level of catalase protein in mouse macrophage J774.1 cells and
human monocyte THP-1 cells but decreased it in human neuroblastoma SH-SY5Y cells and mouse fibroblast 3T3 cells (data not
shown). The production of both NO and superoxide anion by activated macrophages results in peroxynitrite formation (51). It has
been reported that the catalase activity is inhibited by peroxynitrite
(50). These reports suggest that peroxynitrite produced by activated macrophages would inhibit the catalase activity of them. The
balance between the amount of NO and that of peroxynitrite may
decide the susceptibility of activated macrophages to H2O2 toxicity via regulation of catalase activity. It has been demonstrated that
a number of physiological events induced by NO are exerted by an
increase in the cGMP concentration via the activation of soluble
guanylate cyclase (39). It has also been shown that NO protects
astrocytes and cortical neurons from H2O2 toxicity by cGMP-dependent mechanisms (29, 30). However, DBcGMP did not induce
the expression of catalase nor did it inhibit H2O2-induced cell
death in our study (Fig. 7). These results suggest that NO induces
catalase by a cGMP-independent mechanism. The precise mechanism of catalase induction by NO, however, remains unknown.
It was earlier demonstrated that NO inhibits catalase activity and
thus increases intracellular ROS levels in macrophages (52). In the
present study, treatment with SNP or NOC18 increased the level of
catalase and its activity (Fig. 4). This difference may depend on the
concentrations of NO donors used in each experiment. In this context, we observed that treatment with SNP or NOC18 at a high
concentration (1 mM) induced cell death and did not increase the
level of catalase and its activity in RAW264 cells (data not shown).
These observations indicate that the concentration of NO is important in the induction of catalase and its activity in macrophages.
It was earlier demonstrated that GM-CSF-differentiated macrophages exhibit higher catalase activity than M-CSF-differentiated
macrophages and that M-CSF-differentiated macrophages release a
larger amount of H2O2 than GM-CSF-differentiated macrophages
in response to bacterial stimulation (53). It also has been shown
that inhibition of catalase by 3-AT increases H2O2 levels in macrophages (54). NO may attenuate H2O2 production by inducing
catalase. In agreement with this idea, it has been demonstrated that
NO decreases H2O2 production induced by lectin in human polymorphonuclear leukocytes, reduces H2O2 generation by TNF-␣
treatment of carcinomas, inhibits H2O2 production by high glucose
exposure in HUVEC, and decreases the degree of elevation of
H2O2 induced by insulin-like growth factor I in cultured rat aortic
smooth muscle cells (55–58). H2O2 has been shown to act as a
signaling molecule that activates transcriptional factor NF-␬B,
which then mediates the expression of proinflammatory cytokines
in macrophages stimulated by LPS (59 – 61). It also has been reported that NO inhibits LPS-induced activation of NF-␬B and reduces cytokine production by LPS in macrophages (56, 62, 63).
Therefore, the induction of catalase in macrophages by NO may
play a significant role not only in cytoprotection against H2O2
toxicity but also in the regulation of gene expression of cytokines.
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FIGURE 7. DBcGMP did not affect H2O2-induced cell death or the
level of catalase protein. A, RAW264 cells were treated with 0.1–3 mM
DBcGMP, 100 ␮M SNP, or 100 ␮M NOC18 for 24 h. The levels of
catalase protein were determined by Western blotting. B, RAW264 cells
were treated with 0.1–3 mM DBcGMP, 100 ␮M SNP, or 100 ␮M NOC18
for 24 h. Then they were incubated with 1 mM H2O2 for 24 h. Cell viability
was determined by using the colorimetric MTT assay. Results show a
representative blot of four independent experiments (A) and the mean ⫾ SE
obtained from three independent experiments (B). ⴱⴱ, p ⬍ 0.01, significantly different from the values obtained without DBcGMP, SNP, or
NOC18 pretreatment.
4679
4680
NO PROTECTS MACROPHAGES FROM H2O2-INDUCED APOPTOSIS
In summary, this study reveals that NO protected macrophages
from H2O2-induced apoptosis by eliciting the production of catalase. NO may thus help macrophages to survive in an oxidant-rich
environment, such as site of inflammation, by inducing the formation of catalase.
Disclosures
The authors have no financial conflict of interest.
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