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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3823-3831
IMMUNOBIOLOGY
LPS induces apoptosis in macrophages mostly through the
autocrine production of TNF-
Jordi Xaus,
Mònica Comalada,
Annabel F. Valledor,
Jorge Lloberas,
Francisco López-Soriano,
Josep M. Argilés,
Christian Bogdan, and
Antonio Celada
From the Departament de Fisiologia (Biologia del Macròfag) and
Fundació August Pi i Sunyer, Campus de Bellvitge; Departament de
Bioquímica i Biologia Molecular, Facultat de Biologia
Universitat de Barcelona, Barcelona, Spain; and the Institut für
Klinische Mikrobiologie Immunologie und Hygiene, Universität
Erlangen, Erlangen, Germany.
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Abstract |
The deleterious effects of lipopolysaccharide (LPS) during endotoxic
shock are associated with the secretion of tumor necrosis factor (TNF)
and the production of nitric oxide (NO), both predominantly released by
tissue macrophages. We analyzed the mechanism by which LPS induces
apoptosis in bone marrow-derived macrophages (BMDM). LPS-induced
apoptosis reached a plateau at about 6 hours of stimulation, whereas
the production of NO by the inducible NO-synthase (iNOS) required
between 12 and 24 hours. Furthermore, LPS-induced early apoptosis was
only moderately reduced in the presence of an inhibitor of iNOS or when
using macrophages from iNOS -/-mice. In contrast, early apoptosis was
paralleled by the rapid secretion of TNF and was almost absent in
macrophages from mice deficient for one (p55) or both (p55 and p75)
TNF-receptors. During the late phase of apoptosis (12-24 hours) NO
significantly contributed to the death of macrophages even in the
absence of TNF-receptor signaling. NO-mediated cell death, but not
apoptosis induced by TNF, correlated with the induction of p53 and Bax
genes. Thus, LPS-induced apoptosis results from 2 independent
mechanisms: first and predominantly, through the autocrine secretion of
TNF- (early apoptotic events), and second, through the production of
NO (late phase of apoptosis).
(Blood. 2000;95:3823-3831)
© 2000 by The American Society of Hematology.
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Introduction |
Mononuclear phagocytes represent a large family of cell
types that includes tissue macrophages, Kupffer cells in the liver, Langerhans' cells in the epidermis, osteoclasts in the bone, microglia in the brain, and perhaps some of the interdigitating and follicular dendritic cells found in lymphoid organs.1,2 Macrophages exert key functions during the immune response. To perform most of
these functions, macrophages must be activated.3,4 Thus, macrophages are able to kill bacteria, virus, or parasites directly; to
secrete several immune regulators (tumor necrosis factor [TNF- ], interleukin (IL)1- , IL6, etc); to process antigens and present them
to T cells; and finally, to act as scavenger cells and to participate
in tissue remodeling.
However, macrophages do not always play a positive role in the
homeostasis of the immune system. Under some circumstances, macrophages
have deleterious effects. This is the case of septic shock, which is a
severe systemic inflammatory response triggered by the interaction of
lipopolysaccharide (LPS) and some bacterial components with macrophages
and other host cells.5,6 Although this interaction leads to
the progressive release of a variety of proinflammatory cytokines, such
as IL8, IL1-, and IL6,7,8 experimental evidence points
to nitric oxide (NO) and TNF as the primary mediators of the changes
observed during septic shock.9-11 Central to the
pathogenesis of endotoxic shock is the development of circulatory
failure, characterized by hypotension, myocardial dysfunction, and
tissue hypoxia that ultimately leads to multiorgan failure and
death.11,12 Despite major advances in antimicrobial therapy
and critical care, septic shock continues to have a mortality rate of
40%-70% and remains the leading cause of more than 100 000 deaths
per year in the intensive care units of the United States
alone.11,13
Although several reports14-16 suggest that excessive
production of NO by the inducible NO synthase (iNOS) contributes to the circulatory failure during septic shock, the role of this enzyme in
septic shock remains controversial. The use of iNOS -/-mice revealed
the existence of both an iNOS-dependent and -independent pathway for
LPS-induced hypotension.17-19 Deletion of the iNOS gene or
blocking of the activity of iNOS resulted in either no protection,18,20 partial17,19 or total
protection,21,22 or even led to detrimental
effects23,24 during sepsis.
TNF affects the growth, differentiation, and function of many cell
types, and it is a major mediator of inflammatory immune responses.10,25,26 TNF has also been suggested as a key
mediator of the septic shock syndrome induced by either LPS or
bacterial superantigens.27-29 The potent regulatory
abilities of TNF- are transduced by 2 distinct cell-surface
receptors with 55 kd (Type I) and 75 kd (Type II) relative molecular
weights.30,31
Most of the known cellular TNF- responses have been attributed to
the activation of p55 type I TNF-R.32,33 In contrast, little is known about the function of p75 type II
TNF-R.34,35 Activation of the type I TNF-R is
necessary and sufficient for TNF--induced liver failure and
hepatocyte apoptosis36 as well as for cytotoxicity and
apoptosis in other cell types.37-39 Although p55 TNF-R
-/-mice seem to be resistant to endotoxic shock, they succumb to
bacterial infections.40
Thus, LPS-dependent activation of macrophages, exposure to endogenous
or exogenous NO, or treatment with TNF are enough to induce apoptosis
in several cell types.41-43 Apoptosis has been involved in
the ultimate multiorgan failure during septic shock. For this reason,
we have analyzed the mechanisms involved in the LPS-induced apoptosis
of macrophages, since these cells are mainly involved in the secretion
of TNF and NO that plays a crucial role in the pathogenesis of
endotoxic shock.
In this report we provide evidence that macrophage apoptosis induced by
LPS is mediated by both NO and TNF production. However, each of these
agents acts separately. TNF induces the early apoptotic events (3-6 hours), whereas iNOS-dependent apoptotic events occur later (12-24 hours). NO-induced apoptosis, but not TNF--dependent apoptosis,
correlates with the induction of p53 and Bax.
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Materials and methods |
Reagents
LPS was obtained from Sigma Chemical (St Louis, MO). Recombinant
murine TNF- (rmTNF-) was purchased from PrepoTech EC (London, UK). Recombinant murine interferon (IFN)- was kindly provided by
Genentech (South San Francisco, CA). 4'6-diamidino-2-phenylindole (DAPI), (±)-S-nitroso-N-acetylpenicillamine (SNAP), and
S-methylisothiourea sulfate (SMT) were all purchased from
Calbiochem (La Jolla, CA). All other chemicals were of the highest
available purity grade and were purchased from Sigma Chemical.
Deionized water further purified with a Millipore Milli-Q
system (Bedford, MA) was used.
Antibodies
For Western blot analysis, we used a rabbit antibody against mouse
iNOS (M-19; Santa Cruz Biotechnology, Santa Cruz, CA), a sheep
antimouse p53-PAN antibody (Boehringer Mannheim, Mannheim, Germany),
and, as a control, a mouse antimouse -actin antibody (Sigma
Chemical). Peroxidase-conjugated antirabbit immunoglobulin G (IgG;
Cappel, Turnhout, Belgium), anti-goat/sheep IgG (Boehringer), or
antimouse IgG (Cappel) were used as secondary antibodies.
Plasmids and constructions
The plasmid corresponding to the rat iNOS full-length complementary
DNA (cDNA) was kindly provided by Dr A. Felipe (University of
Barcelona, Spain). Murine cDNA probes for TNF- and Bax were kindly
provided by Dr M. Nabholz (ISREC, Epalinges, Switzerland) and Dr R. Merino (University of Cantabria, Spain), respectively. As a control for
RNA loading and transfer, we used an 18S rRNA transcript.44
Cell culture
Bone marrow-derived macrophages (BMDM) were isolated as previously
described.45 Six-week-old BALB/C mice (Charles River Laboratories, Wilmington, MA) were killed by cervical dislocation, and
both femurs were dissected free of adherent tissue. The ends of the
bones were cut off and the marrow tissue was flushed by irrigation with
media. The marrow plugs were dispersed by passing through a 25-gauge
needle, and the cells were suspended by vigorous pipetting and washed
by centrifugation. The cells were cultured in plastic tissue-culture
dishes (150 mm) in 40 mL DMEM containing 20% FBS and 30% L-cell
conditioned media as a source of macrophage colony-stimulating factor
(M-CSF). Macrophages were obtained as a homogeneous population of
adherent cells after 7 days of culture. The cells were incubated at
37°C in a humidified 5% CO2 atmosphere.
BMDM from iNOS or TNF-R knock-out (KO) mice and the corresponding
controls were isolated under the same conditions. TNF-RI KO
mice46 and TNF-RI/II double KO mice47 were
kindly donated by Dr K. Matsushima from Kanazawa University, Japan; and
Dr J. Peschon from University of Kentucky, respectively. The iNOS KO mice were kindly donated by Dr S. Mudgett17 and obtained as previously described.48
Analysis of DNA content with DAPI
Macrophages (106) previously subjected or not to LPS
treatment were resuspended and fixed in ice-cold 70% ethanol. The
cells were then washed in phosphate-buffered saline (PBS), resuspended in 0.2 mL of a solution containing 150 mmol/L NaCl, 80 mmol/L HCl, and
0.1% Triton X-100, and incubated at 0°-4°C for 10 minutes. Afterward, 1 mL of a solution containing 180 mmol/L
Na2HPO4, 90 mmol/L citric acid, and 2 µg/mL
DAPI, pH 7.4, was added to each sample. After incubating the cells at
4°C for 24 hours, their fluorescence was measured with an Epics
Elite flow cytometer (Coulter, Hialeah, FL). For this analysis, we used
an UV laser with an excitation beam of 25 mW at 333-364 nm and
fluorescence was collected with a 525 nm band-pass filter. Cell
doublets were gated out by comparing the pulse area versus the pulse
width. A total of 12 000 cells were counted for each histogram, and
cell-cycle distributions were analyzed with the Multicycle program
(Phoenix Flow Systems, Inc; San Diego, CA).
In parallel experiments, cells stained with DAPI were mounted on a
slide and visualized in a Zeiss fluorescent microscope. Pictures were
taken, using a Kodak camera installed to the microscope. Under these
conditions, condensed DAPI-stained chromatin was visualized in the
nucleus of the apoptotic cells.
Analysis of apoptosis
DNA fragmentation due to internucleosomal cleavage was determined as
described previously.49 Briefly,
3 × 106 macrophages were harvested and washed in
ice-cold PBS. The cells were lysed in 0.5 mL of lysis buffer (50 mmol/L
Tris-HCl, 10 mmol/L EDTA, 1% SDS, pH 8.0) for 16 hours at 4°C, and
the lysates were centrifuged (15 000 × g) to separate
high molecular weight DNA (pellet) from cleaved low-molecular-weight
DNA (supernatant). The DNA supernatants were phenol-extracted twice and
precipitated. The pellets were resuspended in Tris-EDTA buffer
containing 250 µg/mL RNAse (Boehringer Mannheim). The samples were
heated at 65°C for 10 minutes and subjected to electrophoresis in a
2% agarose gel containing ethidium bromide.
Low-molecular-weight apoptotic DNA, obtained as previously described,
was also measured by an enzyme-linked immunosorbent assay (ELISA)
technique (Cell Death Detection ELISA Plus; Boehringer Mannheim) that
is directed against cytoplasmic histone-associated DNA fragments. Each
point was performed in triplicate and the results were expressed as the
mean ± SD.
Determination of NO production
NO production was estimated by measuring nitrate/nitrite in the cell
culture media. Macrophages were cultured in DMEM without phenol-red
(GIBCO Life Technologies, UK) to avoid interference with the Griess
absorbance at 550 nm. Samples were stored at  80°C until
assayed. Nitrate was converted to nitrite with Zea mays nitrate
reductase (Calbiochem). Reduced samples were incubated with an equal
volume of Griess reagent, and the absorbance at 550 nm was measured.
The total nitrate/nitrite concentration was determined by comparison
with a standard curve.
Protein extraction and Western blot analysis
Cells were washed twice in cold PBS and lysed on ice with lysis
solution (1% Triton X-100, 10% glycerol, 50 mmol/L Hepes pH 7.5, 150 mmol/L NaCl, protease inhibitors). The protein concentration of the
samples was determined with the Bio-Rad protein assay. The
proteins from the cell lysates (100 µg) were boiled at
95°C in Laemmli SDS loading buffer, separated on 7.5%
SDS-PAGE for the detection of iNOS or on 10% SDS-PAGE for
p53 immunoblotting. Then, the proteins were electrotransferred to
nitrocellulose membranes (Hybond-ECL; Amersham, Arlington Heights, IL).
The membranes were blocked for at least 1 hour at room temperature in
Tris buffered saline-0.1% Tween-20 (TBS-T) containing 5% nonfat dry
milk and then incubated with TBS-T containing the primary antibody. For iNOS, p53, and -actin immunoblotting, incubation was performed for 1 hour at room temperature. After 3 washes of 15 minutes each in TBS-T,
the membranes were incubated with peroxidase-conjugated anti-goat/sheep
(Boehringer), antirabbit, or antimouse IgG (Cappel) antibodies for 1 hour. After 3 washes of 15 minutes with TBS-T, ECL detection was
performed (Amersham) and the membranes were exposed to x-ray films
(Amersham). Quantitation of the blots was carried out by densitometric analysis.
Northern blot analysis
Total cellular RNA (20 µg), extracted with the acidic guanidinium
thiocyanate-phenol-chloroform method,50 was separated in
1% agarose with 5 mmol/L 3-[N-morpholino]propanesulfonic acid (MOPS), pH 7.0/1 mol/L formaldehyde buffer. The RNA was transferred overnight to a GeneScreen nitrocellulose membrane (Life Science Products, Boston, MA) and fixed by UV irradiation (150 mJ). All probes
were labeled with 32P-dCTP (Amersham) with the
oligolabeling kit method (Pharmacia Biotech, Uppsala, Sweden). To check
for differences in RNA loading, the expression of the 18S rRNA
transcript was analyzed. After incubating the membranes for 18 hours at
65°C in hybridization solution (20% formamide, 5X Denhart's, 5X
SSC, 10 mmol/L EDTA, 1% SDS, 25 mmol/L
Na2HPO4, 25 mmol/L
NaH2PO4, 0.2 mg/mL salmon sperm DNA, and
106 cpm/mL of 32P-labeled probe), they were
exposed to Kodak X-AR films (Kodak, Rochester, NY). The bands of
interest were quantified with a Molecular Analyst system (Bio-Rad Labs,
Richmond, CA).
Determination of TNF- production
The secretion of TNF- was measured with the use a commercial
murine TNF- ELISA kit (Quantikine M; R&D Systems, Minneapolis, MN);
105 cells were cultured in 24-well plates and stimulated
with LPS. Supernatant samples were obtained at the indicated times
and subjected to ELISA analysis.
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Results |
Induction of apoptosis by LPS
Bone marrow macrophages growing in the presence of M-CSF are
unevenly distributed into the different phases of the cell cycle. On
the activation with LPS, macrophages arrest at the
G0/G1 phase of the cell cycle and die through
the induction of apoptosis. This conclusion is supported by several
observations: (1) the staining of the DNA with DAPI revealed that after
6 hours of LPS treatment, 35% of the cells had a subdiploid DNA
content corresponding to that of apoptotic cells, in contrast with 3%
of subdiploid cells in nontreated cell cultures (Figure
1A); (2) macrophages treated with LPS and
stained with DAPI showed condensed chromatin in the nucleus (Figure
1B); and (3) electrophoresis on an agarose gel of the DNA obtained from
macrophages treated with LPS showed the typical laddering observed
after internucleosomal fragmentation of apoptotic DNA (Figure 1C).
Therefore, all these results demonstrate that the treatment of bone
marrow macrophages with LPS induces cell death by apoptosis. Moreover,
apoptosis was quantified with the use of an ELISA kit that measures the
presence of histone-associated DNA fragments. The induction of
macrophage apoptosis by LPS was time- and dose-dependent (Figure 1D-E).
The kinetics of induction of apoptosis was very fast and maximal
induction was observed as soon as 3 hours after the start of LPS
treatment. The levels of apoptosis did not further increase thereafter
and up to 24 hours of stimulation.
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| Fig 1.
LPS induces apoptosis in bone marrow macrophages.
(A and B) 106 macrophages were stimulated with 100 ng/mL of
LPS for 6 hours. DNA was stained with DAPI, and induction of apoptosis
was analyzed by cytometric analysis (A) or visualizing the cells in a
fluorescence microscope (B). Apoptotic cells are marked with arrows. (C
and D) LPS induces apoptosis in a time-dependent manner. Apoptotic DNA
from macrophages treated with LPS (100 ng/mL) for the indicated times
was analyzed by agarose gel electrophoresis (C) or by using an ELISA
technique (D). (E) LPS induces apoptosis in a dose-dependent fashion;
105 macrophages were treated for 12 hours with the
indicated concentrations of LPS. Apoptotic DNA was measured as in (D).
(F) Apoptosis induced by LPS depends on the presence of FBS. The cells
were treated with 100 ng/mL LPS for 12 hours in the presence of the
indicated concentrations of FBS. Fragmentation of DNA was measured by
ELISA. The ELISA experiments were performed in triplicate and
represented as the mean value ± SD. These figures are
representative of 4 independent experiments.
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Maximal induction of apoptosis was obtained at a concentration of 100 ng/mL of LPS (Figure 1E) and was serum-dependent (Figure 1F), which can
be explained by the fact that the recognition of LPS by its high
affinity receptor CD14 requires the previous association of LPS with
the serum protein LBP (LPS-binding protein).51,52
Exogenous NO is toxic to macrophages, but iNOS-derived NO does not
account to LPS-induced early apoptosis
In several cell types, apoptosis induced by LPS has been linked to
the cytotoxic effect of iNOS-derived NO.41,53,54 Because LPS induces the expression of iNOS in macrophages,41,42 we analyzed whether the induction of apoptosis in bone marrow microphages was also mediated by the generation of NO. The toxic effect of NO was
tested by treating bone marrow macrophages with the NO-donor SNAP55 that spontaneously produces NO after being added to
the culture (Figure 2A). SNAP induced
apoptosis in macrophages in a time- and dose-dependent fashion as
determined by measuring DNA fragmentation, by DNA laddering (Figure
2B-C), or by cytometric analysis of DAPI-stained cells (data not
shown). Therefore, as has been observed for other types of
macrophages,41,42 exogenous NO is toxic for bone marrow
macrophages.
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| Fig 2.
Exogenous NO induces apoptosis in bone marrow
macrophages.
(A) NO production by SNAP; 105 macrophages were treated
with 50 µmol/L of SNAP for the indicated times and the production of
NO was determined. (B) Time course of SNAP-induced apoptosis;
105 macrophages were treated with 50 µmol/L SNAP at the
indicated times. Apoptosis was measured by ELISA. (C) The cells were
treated for 24 hours with the indicated concentrations of SNAP, and
apoptosis was detected as indicated above. Each experiment was
performed in triplicate and the results of 1 representative of 2 independent experiments are represented as the mean value ± SD.
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LPS induces the expression of iNOS in bone marrow macrophages.
However, whereas mRNA expression was maximal after 6 hours of
LPS treatment (Figure 3A), the expression
of iNOS protein was a late event not observed until 12 hours
of LPS treatment, reaching a maximum level after 24 hours
(Figure 3B). The synthesis of iNOS protein correlated with NO
production, which was not detected until 12-24 hours of LPS treatment
(Figure 3C).
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| Fig 3.
LPS induces the expression of iNOS and the production of
NO.
The expression of iNOS induced by LPS was measured by Northern blot (A)
or Western blot (B) as described in the "Materials and methods"
section. (C) LPS induces the production of NO; 106 BMDM
cultured in media without phenol-red were stimulated with 100 ng/mL
LPS, and the supernatants were harvested at the indicated times. NO
production was measured as the nitrite/nitrate levels. Each point was
performed in triplicate and represented as the mean ± SD. These
figures are representative of 3 independent experiments.
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These results suggest that, although exogenous NO may induce macrophage
apoptosis, the early apoptotic events induced by LPS (3-6 hours) are
not related to the production of endogenous NO derived from iNOS. To
further determine the role of endogenous NO in LPS-induced apoptosis,
we blocked the LPS-induced production of NO by using the iNOS inhibitor
SMT.56,57 SMT did not affect the induction of iNOS mRNA
expression by LPS (Figure 4A). However, SMT
totally blocked the NO production induced by LPS (Figure 4B). The
treatment with SMT had a very weak effect on the LPS induction of
apoptosis in macrophages (14% inhibition after 6 hours, 23% inhibition after 24 hours of LPS treatment) (Figure 4C). All this suggests that early macrophage apoptosis induced by LPS is not mediated
by the production of endogenous NO.
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| Fig 4.
Treatment of macrophages with SMT inhibits LPS-induced NO
production but not apoptosis.
(A) The expression of iNOS was measured by Northern blot in macrophages
treated with 100 ng/mL of LPS in the presence or absence of SMT, an
iNOS inhibitor (20 µmol/L). (B) SMT inhibits LPS-induced NO
production; 106 macrophages were treated with 100 ng/mL of
LPS for the indicated times in the presence or absence of SMT (20 µmol/L). The production of NO was assessed by determination of the
nitrate/nitrite levels. (C) SMT did not inhibit LPS-induced apoptosis.
The cells were treated with 100 ng/mL of LPS for the indicated times in
the presence or absence of SMT (20 µmol/L). Each experiment was
performed in triplicate, and the results of 1 representative of 2 independent experiments are represented as the mean value ± SD.
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Early apoptosis in LPS-stimulated macrophages is due to endogenous
TNF-
Because tissue macrophages are major producers of
TNF-,7 we analyzed the role of this cytokine in the
LPS-induced apoptosis in macrophages. At 30 minutes after LPS
stimulation, bone marrow macrophages already expressed high levels of
TNF- mRNA (Figure 5A). The protein
levels of TNF- increased very rapidly in the culture supernatants
and reached a concentration of 2490 pg/mL after 2 hours. These levels
of TNF- are sufficient to induce apoptosis in BMDM, since doses
between 1 and 10 ng/mL of rmTNF- induced significant levels of
apoptosis in these cells (Figure 5C). Moreover, the kinetics of
induction of apoptosis by rmTNF- is very similar to that triggered
by LPS (Figure 5D), with a significant induction of apoptosis within
the first 6 hours. Finally, the presence of the iNOS inhibitor SMT did
not inhibit the apoptosis induced by TNF- (data not shown),
demonstrating that in bone marrow macrophages the apoptosis induced by
TNF- is not mediated through the production of NO.
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| Fig 5.
LPS induces the autocrine secretion of TNF-, which
produces apoptosis.
(A) LPS induces the mRNA expression of TNF-. Total RNA (20 µg per
lane) from macrophages was treated with 100 ng/mL of LPS for the
indicated times was analyzed by Northern blotting. (B) LPS induces the
secretion of TNF-. The concentration of TNF- in the culture
supernatants was analyzed by ELISA. Each experiment was performed 3 times and represented as the mean value ± SD. (C) TNF- induces
apoptosis in bone marrow macrophages. Macrophages were stimulated for
12 hours with the indicated concentrations of rmTNF-. Induction of
apoptosis was measured by ELISA. (D) Time course of rmTNF--induced
apoptosis. Macrophages were treated with rmTNF- (100 ng/mL) for the
indicated periods of time. Each experiment was performed in triplicate
and represented as the mean ± SD, and 1 of 3 independent experiments
is shown in this figure.
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So far we have shown that LPS induces TNF- production and that
TNF- induces apoptosis in macrophages. LPS-induced secretion of
TNF- and apoptosis occurred almost simultaneously. Therefore, we
wanted to determine the role of the autocrine production of TNF- in
the LPS-induced apoptosis of BMDM. For these experiments, we used
macrophages from mice with both TNF- receptors disrupted by genetic
recombination (TNF-R KO).46,47 The data presented were
obtained from TNF-RI/II double KO mice. Although not shown, most
experiments were repeated with mice with the single type I TNF-
receptor disrupted, from which identical results were obtained.
Macrophages from the TNF-R KO mice did not undergo apoptosis on
exposure to TNF- (Figure 6A). Unlike
exogenous TNF-, LPS induced significant apoptosis in these
macrophages (Figure 6A). After 24 hours of LPS treatment, induction of
apoptosis in the TNF-R KO macrophages was only 36% lower than that
observed in the control mice. However, the time course of apoptosis
induction was very different. LPS-induced apoptosis within the first 6 hours in macrophages from normal mice, whereas induction of apoptosis in the TNF-R KO mice only started at 12 hours and increased up to 24 hours. These results suggest that in wild-type macrophages treated with
LPS, the autocrine production of TNF- is the major mediator of early
apoptosis.
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| Fig 6.
LPS-induced apoptosis in macrophages from TNF-R KO
mice.
(A) LPS, but not TNF-, induces apoptosis in macrophages from
TNF-R KO mice. Macrophages (105) from either wild-type
or TNF-R KO mice were treated for 24 hours with LPS (100 ng/mL) or
TNF- (100 ng/mL). DNA fragmentation was evaluated by measuring
histone-associated DNA fragments by ELISA. (B) Time course of
LPS-induced apoptosis in macrophages from TNF-R KO mice. Cells from
control and KO mice were treated with LPS (100 ng/mL) for the indicated
periods of time. Apoptosis was determined as indicated previously. (C)
Macrophages from TNF-R KO mice express iNOS in response to LPS.
Macrophages were treated with 100 ng/mL of LPS for the indicated times;
20 µg of total RNA per lane was analyzed by Northern blotting. (D)
Production of NO in macrophages from TNF-R KO mice. The production
of NO was measured in cultures of macrophages from each group of mice
stimulated with 100 ng/mL of LPS for the indicated times in the
presence or absence of 20 µmol/L SMT. (E) SMT blocks LPS-induced
apoptosis in macrophages from TNF-R KO mice but not in control
macrophages. Macrophages (105) from control and KO mice
were treated with LPS (100 ng/mL) for the indicated periods of time in
the presence or absence of SMT (20 µmol/L). Apoptosis was determined
by ELISA. Each experiment was performed in triplicate and represented
as the mean ± SD. (F) LPS-induced apoptosis in macrophages from
TNF-R KO mice is mediated by NO production. DNA fragmentation was
analyzed in a 2% agarose gel electrophoresis. Macrophages of each
group were treated with 100 ng/mL of LPS in the presence or absence of
either 20 µmol/L SMT (iNOS inhibitor), 50 µmol/L SNAP (NO donor),
or 100 ng/mL rmTNF-.
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Apoptosis mediated by autocrine TNF- or endogenous NO is
independent event in LPS-stimulated macrophages
To determine if the apoptosis mediated by the autocrine production
of TNF- is due to the production of NO, we studied the induction of
iNOS and the secretion of NO in macrophages from TNF-R KO mice. In
these macrophages, LPS induced a pattern of expression of iNOS mRNA and
NO production similar to that in control macrophages (Figure 6C-D).
Furthermore, treatment with SMT also inhibited the production of NO to
the same degree in both populations of macrophages (Figure 6D). These
results suggest that in BMDM the autocrine secretion of TNF- is not
involved in the control of NO production in response to LPS. Moreover,
the treatment with rmTNF- did not induce NO secretion in control
macrophages (data not shown).
The role of NO induction in late apoptosis was also tested by adding
the iNOS inhibitor SMT. In the presence of this inhibitor, the
LPS-induced apoptosis in macrophages from TNF-R KO mice decreased 79% (Figure 6E). In control macrophages treated with LPS for 24 hours,
SMT only resulted in a 23% reduction of apoptosis. These results were
also confirmed by agarose electrophoresis of the apoptotic DNA (Figure
6E). Therefore, these results suggest that TNF- secretion by LPS
plays a major role in the induction of early apoptosis, whereas NO
production induced by LPS only induces the late apoptotic events. In
control macrophages, LPS and TNF-, induced the typical DNA laddering
associated with apoptosis, which was not blocked by SMT. In contrast,
LPS, but not TNF-, induced DNA fragmentation in macrophages from
TNF-R KO mice. In this case, SMT totally blocked the effect of LPS,
thus suggesting that the LPS-induced apoptosis of TNF-R KO
macrophages was mediated only through NO production.
We also analyzed the apoptosis induced in macrophages from iNOS KO
mice. After LPS stimulation, these cells expressed similar levels of
TNF- mRNA (Figure 7A) and secreted
similar amounts of this cytokine than macrophages from wild-type or
TNF-R KO mice (Figure 7B). The apoptosis induced by LPS in
macrophages from iNOS KO mice was almost identical to that observed in
macrophages from control mice and followed similar kinetics (Figure
7C). Only a slight reduction of apoptosis was observed after 24 hours
of LPS treatment (26% reduction), at a time when NO-mediated apoptosis is significant in normal macrophages treated with LPS.
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| Fig 7.
LPS-induced apoptosis in macrophages from iNOS KO mice.
(A) LPS induces TNF- expression in iNOS KO macrophages. The
expression of iNOS and TNF- mRNA was analyzed by Northern blotting.
(B) LPS induces TNF- expression in iNOS KO macrophages. The
secretion of TNF- was analyzed by ELISA in macrophage cultures from
each group of mice. Each experiment was performed 3 times and
represented as the mean value ± SD. (C) LPS induced similar rates of
apoptosis in macrophages from iNOS KO and in control macrophages.
Macrophages (105) from control and KO mice were treated
with LPS (100 ng/mL) for the indicated periods of time in the presence
or absence of SMT (20 µmol/L). Apoptosis was determined by ELISA.
Each experiment was performed in triplicate and represented as the mean ± SD.
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Finally, we were interested in determining the signaling
mechanisms involved in the induction of macrophage apoptosis
by either TNF- or NO. Thus, we analyzed the expression of p53
and Bax, 2 genes involved in several apoptotic
processes.58-61 The expression of
p53 and Bax was detected after 24 hours of treatment with LPS, but not
after 6 hours (Figure 8A-B). Therefore,
the early apoptotic events (those that take place after 6 hours
of treatment with LPS) occurred in the absence of p53 and
Bax induction. However, the expression of these pro-apoptotic genes
took place at the time at which macrophage apoptosis is dependent on NO
production (24 hours of LPS stimulation). Therefore, we determined
whether the expression of these genes could be induced by
treatment with a NO donor or TNF- in
macrophages. Treatment with the NO donor SNAP, but not with
recombinant TNF-, induced the expression of p53 and Bax
(Figure 8C-D). These results show that the early
(TNF--dependent) and late (NO-dependent) mechanisms
induced by LPS to produce apoptosis follow 2 different
independent pathways.
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| Fig 8.
NO-dependent, but not TNF--dependent, LPS-induced
apoptosis was due to p53 and Bax expression.
Macrophages from control mice were treated with LPS (100 ng/mL) for the
indicated periods of time. Expression of p53 was analyzed by Western
blotting (A), whereas expression of Bax mRNA was analyzed by Northern
blotting (B). The expression of p53 (C) and Bax (D) was analyzed in
macrophages treated with either 50 µmol/L SNAP or 100 ng/mL rmTNF-
for 24 hours. Northern and Western blotting were performed as described
in the "Materials and methods" section.
|
|
| |
Discussion |
Endotoxic shock is a potentially lethal complication of
systemic infection by gram-negative bacteria.5,6 The toxin
responsible for the induction of endotoxic shock is the glycolipid LPS,
the major component of the gram-negative bacterial wall. The release of
LPS into the circulation activates a series of tissue responses that in
their most severe forms lead to septic shock and death. Tissue
macrophages play a major role in the generation of the endotoxic
response. NO production and TNF- secretion produced by these cells
have been proposed as the primary mediators of this event.
Although an enhanced generation of NO by iNOS has been
involved in the pathophysiology of septic shock,9,11 the
inhibition of iNOS in rodent and human models for sepsis and the
analysis of iNOS KO mice has produced conflicting
results.17-24 Moreover, the involvement of TNF-
secretion in septic shock came from observations that antibodies or
soluble receptors against TNF- inhibited the deleterious actions of
LPS during sepsis.27-29 However, the use of TNF-R KO
mice as an experimental model also gave contradictory results. Although
mice deficient for the p55 kd TNF-R seemed to be resistant to
endotoxic shock, they still succumbed to bacterial infection.40
We have analyzed the role of both NO and TNF- secretion in the
LPS-induced apoptosis in BMDM. Our results demonstrate that LPS induces
apoptosis in macrophages by 2 independent mechanisms: one is mediated
by the autocrine production of TNF- and the other is triggered by
the production of NO. Although both mechanisms are involved in the
apoptosis induced by LPS, they act independently, with different
kinetics, and through separate pathways (Figure 8).
The cytotoxic effects of TNF- in most cell types are only evident
when RNA or protein synthesis are inhibited, suggesting that de novo
RNA or protein synthesis protects cells from TNF- cytotoxicity,
probably by the induction of protective genes.62-64 In bone
marrow macrophages (as is the case in other inflammatory cell types,
including neutrophils and granulocytes or in endothelial cells and
oligodendrocytes), TNF- alone is sufficient to induce DNA
fragmentation and cell death by apoptosis.43,65-67
The TNF- signal is transduced by 2 distinct cell surface receptors,
TNF-RI and TNF-RII.32,34 In this work we have
reported the experiments performed with macrophages from the
TNF-RI/II double KO mice,47 but experiments using
macrophages from the TNF-RI KO mice46 produced identical
results. Thus, this confirms that Type I p55 TNF- receptor mediated
the TNF--induced apoptosis in macrophages.33,36,37
TNF- did not induce the expression of iNOS or NO production in
macrophages.68 Macrophages from TNF-R KO mice showed
levels of LPS-induced expression of iNOS and NO production similar to those measured in control macrophages. In fact, recombinant TNF- did
not induce NO production in macrophages from control mice. Moreover,
TNF--dependent apoptosis was not blocked by the iNOS inhibitor SMT
or in macrophages from iNOS KO mice. Therefore, we conclude that the
apoptosis induced by the autocrine production of TNF- is independent
of the production of NO. These results are in disagreement with other
previous observations69,70 in which TNF-
has been associated to the production of NO.
Other studies71-73 have clarified the mechanism by which
the 55 kDa TNF- receptor signals toward the apoptotic response. This receptor contains a carboxy-terminal death-domain that appears to be
required for the transmission of the apoptotic signal. Binding of
TNF- to the receptor triggers the formation of a multiprotein complex in which cytoplasmic proteins and the receptor interact through
their respective death-domain motifs. On TNF- stimulation, the
receptor death domain binds to the death domain of a cytoplasmic protein called TRADD (TNF receptor I-associated death domain), which in
turn binds to the death domain of another cytoplasmic protein, termed
FADD/MORT-1. This protein also contains a death effector domain (DED)
motif, which binds to the DED motif of ICE/Ced-3 protease
FLICE/MACH-1 (Caspase 8). It has been suggested that activation of
Caspase 8 initiates the activation of a cascade of caspases, which
is the effector system for the apoptotic destruction of the cell.
This model suggests that ligand binding to the TNF- receptor
activates the final death effector pathway apparently without any
second messengers.
Besides, the LPS-induced apoptosis that is mediated by the production
of NO occurs slowly and uses a different signaling pathway. In bone
marrow macrophages, NO-dependent apoptosis correlated with the
expression of p53 and Bax. Probably the DNA alterations induced by
increasing levels of NO induced the expression of p53.61 Moreover, the p53 expression induced by LPS in macrophages has been
observed that could be blocked by iNOS inhibitors, such as arginine
analogs.74 p53 regulates the transcription of the
Bcl-2-related pro-apoptotic gene Bax.75 The expression of
Bax is sufficient to produce the release of cytochrome C from the
mitochondria76 and the activation of the mitochondrial
apoptotic pathway,77,78 leading to apoptosis.
In summary, we have shown that LPS-induced apoptosis is
mediated mostly through the autocrine production of TNF-.
However, when this pathway is inhibited, the apoptosis induced by
LPS occurs through the induction of NO. The existence of 2 independent
pathways activated by LPS may explain the inefficiency of several
strategies directed against one of these mechanisms to prevent the
deleterious effects of LPS during endotoxic shock. This
finding underscores that salvage from ongoing
septic shock may require the simultaneous interruption of more than one
final pathway, each of them lethal for the host organism.
| |
Acknowledgments |
We thank Dr Antonio Felipe (University of Barcelona, Spain), Dr Ramon
Merino (University of Cantabria, Santander, Spain), and Dr M. Nabholz
(ISREC, Switzerland) for their gift of cDNAs used in this work. We are
also grateful to Dr K. Matsushima (University of Kanazawa, Japan) and
to Dr J. Peachon (University of Kentucky, USA) for the generous gift of
the TNF-R I and I/II double KO mice. We acknowledge the help
received from Jaume Comas and Rosario González from the flow
cytometry facility of the Serveis Científico Tècnics de
la Universitat de Barcelona. We also thank Martí Cullell-Young
for his editorial assistance.
| |
Footnotes |
Submitted October 21, 1999; accepted February 8, 2000.
Supported by grants from CICYT (SAF98-102 and PM 98/0200) to A.C. and
by the Deutsche Forschungsgemeinschaft to C.B. (SFB 263, A5). J.X. and
A.F.V. are recipients of a fellowship from the Comisió
Interdepartamental de Recerca i Innovació Tecnològica (CIRIT). M.C. is recipient of a fellowship from Fundació August Pi i Sunyer.
J.X. and M.C. contributed equally to this work.
Reprints: Antonio Celada, Departament de Fisiologia,
Facultat de Biologia, Av. Diagonal 645, 08028 Barcelona, Spain; e-mail:
acelada{at}bio.ub.es.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction