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Division of Developmental Genetics, Center for Biomedical Science, Chiba University School of Medicine, Chiba, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and LPS. Bcl-xL, but not bcl-2 and
bax mRNA, was highly inducible within 3 h after
stimulation. The induction required new protein synthesis, but was
independent of effects of synthesized NO. Since activated RAW264 were
more resistant to NO-induced apoptosis mediated by the exposure to
S-nitroso-N-acetyl-penicillamine (SNAP) than
nonactivated RAW264, the inducible Bcl-xL may play a role
in the protection from NO toxicity. To confirm the protective function,
RAW264 were stably transfected with bcl-xL.
Those transfectants activated with IFN- and LPS appeared highly
resistant to NO-induced cell death detected within 24 h after
stimulation, although their NO production was similar to those of
parental RAW264 and neomycin control-transfected cells. Furthermore,
bcl-xL transfectants displayed substantial
protection from SNAP-induced apoptosis. These results establish a link
between self-defense to the synthesized NO and the induction of
Bcl-xL in activated macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bcl-2 family genes play a role in regulating apoptosis (14, 15) and may be a key molecule for self-defense mechanisms in activated macrophages. Bcl-2 and its relatives, bcl-xL and bax, encode intracellular membrane-bound proteins. Bcl-2 and Bcl-xL protein are functionally equivalent (16, 17, 18). They enhance the survival of several cell types and prevent apoptosis induced by a wide range of agents, including oxidative stress (16, 19, 20). However, expression of bcl-xL appears to be more restricted than that of bcl-2, and mitogenic activation of mature cells strongly stimulates expression of bcl-xL, but only modestly increases that of bcl-2 (21). These differences may reflect their functional roles in cell survival. On the other hand, Bax antagonizes the survival function of Bcl-2. Bax forms homodimers that comprise an active trigger for cell death. Bax also makes heterodimers with Bcl-2 and Bcl-xL (22) that prevent the formation of toxic Bax homodimers, resulting in survival. Therefore, the ratio of Bcl-2 or Bcl-xL to Bax appears to determine the fate of cells (14, 15, 22).
Expression of iNOS is strongly induced in RAW264.7 murine macrophage
cell line activated with IFN- and LPS within 3 h after
stimulation and remains elevated for a few days (5, 23). Continuous NO
production generated by the iNOS gradually induces apoptosis in some of
the RAW264.7 within 24 h after stimulation. This NO-mediated
apoptosis can be blocked by overexpression of bcl-2 (23),
suggesting that Bcl-2 might play a role in the self-defense mechanisms.
However, the protective activity of Bcl-2 is not physiologic since the
endogenous bcl-2 gene is not up-regulated in activated
RAW264.7 (23). To elucidate the mechanisms in a physiologic condition,
we analyzed expression of bcl-2 family genes in peritoneal
macrophages and RAW264 macrophage cell line activated with IFN- and
LPS. We show in this study that expression of
bcl-xL was highly inducible in activated
macrophages. We discuss the function of Bcl-xL as a
physiologic molecule that plays a key role in the self-defense
mechanisms against NO toxicity in macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6CrSlc mice were purchased from Japan SLC Co. (Hamamatsu, Japan).
Reagents
Murine rIFN- with sp. act. of 1 x 107
U/mg was purchased from Genzyme Corp. (Cambridge, MA). LPS (from
Escherichia coli, serotype 0111:B4) and cycloheximide (CHM)
were purchased from Sigma Chemical Co. (St. Louis, MO). NO inhibitor,
L-N5-iminoethyl ornithine dihydrochloride
(L-NIO) and NO generation agent,
S-nitroso-N-acetyl-D,L-penicillamine
(SNAP) were purchased from Cayman Chemical Co. (Ann Arbor, MI). IL-4
(104 U/ml) was prepared from culture supernatant of
X63Ag8-653 cells transfected with murine IL-4 gene (24).
Macrophage cell culture
Macrophages from mice were prepared from resident peritoneal cells. Briefly, peritoneal cells were harvested without elicitation and cultured in six-well tissue culture plates for 2 h at 37°C in 5% CO2. After nonadherent cells were removed by extensive washing, adherent cells were harvested by trypsinization and used as peritoneal macrophages. RAW264 murine macrophage cell line was obtained from RIKEN Cell Bank (Tsukuba, Japan). Macrophages were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 100 µg/ml streptomycin sulfate (Wako Chemical Co., Osaka, Japan), 100 U/ml penicillin G potassium (Banyu Pharmaceutical Co., Tokyo, Japan), and 10% (v/v) heat-inactivated FCS (Bioserum, Victoria, Australia).
Assay for NO synthesis
Synthesis of NO by activated macrophages was measured by the assay for nitrite (NO2-), a stable NO oxidation product, in culture supernatants, as previously described (25). Briefly, 100 µl of culture supernatants were mixed with an equal volume of Griess reagent (0.5% sulfanilamide, 0.05% N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5% H3PO4) in a 96-well tissue culture plate for 10 min at room temperature. The absorbance of samples was measured on an ELISA plate reader (Bio-Rad, Richmond, CA) at 570 nm. The nitrite concentration was calculated using sodium nitrite as a standard.
Northern blot analysis
Total RNA was isolated from cultured macrophages using the TriZOL RNA isolation reagent (Life Technologies). Total RNA (5–10 µg) was loaded on a 1% agarose gel in MOPS buffer containing 6% formaldehyde, transferred to a nylon membrane (Boehringer Mannheim GmbH, Mannheim, Germany), and fixed by cross-linking with UV irradiation and by baking at 80°C for 3 h. The filter was hybridized with digoxigenin-labeled probe overnight at 50°C. Following hybridization, the filter was washed twice with 0.1x SSC and 0.1% SDS at 55°C for 15 min. The probe on the filter was detected with sheep anti-digoxigenin Abs conjugated with alkaline phosphatase. The Ab detection reaction was performed using an enhanced chemiluminescent detection system (Boehringer Mannheim). Full-length murine iNOS cDNA (26) (a gift from Dr. K. Nishina, Kobe University, Kobe, Japan), murine bcl-2 cDNA (27), murine bax cDNA (22), and rat bcl-xL cDNA (28) (a gift from Dr. S. Ohta, Nippon Medical School, Kawasaki, Japan) were subcloned into pGEM vectors and labeled by digoxigenin, using PCR with T7 and SP6 primers, then used as a probe. Amounts of the mRNAs were measured by the densitometer (AE-6920-MF; Atto, Tokyo, Japan).
Transfection of bcl-xL cDNA into a RAW264 cell line
The bcl-xL cDNA was subcloned into an
XbaI site of the plasmid pEF-BOS, which harbors an
elongation factor 1
promoter (29). The
pEF-BOS/bcl-xL (20 µg) was transfected into
RAW264 with 1 µg of the pST-neoB, which carries a neomycin-resistant
gene (30). Transfection was performed by electroporation with a Gene
Pulser (Bio-Rad) at 0.35 kV in a 0.4-cm cell. After selection with 0.5
mg/ml geneticin G418 (Life Technologies), five clones that stably
expressed the exogenous bcl-xL gene were established. The
clone 35-1 with the highest level and the clone 35-3 with the lowest
level of bcl-xL mRNA among five transfectants
(data not shown) were used for following experiments. Parental RAW264
as well as two clones of pST-neoB-transfectants (neo-1 and neo-2) were
used as controls. Cell morphology, proliferative behavior, and
expression of bcl-2 and bax mRNA were comparable
between bcl-xL transfectants and parental RAW264
(data not shown).
FACS analysis for apoptotic cell death
Cell viability was examined by the propidium iodide (PI) exclusion method, as described (31). Briefly, cultured macrophages were harvested by trypsinization and resuspended in staining buffer (0.1% sodium azide, 3% FCS in PBS) with 2 µg/ml PI. PI uptake in each cell was analyzed on FACSCalibur (Becton Dickinson, Mountain View, CA) using Cell Quest software. Data were displayed as percentages of PI-stained cells.
Since the nuclei in apoptotic cells show a uniform reduction in DNA stability with PI, which is indicated by the appearance of a subdiploid fraction of cells on the DNA histogram (32), FACS analysis of PI-stained nuclei was performed to detect apoptotic cells. Briefly, harvested macrophages were incubated in hypotonic lysing buffer (0.1% sodium citrate, 0.01% Triton X, and 0.1 mg/ml PI) at 4°C for 4 h. DNA content in each cell was analyzed on FACSCalibur using Cell Quest software for Macintosh. Data were displayed as percentages of apoptotic (hypodiploid) nuclei.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
LPS
Expression of iNOS and bcl-2 family genes
(bcl-2, bcl-xL, and
bax) was analyzed in peritoneal macrophages
stimulated with IFN- and LPS by Northern blot (Fig. 1
A). Expression of iNOS
was induced within 3 h and reached to the maximum at 6 h
after stimulation. Bcl-xL mRNA was faintly detectable
before stimulation. The expression was up-regulated within 3 h,
reached the plateau level (5.2-fold increase) from 6 h, and was
sustained for at least 24 h after stimulation. Bcl-2 mRNA was
detected before stimulation. However, it was not up-regulated at all.
Bax mRNA was observed at relatively high levels before stimulation and
up-regulated (1.8-fold) from 6 h after stimulation.
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and LPS, the similar kinetics
of those gene expressions was observed (Fig. 1
B).
RAW264 without stimulation did not express a detectable level of iNOS
mRNA. Expression of iNOS was induced from 3 h after stimulation,
and detectable until 48 h after stimulation. Bcl-xL
was rapidly up-regulated within 1 h and maintained the high level
(26-fold increased) until at least 48 h after stimulation. As is
different from peritoneal macrophages, bcl-2 expression was
not observed at all. Bax was also detected before stimulation,
up-regulated (twofold) from 12 h after stimulation, and maintained
until 48 h after stimulation.
The up-regulation of bcl-xL mRNA in RAW264
stimulated with IFN- and LPS is not controlled by synthesized NO
Expression of iNOS and bcl-xL was induced
simultaneously in activated macrophages, suggesting that their
induction mechanisms are very similar. The similarity was further
analyzed in RAW264 stimulated with IFN- or LPS (Fig. 2). Expression of iNOS and
bcl-xL was induced in RAW264 stimulated with LPS
in a dose-dependent manner. Those expressions were also induced by
stimulation of IFN- alone. When RAW264 were costimulated with
IFN- (40 U/ml) and LPS (1 µg/ml), expression of iNOS was
synergistically up-regulated. When the amount of iNOS mRNA in RAW264
activated with IFN- was arbitrary 1, the relative amounts in RAW264
stimulated with LPS or with IFN- and LPS were 3.5 or 13. However,
the synergistic response such as the iNOS expression was not obvious in
expression of bcl-xL, since the relative amounts
of bcl-xL mRNA in the cells activated with LPS
or with IFN- and LPS were 3.7 or 4.6.
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and LPS, the up-regulation of
bcl-xL was examined in activated RAW264 without
NO generation. L-NIO, L-arginine analogue, is a
potent, fast acting, and irreversible inhibitor of NO generation (33).
When RAW264 were stimulated with IFN- and LPS in the presence of
L-NIO, the up-regulation of bcl-xL
mRNA was not affected (Fig. 3A), while
NO2- production in culture supernatants was
drastically repressed (Fig. 3B). This was repeated
using another NO inhibitor, IL-4. IL-4 inhibits iNOS production at the
transcriptional level (4, 34). When RAW264 were cultured with IL-4 for
1 h before stimulation with IFN- and LPS, both iNOS mRNA (Fig. 3A) and NO2- production (Fig. 3C) were significantly reduced in a dose-dependent
fashion. However, the up-regulation of bcl-xL
was not affected (Fig. 3A). These data indicate that
the up-regulation of bcl-xL is not controlled by
synthesized NO.
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and LPS
Since the bcl-xL mRNA was rapidly up-regulated in activated
macrophages after stimulation (Fig. 1), expression of
bcl-xL may be induced without new protein
synthesis in the macrophages such as an immediate early gene (35). To
examine the possibility, RAW264 were precultured with CHM, a reversible
protein synthesis inhibitor (36), for 30 min and then stimulated with
IFN- and LPS. Expression of c-fos mRNA (one of the
immediate early genes) in the RAW264 was strongly up-regulated by the
CHM treatment (data not shown). However, the CHM treatment blocked the
up-regulation of bcl-xL as well as that of iNOS
(Fig. 4). Thus, both inductions required
the de novo protein synthesis in RAW264 stimulated with IFN-
and LPS.
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To examine the activity of Bcl-xL in activated
RAW264 to protect from NO toxicity, RAW264 were stimulated with IFN-
and LPS for 2 h to up-regulate Bcl-xL and then exposed
to NO donor, SNAP. Percentages of dead cells in the activated RAW264
were analyzed at 8 h after SNAP exposure by the PI exclusion with
FACS. As shown in Figure 5, approximately
23% of nonactivated RAW264 were dead by the SNAP exposure, as expected
(23). The pretreatment of RAW264 with IFN- and LPS clearly reduced
the number of dead cells (7%), suggesting that the up-regulation of
Bcl-xL protects RAW264 from NO-induced cell death.
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and LPS for 24 h since some of RAW264 stimulated with IFN- and
LPS are known to die by apoptosis within 24 h after stimulation
(23). As shown in Figure 6A,
approximately 30% of cells were dead in activated RAW264 and pST-neoB
transfectants (neo-1 and neo-2) at 24 h after stimulation. The
cell death was due to the synthesized NO since the cell death was
blocked by the addition of NO inhibitor (L-NIO) in the
culture. Percentages of dead cells in the bcl-xL
transfectants were lower than those in the parental RAW264 and the
pST-neoB transfectants. These results indicate that the
bcl-xL transfectants were protected from
NO-induced cell death because NO production (Fig. 6B)
and expression of bcl-2 and bax mRNA (data not
shown) from the bcl-xL transfectants were
similar to those from the controls. In parallel, the presence of
apoptotic (subdiploid) cells in the activated 35-1 and parental RAW264
at 24 h after stimulation was examined by PI staining with FACS.
The overexpression of Bcl-xL partially protected apoptotic
cell death of activated RAW264 (Fig. 6C), despite a
concomitant production of NO2- (Fig. 6B). Although the exogenous Bcl-xL
protected RAW264 from NO-induced cell death, the Bcl-xL did
not influence the level of NO produced by the RAW264 (Fig. 6B).
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), the protective
activity of exogenous Bcl-xL in RAW264 was examined to the
SNAP-induced apoptosis. Figure 7A shows that 28% of parental
RAW264 were dead at 8 h after SNAP exposure. Similar results were
obtained from neo-1 and neo-2. In contrast, 35-1 and 35-3 displayed
better survival. Percentages of apoptotic cells in 35-1 were
substantially less than those in control (Fig. 7B).
The 35-1 showed only 4.3% DNA fragmentation, whereas the parental
RAW264 showed 23.5% at 8 h, and 16.7 and 43.6% at 24 h
after SNAP exposure, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and LPS (Fig. 1), although
bcl-2 was slightly expressed in resting peritoneal
macrophages (Fig. 1A). Since Bcl-xL
transiently induced in differentiating lymphocytes plays a role in
their selection processes (41, 42) and kinetics of the up-regulation in
bcl-xL was coincident with that in iNOS (Fig. 1), Bcl-xL induced in activated macrophages may also play a
role in their transient protection from NO-induced apoptosis.
Bcl-xL is a potent repressor of apoptosis induced by NO and
reactive oxygen species (19), suggesting that Bcl-xL
controls a common pathway for apoptosis mediated by NO and oxidants
(16, 20). Bcl-xL homodimers have been suggested to form
protective complexes against apoptosis. Since Bcl-xL can
form heterodimers with several other proteins, including BAG-1, Bax,
Bad, and Bcl-xS, its protective effects can be further
enhanced by BAG-1 and diminished by Bax, Bad, and Bcl-xS
(14, 15). Bax homodimers comprise an active trigger for cell death (15, 22). Expression of bax was up-regulated (within twofold) in
macrophages from 6 to 12 h after activation, and was not
coincident with the induction in iNOS and bcl-xL
(Fig. 1). Furthermore, the inducibility (5-fold in peritoneal
macrophages and 26-fold in RAW264) of bcl-xL was
much larger than that of bax in activated macrophages until
12 h after stimulation. Therefore, amounts of Bcl-xL
homodimers and Bcl-xL/Bax heterodimers may increase and
that of Bax homodimers may decrease to result in the protection from
NO-induced apoptosis. Since some of activated macrophages became
apoptotic from 12 to 24 h after stimulation (Fig. 6A), this NO-induced self-destruction may be
explained by the up-regulation of bax in activated
macrophages from 6 to 12 h after stimulation (Fig. 1).
NO is toxic for macrophages via several mechanisms, including
inhibition of mitochondrial respiration and DNA synthesis, and
initiation of DNA strand breaks (1, 3, 4, 5). In some instances, NO has
been shown to enhance cellular oxidative injury (4). Several molecules
other than Bcl-xL may be able to regulate susceptibility of
macrophages against NO toxicity. Kim et al. demonstrated the existence
of inducible cellular resistance mechanism by hsp32 (heme oxygenase) in
hepatocytes against NO (11). Furthermore, many of the stimuli that
induce iNOS expression are known to increase expression of
metallothionein (43), and overexpression of metallothionein reduces the
sensitivity to cell injury and DNA double strand break by NO in
National Institutes of Health 3T3 cells (44). Thus, metallothionein may
play a physiologic role in activated macrophages since normal
macrophages can produce metallothionein (43). Heat shock as well as
overexpression of a heat-shock protein (hsp70) also induce resistance
to NO toxicity in islet cells (45). Indeed, an NO donor has been shown
to induce hsp70 expression in a hepatoblastoma cell line and in various
organs of animals (46). Moreover, pretreatment or repeated treatments
of macrophages with nontoxic doses of IFN- and LPS increase
expression of hsp70 and result in cell resistance to NO toxicity (47, 48). Since those IFN- and LPS treatments also up-regulated
bcl-xL in macrophages (Fig. 2) and a single
stimulation could induce enough amounts of Bcl-xL to
protect RAW264 from NO-induced apoptosis (Fig. 5), Bcl-xL
may play a physiologic role in inducible self-defense mechanisms in
macrophages against NO toxicity.
Induction mechanisms of bcl-xL initiated by LPS
stimulation seem to be very similar to those of iNOS in activated
macrophages since kinetics (Fig. 1) and sensitivity to LPS stimulation
(Fig. 2) for those inductions were very similar. Therefore, the
signal-transduction pathways initiated by LPS stimulation may be shared
between inductions of bcl-xL and iNOS. However,
the costimulation of RAW264 with IFN- and LPS made different
inducibilities between them (Fig. 2). Many DNA elements homologous to
consensus sequences for the binding of transcription factors such as
nuclear factor-
B and AP-1 are located in promoter regions of the
iNOS gene (49), but not in those of the bcl-xL
gene (50). Those elements in the iNOS promoter may be responsible for
the synergistic augmentation. This is supported by the result that the
induction of iNOS expression required synthesis of proteins such as
transcription factors in activated macrophages (Fig. 4). Although
Bcl-xL seemed to be up-regulated in activated macrophages
for the protection of NO toxicity, neither iNOS nor synthesized NO
affected expression of bcl-xL (Fig. 3). Further
study is required to elucidate the induction mechanisms of
bcl-xL in activated macrophages.
In summary, bcl-xL, but not bcl-2,
was strongly induced in murine peritoneal macrophages stimulated with
IFN- and LPS, and the induction was coincident with that in iNOS.
Furthermore, overexpression of bcl-xL in RAW264
macrophage cell line inhibited or delayed apoptosis induced by the
endogenous or the exogenous NO. These findings suggest that
Bcl-xL is a physiologic molecule to contribute to the
self-defense mechanisms in macrophages against NO toxicity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Takeshi Tokuhisa, Division of Developmental Genetics, Center for Biomedical Science, Chiba University School of Medicine, Chiba 260-8670 Japan. E-mail address:
3 Abbreviations used in this paper: NO, nitric oxide; CHM, cycloheximide; iNOS, inducible nitric oxide synthase; L-NIO, L-N5-iminoethyl ornithine dihydrochloride; NO2-, nitrite; NOS, nitric oxide synthase; PI, propidium iodide; SNAP, S-nitroso-N-acetyl-penicillamine.
Received for publication July 16, 1997. Accepted for publication November 17, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. Orlofsky, R. D. Somogyi, L. M. Weiss, and M. B. Prystowsky The Murine Antiapoptotic Protein A1 Is Induced in Inflammatory Macrophages and Constitutively Expressed in Neutrophils J. Immunol., July 1, 1999; 163(1): 412 - 419. [Abstract] [Full Text] [PDF]
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L. Sevilla, A. Zaldumbide, F. Carlotti, M. A. Dayem, P. Pognonec, and K. E. Boulukos Bcl-xL Expression Correlates with Primary Macrophage Differentiation, Activation of Functional Competence, and Survival and Results from Synergistic Transcriptional Activation by Ets2 and PU.1 J. Biol. Chem., May 18, 2001; 276(21): 17800 - 17807. [Abstract] [Full Text] [PDF]
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Y.-G. Kwon, J.-K. Min, K.-M. Kim, D.-J. Lee, T. R. Billiar, and Y.-M. Kim Sphingosine 1-Phosphate Protects Human Umbilical Vein Endothelial Cells from Serum-deprived Apoptosis by Nitric Oxide Production J. Biol. Chem., March 30, 2001; 276(14): 10627 - 10633. [Abstract] [Full Text] [PDF]
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J. Lee, J. Hur, P. Lee, J. Y. Kim, N. Cho, S. Y. Kim, H. Kim, M.-S. Lee, and K. Suk Dual Role of Inflammatory Stimuli in Activation-induced Cell Death of Mouse Microglial Cells. INITIATION OF TWO SEPARATE APOPTOTIC PATHWAYS VIA INDUCTION OF INTERFERON REGULATORY FACTOR-1 AND CASPASE-11 J. Biol. Chem., August 24, 2001; 276(35): 32956 - 32965. [Abstract] [Full Text] [PDF]
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