Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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ABSTRACT |
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Apoptosis is a critical event for eliminating activated macrophages. Here we show that Fas-mediated apoptosis may participate in the mechanism of negative feedback regulation of activated macrophages. Cytokine-activated macrophages released high levels of nitric oxide (NO) that induced apoptosis in macrophages themselves. This NO-induced macrophage apoptosis was inhibited by a Fas-Fc chimeric molecule that binds to Fas ligand (FasL) and prevents its interaction with endogenous cell surface Fas. High levels of NO stimulated the release of the soluble form of FasL that was inhibited by a matrix metalloproteinase inhibitor KB-8301. High levels of NO also upregulated the expression of Fas mRNA in macrophages. In addition, macrophages isolated from Fas-lacking mice were resistant to NO-induced apoptosis. Finally, inhibition of apoptosis by a caspase inhibitor augmented peroxide production from activated macrophages. These findings suggest that high levels of NO released from activated macrophages may promote the Fas-mediated macrophage apoptosis that may be a negative feedback mechanism for elimination and the downregulation of activated macrophages in the vessel wall.
inducible nitric oxide synthase; Fas ligand; metalloproteinase; lpr
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INTRODUCTION |
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THE INFILTRATION OF MONOCYTES/MACROPHAGES into the vessel wall is critical for the progression of atherosclerosis (22). Recent pathological studies of human postmortem specimens have demonstrated that ruptured plaques have a larger extracellular lipid-rich core with an increased macrophage density and a reduced collagen and smooth muscle cell content in their fibrous cap compared with intact plaques (5, 7, 26). Human monocyte-derived macrophages were shown to induce collagen breakdown in fibrous caps of human atherosclerotic plaques (24), and tissue factor content is increased in unstable angina and correlates with areas of macrophages (16). In addition, the chemoattractant proteins (chemokines) monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 are found in human atheroma, and mice lacking receptors for these chemokines (3) or mice lacking MCP-1 itself (8) are less susceptible to atherosclerosis and have fewer monocytes in vascular lesions. These findings suggest that blockade of monocyte/macrophage recruitment is useful for the inhibition of atherogenesity.
Apoptosis is a physiological cell death pathway that removes damaged or unwanted cells. Recent pathological studies have shown that apoptosis was common in the atherosclerotic lesion, especially in vascular smooth muscle cells and inflammatory cells such as macrophages and T cells (2). Fas ligand (FasL; also called CD95 ligand or APO-1 ligand) is a cytokine that mediates apoptosis by binding to its receptor, Fas (also called CD95 or APO-1), through activating caspases (18). The Fas/FasL system is implicated in the elimination of activated T cells after they have responded to foreign antigens (12). Apoptosis regulates inflammatory cell survival, and its reduction contributes to the chronicity of an inflammatory process (27). On the basis of the expression of both Fas and FasL on macrophages (10), we hypothesized that Fas-mediated apoptosis may represent the mechanism for eliminating activated macrophages. High levels of nitric oxide (NO) released from activated macrophages have been postulated to play important roles in host defense mechanisms against tumor cells and organisms.
However, these high levels of NO also induce apoptosis in macrophages themselves (1, 23). In this study, we examined, therefore, whether NO-induced macrophage apoptosis is mediated through the Fas/FasL system. We also examined whether inhibition of apoptosis can modulate the production of reactive oxygen from macrophages. We found that high levels of NO stimulated the release of the soluble form of FasL (sFasL) and sensitized macrophages to Fas-mediated apoptosis and that inhibition of apoptosis augmented peroxide production from macrophages.
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MATERIALS AND METHODS |
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Materials.
Interleukin-1 (IL-1
), tumor necrosis factor-
(TNF-
),
interferon-
(INF-
), and
NG-monomethyl-L-arginine
(L-NMMA) were purchased from Sigma Chemical (St. Louis,
MO). Fas-Fc was generously provided by Dr. Shigekazu Nagata (Osaka
Bioscience Institute, Osaka, Japan). KB-8301, a matrix
metalloproteinase inhibitor, was donated by Kanebo (Osaka, Japan).
N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) were purchased from Dojin Laboratory (Kumamoto, Japan). Hoechst 33258 was
purchased from Flow Laboratories (McLean, VA). 2',7'-Dichlorofluorescin diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR).
DEVD-fmk (Z-Asp-Glu-Val-Asp-FMK), a caspase family inhibitor, was
purchased from Medical and Biological Laboratories (Nagoya, Japan).
Cells. Male MRL/MPJ lpr/lpr mice and MRL/MPJ +/+ mice were purchased from breeding colonies at Japan SLC (Shizuoka, Japan). Resident peritoneal macrophages were collected from peritoneal cavities of each mouse and were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (75 U/ml), and streptomycin (0.05 µg/ml). The cells were then incubated for about 120 min at 37°C in a CO2 incubator to allow them to adhere to the plates. The nonadherent cells were then removed by three vigorous washings with prewarmed PBS solution. More than 95% of the adherent cells were judged to be macrophages by both Giemsa staining and carbon-particle uptake. These macrophages were cultured in RPMI 1640 medium and then used in the experiments.
Nitrite assay. The nitrite level in the medium, which is used as a reflection of NO production, was determined using Greiss reagent consisting of 1% sulfanilamide, 0.1% naphthylene-diamine-dihydrochloride, and 2% H3PO4. Absorbance at 560 nm was measured.
Apoptosis analysis. Apoptosis was monitored by measuring the population distribution of DNA content. The treated cells were harvested, suspended in 100 µl of PBS, fixed in 900 µl of ice-cold 100% ethanol, and then resuspended in staining buffer (1 mg/ml RNase, 20 mg/ml propidium iodide, and 0.01% Nonidet P-40). After staining, flow cytometric analysis was performed immediately in a Becton Dickinson FACScan. To analyze fragmented apoptotic nuclei, macrophages were fixed with methanol-acetic acid, 3:1 (vol/vol), and stained with fluorescent dye (Hoechst 33258, 10 µmol/l). After incubation for 30 min in the dark at 37°C, photographs were obtained with a Nikon EFD2 fluorescence microscope (×400).
Analysis of Fas mRNA.
Total RNA from macrophages was extracted by a guanidine
isothiocyanate/acid phenol method. RT-PCR was performed using a One Step RNA PCR kit from Takara Biomedicals (Tokyo, Japan). The primers used for amplification of the Fas mRNA were described below,
respectively: Fas, upper primer sequence 5'-CATGTCTTCAGCAATTCTCGG-3',
lower primer sequence 5'-GGCTGTGAACACTGTGTTCG-3'. The efficiency of the
RT and the amount of RNA used in the RT-PCR were verified by detection
of the mouse -actin mRNA with RT-PCR. One microgram of total RNA was
reverse transcribed to cDNA by avian myeloblastosis virus RT
(RTase) for 30 min at 50°C, and RTase was inactivated for 2 min at
94°C. The cDNA were then amplified with 5 units of Ampli
Taq DNA polymerase and 0.4 mmol/l of both upstream and
downstream primers in a volume of 50 µl. The 35 PCR cycles consisted
of a denaturation step (94°C, 30 s), an annealing step (56°C,
30 s), and an elongation step (72°C, 2 min). The PCR products
were analyzed on a 1.3% 40 mmol/l Trizma-base, 20 mmol/l sodium
acetate 3H2O, 1 mmol/l EDTA Na2
2H2O agarose gel.
Immunoblot analysis of FasL expression. After incubation for 24 h with NOC-12 in the presence or absence of KB-8301, each supernatant was collected and cells were then treated with 500 µl of lysis buffer [50 mmol/l Tris·HCl (pH 8.0), 20 mmol/l EDTA, 1% SDS, and 100 mmol/l NaCl]. The supernatants (4 ml) from cells were concentrated to 100 µl with an Ultrafree-15 centrifugal filter device (Millipore, Bedford, MA). The cell lysates or concentrated supernatants were then melted by sample buffer (without 2-mercaptoethanol) and then analyzed by SDS-PAGE using a 10% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore). After blocking, the membrane was incubated with an anti-mouse FasL goat polyclonal antibody (1:100; Santa Cruz Biotechnology). Membranes were then washed and incubated with a horseradish peroxidase-conjugated goat antibody. After washing, FasL expression was detected using enhanced chemiluminescence (Amersham International, Buckinghamshire, England).
Immunohistochemical studies. At 20 wk of age, mice were anesthetized with ether and killed by exsanguination. Specimens of kidneys were embedded in optimum cutting temperature compound and frozen on dry ice. Serial 10-µm-thick cryostat sections were collected on poly-D-lysine-coated slides and fixed in acetone. To examine cell surface markers of macrophages, we used the avidin-biotin-peroxidase complex technique. In brief, 10-µm-thick cryostat sections were incubated with monoclonal rat anti-mouse Mac-1 antigen (Immunotech) and then incubated with biotinylated rabbit anti-rat IgG (Dako). After they were combined with streptavidin peroxidase, sections were visualized with DAB substrates. In some experiments, sections were stained with hematoxylin and eosin.
Assay for peroxide production. Production of peroxide by the cells was quantified by flow cytometry using the peroxide-sensitive dye DCFH-DA (9). Cells treated with cytokines in the presence or absence of a caspase inhibitor were incubated with DCFH-DA (5 µmol/l). After 30 min, fluorescence intensity, which is directly proportional to peroxide content, was then analyzed by flow cytometry on the FL1 channel.
Statistical analysis. The differences between means were evaluated using Student's t-test. Significant levels were established when P values were <0.05.
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RESULTS |
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Large amounts of NO-induced macrophage apoptosis is partially
mediated through activation of the Fas/FasL system.
Incubation of macrophages with IFN- (400 U/ml), TNF-
(40 ng/ml),
and IL-1
(10 ng/ml) induced high levels of NO release in a
time-dependent manner (Fig.
1A). Incubation for
72 h with these cytokines induced apoptosis in macrophages.
However, L-NMMA, an inhibitor of NO
synthase, partially suppressed the cytokine-induced apoptosis (Fig.
1B). NOC-12, a NO donor, at a high concentration of 0.5 mg/ml, also induced macrophage apoptosis (Fig. 1C). Nuclear staining with Hoechst 33258 showed condensed and fragmented apoptotic nuclei in NOC-12-treated cells (Fig. 2),
suggesting that large amounts of NO released from macrophages induce
apoptosis in macrophage themselves. To address the mechanism of
NO-induced macrophage apoptosis, we next examined the effect of a
Fas-Fc chimeric molecule that binds to FasL and prevents its
interaction with endogenous cell surface Fas (19) on
macrophage apoptosis.
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High levels of NO upregulates the Fas/FasL expression in
macrophages.
We next examined whether NO modulates the Fas/FasL expression in
macrophages. RT-PCR analysis showed that incubation for 2 and 4 h
with 0.5 mg/ml NOC-12 induced an increase in the levels of Fas mRNA in
macrophages (Fig. 3A). In
addition, immunoblot analysis with an anti-FasL antibody showed that
NOC-12 stimulated the release of sFasL from macrophages into the
supernatant (Fig. 3B). KB-8301, a metalloproteinase
inhibitor, inhibited this sFasL release. Although NOC-12 alone did not
affect the levels of the cellular FasL, NOC-12-induced upregulation of
the cellular FasL was unmasked in the presence of KB-8301 (Fig.
3C).
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Macrophages isolated from lpr mice are resistant to NO-induced
apoptosis.
To further confirm the mechanistic link between NO-induced apoptosis
and the Fas/FasL system, we next examined the levels of NO-induced
apoptosis in macrophages isolated from lpr mice that lack
functional Fas (18) compared with macrophages isolated from wild-type mice. Macrophages from lpr mice were
resistant to NOC-12-induced apoptosis compared with those from
wild-type mice (Fig. 4). In addition,
immunohistochemical examination showed that macrophages were much more
accumulated in a perivascular space of the renal artery in
lpr mice compared with wild-type mice (Fig.
5). These findings indicate that
Fas-mediated apoptosis may be important in the regulation of macrophage
elimination in the vessel wall.
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Caspase inhibitor augments peroxide production from activated
macrophages.
To address whether apoptosis can modulate the levels of reactive oxygen
production from macrophages, we next measured the levels of peroxide
production from cytokine-activated macrophages in the presence or
absence of an inhibitor of the caspase cascade that is a downstream
apoptotic signal for not only the Fas/FasL system but also other
apoptotic inducers (18). Consistent with previous reports,
incubation for 24 h with IL-1, TNF-
, and IFN-
induced an
increase in the levels of peroxide production from macrophages (Fig.
6A). However, the progression
of cytokine-induced apoptosis at 48 h was associated with a
decrease in the levels of peroxide production (Fig. 6B).
Interestingly, in the presence of 20 µmol/l DEVD-fmk, a caspase
family inhibitor, cytokine-induced peroxide production was augmented
and restored (Fig. 6, A and B).
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DISCUSSION |
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Recent histological studies have demonstrated that the vulnerable
plaque from patients with acute coronary syndromes contains many more
monocytes/ macrophages compared with the stable plaque (17). Because monocytes/macrophages release many
inflammatory mediators including cytokines, proteinases, and free
radicals such as NO and superoxide, suppression of monocyte/macrophage accumulation may be a useful therapeutic strategy for the prevention of
acute coronary syndromes. Although pathological accumulation of
monocytes/macrophages has been attributed to the migration and
proliferation of these cells, inhibition of apoptosis may also
participate in the mechanism of monocyte/macrophage accumulation. It
has been shown that monocyte/macrophage apoptosis is controlled by a
variety of inflammatory cytokines and bacterial products (13, 21). However, the precise mechanism of
macrophage apoptosis is not well known. Large amounts of NO released
from cytokine-activated macrophages exert two separate effects on
macrophages, stimulating effector functions while simultaneously
inducing apoptosis (1, 23). In addition to
well-established proapoptotic effects of NO, low levels of NO function
as an important inhibitor of apoptosis by interference with signal
transduction pathways that control apoptotic cell death
(14, 15). The present study demonstrated that
NO-induced macrophage apoptosis is partially mediated through activation of the Fas/FasL system. Kiener et al. (11)
reported that monocyte-derived macrophages that express both Fas and
FasL are resistant to Fas-mediated apoptosis. However, Brown and Savill (4) recently reported that phagocytosis triggers
macrophage release of FasL and induces apoptosis of bystander
monocytes. More recently, Perlman et al. (20)
reported that monocyte differentiation into macrophages was associated
with upregulation of Fas-associated death domain-like
IL-1-converting enzyme-inhibitory protein (Flip) and a decrease in
Fas-mediated apoptosis.
In this study, we demonstrated for the first time that high levels of NO induced stimulation of sFasL release as well as upregulation of Fas expression in macrophages. In addition, fluorescence-activated cell sorting analysis revealed that neither cytokines nor NOC-12 increased the levels of cell surface FasL expression in macrophages (data not shown). Furthermore, macrophages isolated from Fas-lacking mice were resistant to NO-induced apoptosis. Taken together, these findings suggest that high levels of NO may sensitize macrophages to Fas-mediated apoptosis and that macrophages can serve as a source of sFasL, which may function in a paracrine pathway to mediate apoptosis in macrophages themselves.
Yaoita et al. (28) recently reported that a caspase inhibitor was effective in reducing myocardial ischemia-reperfusion injury in rats. More recently, Daemen et al. (6) also reported that administration of the anti-apoptotic agents insulin-like growth factor I and a caspase inactivator prevented the early onset of apoptosis, inflammation, and tissue injury, suggesting that inhibition of apoptosis by the caspase inhibitor may have beneficial effects. On the other hand, it has also been shown that airway inflammation is associated with an enhanced survival of inflammatory cells caused by reduced apoptosis (25, 27). In the present study, we demonstrated that inhibition of caspase activation augmented cytokine-induced peroxide generation from macrophages. These findings could raise the possibility that suppression of caspase activation may enhance proinflammatory products from macrophages and facilitate inflammation in certain diseases. Thus it might need to be considered carefully in the therapeutic potential of caspase inhibitors.
In conclusion, we demonstrated that high levels of NO released from cytokine-activated macrophages may sensitize macrophages themselves to Fas-mediated apoptosis. This may be a negative feedback loop serving to promote resolution of inflammation by accelerating deletion of macrophages by apoptosis. In addition, inhibition of macrophage apoptosis by caspase inhibitors may facilitate inflammation through enhancing reactive oxygen production.
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ACKNOWLEDGEMENTS |
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We thank Taeko Kaimoto for technical assistance and Tomoko Hironaka for secretarial assistance.
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FOOTNOTES |
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This work was supported by a grant from the Ministry of Education, Science, and Culture of Japan.
Address for reprint requests and other correspondence: K. Fukuo, Dept. of Geriatric Medicine, Osaka Univ. Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan (E-mail: fukuo{at}geriat.med.osaka-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 19 November 1999; accepted in final form 8 March 2000.
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