From the Cardiovascular Division, Brigham and Women's Hospital, Boston, Massachusetts 02115
Received for publication, October 6, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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Growing evidence from patients with heart failure
and from experimental animal models implicates effectors of innate
immunity in the pathogenesis of this syndrome. The expression of the
innate immunity signaling protein, Toll-like receptor 4 (TLR4), is
increased in cardiac myocytes in situ and in failing
myocardium, but the mechanism by which TLRs may be activated in the
failing heart remains unclear. We report that TLR2, which is expressed
in cardiac myocytes, participates in the response of these cells to
oxidative stress, a major contributor to the pathogenesis of cardiac
dysfunction. Hydrogen peroxide increased nuclear factor The mammalian toll-like receptors
(TLRs)1 are a recently
recognized family of proteins implicated in directing innate immune responses by components of diverse pathogens (1, 2). So far, six human
TLRs have been cloned (3, 4), and several others are suggested to be
present. Medzhitov et al. (5) were the first to show that
constitutively active TLR4 induces the activation of NF- Most studies have focused on TLR expression and function in mononuclear
cells and monocyte-like cell lines; however, TLRs differentially
exhibit a wider pattern of tissue expression, including the lung,
heart, and brain (2). Thus, TLRs may additionally serve as
pro-inflammatory receptors in cell types without a dedicated immune
function. TLR4, for example, is expressed by human dermal endothelial
cells and can activate NF- Oxidative stress in the post-ischemic heart has been identified as an
important event in myocardial dysfunction, linked to both myocardial
hypertrophy and apoptosis. Oxidative stress is known to induce cell
death by apoptotic and necrotic pathways in isolated cardiac myocytes
(20) and to trigger pro-inflammatory signaling pathways that activate
NF- Chemicals--
Human recombinant TNF Cell Lines, Cell Isolation, and Culture--
Chinese hamster
ovary K1 (CHO-K1) fibroblasts were obtained from D. T. Golenbock
(23). The cell lines stably express CD-14, an
NF-
Neonatal rat ventricular myocytes (NRVM) were isolated from 1-day-old
Harlan Sprague-Dawley pups as described (17). NRVM were cultured in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum
(Life Technologies, Inc.). After 48 h, the medium was changed to
Dulbecco's modified Eagle's medium /F-12 medium (Life Technologies,
Inc.) containing 1% insulin, transferrin, selenium media supplement
(ITS, Sigma) with antibiotics. Treatments were initiated 12 h later.
CHO fibroblasts and NRVM were pretreated with a TLR-2 blocking antibody
raised to a peptide that maps to the amino terminus of human TLR2 (5 or
10 µg/ml, number N-17, Santa Cruz Biotechnologies Inc., Santa Cruz,
CA) for 1 h prior to addition of experimental treatments.
RT-PCR--
Expression of rat TLR 1-6 were assessed using total
RNA by RT-PCR from NRVM after treatment with deoxyribonuclease 1 (Life Technologies, Inc.) as described in the manufacturer's protocol. cDNA was generated using Superscript II (Life Technologies, Inc.). As a negative control, no reverse transcriptase was added. PCR primers
were for TLR1 (383 bp) 5'-AAA CGG TCT CAT CCA CGT TC and 5'-GAG CAA TTG
GCA GCA CAC TA, for TLR 2 (298 bp) 5'-GGC CAG CAA ATT ACC TGT GT and
5'-TTC TCC ACC CAG TAG GCA TC, for TLR3 (469 bp) 5'-TGC CTT GGT CCC AAG
CCT TCA ACG A and 5'-TGG CCC GAA AAC CTT CTT CTC AAC GGA, for TLR4 and
the positive control (555 bp) 5'-CGC TTT CAG CTT TGC CTT CAT TAC and
5'-TGC TAC TTC CTT GTG CCC TGT GAG, for TLR5 (280 bp) 5'-AAG AGG GAA
ACC CCA CAG AA and 5'-GGG GAC TAA GCC TCA ACT CC, and for TLR6 (593 bp)
5'-GGG GCT TTC CTC TGT CTC TA and 5'-CCC AAG TTT CAA GTT TTC ACA T. All
PCR products were size-fractionated by a 2% agarose gel
electrophoresis, and DNA bands were visualized by staining the gel with
ethidium bromide.
Immunocytochemistry--
NRVMs were grown in chamber slides
(Nunc, Naperville, IL). Cells were fixed with 4% formaldehyde in
phosphate-buffered saline (PBS) for 10 min, washed, and exposed for 10 min to 0.3% Triton X-100 in PBS. After 10-min exposure to 0.1 M glycine in PBS, cells were blocked with 5% normal horse
serum (Vector Laboratories, Burlingame, CA). Cells were labeled by the
sequential application of the primary human TLR2 antibody, an anti-goat
FITC-conjugated secondary antibody, a second primary rat caveolin 3 antibody (Transduction Laboratories, Lexington, KY), and a secondary
Alexa 568-tagged anti-mouse antibody (Molecular Probes, Eugene, OR).
Images were taken with a confocal laser scanning microscope (model
MRC-1024, Bio-Rad Laboratories, Hercules, CA). The specificity of the
primary goat polyclonal anti-hTLR2 antibody was assessed by
preincubating the antibody with the hTLR2 matching peptide (Santa Cruz
Biotechnologies Inc.).
Western Blotting--
For Western blots, cells were harvested in
SDS sample buffer (62.5 mM Tris base, 2% SDS, 10%
glycerol, 50 mM dithiothreitol). After cell extracts were
boiled, equal amounts of the denatured protein per lane were loaded,
separated on a 7.5 or 15% SDS-polyacrylamide gel electrophoresis, and
transferred to polyvinylidene difluoride membranes. After blocking with
5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST),
membranes were incubated with anti-hTLR2 (1:500) or anti-p38/phospho
p38 (1:100, New England BioLabs, Boston, MA) overnight. After several
washes the membranes were incubated for 1 h with a matching
secondary antibody (Jackson Laboratories, Bar Harbor, ME; 1:10,000
dilution). After additional washes, membranes were incubated with a
chemiluminescent (Renaissance, PerkinElmer Life Sciences, Boston,
MA) and autoradiographed.
Flow Cytometric Analysis of NF- Electromobility Shift Assay--
Cells were treated as indicated
for 2 h following pretreatment with the blocking antibody for
1 h. Electromobility shift assays were performed as described
elsewhere (24). Binding reactions were performed with 10 µg of
nuclear protein. The binding reactions for AP-1 activity contained an
additional 5 mM MgCl2. Control reaction
mixtures contained a 100-fold excess of unlabeled oligonucleotide and
were incubated with nuclear extracts as indicated. DNA complexes were
separated on a 5% nondenaturing polyacrylamide gel in Tris-borate EDTA
buffer. NF- Flow Cytometry Analysis for the Detection of Sub-G1
and Phosphatidylserine--
FACS was used to detect cells with a
hypodiploid content of DNA and surface expression of
phosphatidylserine by staining with FITC-annexin V. NRVM were
collected by trypsinization, pooled with nonattached cells, and fixed
with ice-cold 70% ethanol/PBS. After being rinsed with PBS, cells were
incubated with a propidium iodide (20 µg/ml) solution containing
RNase A (5 Kunitz units/ml) at room temperature for 30 min. NRVM in the
sub-G1 region were identified using ModFit software,
and data are expressed as the percentage of cells in
sub-G1. For detection of surface phosphatidylserine, NRVM
were collected by trypsinization and directly stained with FITC-annexin
V, according to the manufacturer's direction
(CLONTECH, Palo Alto, CA). FACS analysis was
performed on 10,000 cells/sample using a FACscan cytometer (Becton Dickinson).
Statistical Analyses--
All replicate data are expressed as
mean and standard error of mean. In experiments with comparison of
treatments, an unpaired Student's t test was performed. In
experiments with time courses, analysis of variance was used followed
by Fisher's post-hoc test. Statistical significance was achieved when
p Expression Pattern of TLRs in Neonatal Rat Cardiac
Myocytes--
Six human TLR cDNAs have been identified (3, 4).
However, their distribution in cardiac tissue is unknown. TLR2, TLR3, TLR4, and TLR6 were detectable by reverse transcription-PCR in primary
NRVM cultures (Fig. 1a).
Signals for TLR1 and TLR5 were absent in cardiac tissue, whereas spleen
showed the expected TLR1 and TLR5 bands (data not shown).
We previously reported that TLR4 protein is expressed by cardiac
myocytes (17) but did not explore the expression of TLR2. Using a
polyclonal TLR2 antibody, a single band was detected in a
TLR2-overexpressing cell line (CHO/TLR2) but not in control (CHO/control) or TLR4-overexpressing (CHO/TLR4) cell lines. A corresponding band could be detected in lysates of neonatal cardiac myocytes (Fig. 1b). Immunocytochemical staining with the
same antibody confirmed the expression of TLR2 in myocytes (Fig.
1c). Primary isolates of neonatal rat ventricular myocytes
in vitro exhibited sarcolemmal membrane staining that was
inhibited by preincubation with the corresponding TLR2 peptide used to
generate the primary antibody. That TLR2-positive cells also expressed caveolin 3 verified their identity as cardiac myocytes.
TLR2 Enhances NF- TLR2 Is Involved in Nuclear Translocation of NF-
Oxidative stress is also known to rapidly induce the phosphorylation of
p38 MAPK in cardiac myocytes (26). However, blocking of TLR2 had no
effect on hydrogen peroxide-induced p38 phosphorylation after 15 min of
treatment (Fig. 3b).
An Anti-apoptotic Role of TLR2 after Oxidative Stress in Cardiac
Myocytes--
NF-
Annexin V has a strong, specific affinity for phosphatidylserine (PS).
Shortly after cells are induced into apoptosis, PS redistributes from
the inner to the outer layer of the cell membrane, thus providing a
readily detectable marker of dying cells (29). Basal levels of annexin
V-positive cells in these experiments were found to be high, averaging
~25%. Hydrogen peroxide stimulation had no effect at 30 min but
modestly increased annexin V staining of NRVM after 2 h
incubation, to 35%. As shown in Fig. 4b, blockade of TLR2
increased the response to hydrogen peroxide at 30 min (H2O2, 25% versus
H2O2+ The innate immunity system is an evolutionarily ancient and
conserved form of the immune system that is triggered by soluble and
membrane bound "pattern recognition receptors" (PRR). In the expanded self-nonself model of innate immunity, PRRs have evolved to
distinguish between dangerous and innocuous nonself antigens. Therefore, innate immunity recognizes invariant patterns
(i.e. pathogen-associated molecular patterns) shared by
groups of microorganisms but not by host tissues (30, 31). A typical
pathogen-associated molecular pattern would be bacterial
lipopolysaccharides (LPS), for example. However, Matzinger (19, 32) has
suggested a different explanation for function and triggers of the
innate immune system. In the "Danger" model, the immune system
would be activated by endogenous "alarm" or "danger" signals,
which originate from stressed, injured, or necrotic cells and not by
highly conserved structured motifs in pathogens. These danger signals
are recognized by the pattern recognition receptors and activate the
innate immune system. Therefore, PRRs of the innate immune system would
recognize specific epitopes or ligands that indicate injured, dying, or
dead cells (i.e. injured/dead self) even in the absence of
infection. Signals of "stressed" cells could be sensed by changes
in the lipid and/or carbohydrate moieties expressed on the surface of
cells or by the presence of proteins not normally found in the outer
cell membrane.
Increased myocardial expression of inflammatory cytokines, such as TNF,
IL-1 In this report, we provide evidence for a function of the TLR2
receptor, which is also expressed in unstimulated cardiac myocytes. Reactive oxygen species (ROS) are proposed to be a major pathogenic factor for hypoxic and ischemic damage in the heart. To confer oxidative stress in vitro, hydrogen peroxide was used in
neonatal rat ventricular myocytes. Several lines of evidence are
developed that document TLR2's role in the nuclear translocation of
NF- Oxidative stress has long been known to induce NF- What is the mechanism by which H2O2 activates
TLR2? It seems unlikely that H2O2 would
directly interact with TLR2 in a classical ligand/receptor complex.
However, hydrogen peroxide can modify membrane components and is a
known inducer of apoptosis and necrosis in cardiac myocytes (28, 37).
We suggest that cell death or injury could have caused the release of
one or more factors that interact with and activate TLR2. Such a
mechanism complements the delayed time course of
H2O2-induced NF- What function could H2O2-induced activation of
TLR have in cardiac myocytes? Nuclear translocation of NF- In summary, TLR2 participates importantly to the mechanism of hydrogen
peroxide-induced activation of NF-B
(NF-
B) activation in Chinese hamster ovary fibroblasts that
overexpress TLR2 but not in normal or TLR4-overexpressing Chinese
hamster ovary cells, an effect that was abrogated by an
-TLR2
antibody. In neonatal rat ventricular myocytes, the
-TLR2 antibody
inhibited hydrogen peroxide-induced nuclear translocation of NF-
B
and activator protein-1 (AP-1). Inhibition of TLR2 had no effect on
tumor necrosis factor
-induced NF-
B or AP-1 activation, on the
DNA binding of the basal transcription factor Oct-1, or on hydrogen
peroxide-induced phosphorylation of p38 MAP kinase. Importantly,
oxidative stress-induced cytotoxicity was enhanced by blocking TLR2.
Given the importance of cytotoxicity and apoptosis to the pathology
of the ischemic heart, an anti-apoptotic effect of TLR2 in cardiac
myocytes exposed to elevated levels of ROS may limit further cardiac dysfunction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B with the
subsequent expression of IL-1, IL-6, IL-8, and the costimulatory
molecule B7.1. Active TLR4 can also induce c-Jun
NH2-terminal kinase (JNK) (6). The role of TLRs as innate
immunity receptors has been extensively studied. TLR2, for example, is
activated by components of Gram-positive bacteria (7), mycobacteria (8,
9), and Borrelia (10). The most convincing evidence exists
for a role of TLR4 in the recognition of bacterial LPS
(lipopolysaccharides). Mice with either a spontaneous mutation of the
TLR4 gene or targeted disruption of the gene have no response to
lipopolysaccharides and are thus resistant to endotoxic shock (11-13).
In contrast, mice with targeted disruption of TLR2 have no deficit in
the recognition of Gram-negative bacteria (14). Interestingly, missense
mutations affecting the extracellular domain of the TLR4 receptor are
associated with a blunted response to inhaled LPS in humans (15).
B (16). We previously demonstrated (17)
that TLR4 is expressed in the heart and that injured human and murine
myocardium exhibit focal areas of intense TLR4 expression. The reason
for this differential expression of TLR4, as well as the function of
TLR4 in the injured heart, in the absence of infection, remains
unknown. Although the identity of endogenous ligands, analogous to
Drosophila spatzle, for the vertebrate TLRs remains elusive,
a recent report suggests that the stress-associated factor heat-shock
protein 60 signals through TLR4 (18). Thus, innate immunity receptors
may not only be activated by microorganisms, but also by endogenous
signals that originate from injured cells that emanate "danger
signals" (19).
B and AP-1 transcription factors (21, 22). Numerous mechanisms
are suggested to underlie these effects of hydrogen peroxide, which are
likely to be cell type-specific. Herein, we show a requirement of TLR2
for the activation of NF-
B and AP-1 by hydrogen peroxide and provide
evidence that TLR2 imparts an anti-apoptotic effect in stressed or
injured cardiac myocytes.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was purchased from
Endogen (Woburn, MA). All other chemicals, including hydrogen peroxide,
were purchased from Sigma Chemical Co. unless noted otherwise.
B-dependent CD25 reporter construct, and empty vector
(CHO/control), human TLR2 (CHO/TLR2), or human TLR4 (CHO/TLR4). The
cells were maintained as described (23). Cells were stimulated as
indicated in Ham's F-12 medium containing 10% fetal calf serum (Life
Technologies, Inc.).
B Activity--
CHO cells were
stimulated as indicated in Ham's F-12 medium supplemented with 10%
fetal calf serum for 24 h. Cells were harvested with trypsin-EDTA
and labeled with an FITC CD25 monoclonal antibody (Becton Dickinson
Immunocytometry Systems, San Jose, CA). Analysis was performed on
10,000 cells/sample using a FACscan.
B, AP-1, and Oct-1 oligonucleotides were purchased from
Santa Cruz Biotechnologies.
0.05. Statistical analyses were carried out using
StatView statistics program (Abacus Concepts, Inc., Berkley, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
TLR expression in neonatal rat ventricular
myocytes. a, TLR2, TLR3, TLR4, and TLR6 are
expressed in neonatal rat cardiac myocytes, measured by RT-PCR. No
signal could be detected for TLR1 and TLR5, although both were
detectable in spleen as a control tissue. The ± symbol
indicates the presence/absence of reverse transcriptase prior to
glyceraldehyde-3-phosphate dehydrogenase amplification. b,
polyclonal TLR2 antibody detected selectively TLR2 in
TLR2-overexpressing CHO fibroblasts but not in control or
TLR4-overexpressing cells. c, immunocytochemistry confirmed
TLR2 expression in neonatal rat cardiac myocytes (green
fluorescence). Caveolin 3 was used as a myocyte marker (red
fluorescence). Absorption with the peptide used to generate the
primary antibody was used as control.
B Activity after Stimulation with Hydrogen
Peroxide in a CHO Cell Line--
Reactive oxygen intermediates play a
key role in cardiac damage following ischemic injury. To investigate a
potential role of TLRs in the response to oxidative stress,
TLR-expressing CHO cell lines were used. CHO fibroblasts do not express
mRNA for full-length and functional TLR2 (25). The obtained cell
lines have previously been characterized (23) and overexpress CD14, inducible membrane CD25 under the control of an NF-
B promoter, and
empty vector (CHO/control), human TLR2 (CHO/TLR2), or human TLR4
(CHO/TLR4). Oxidative stress was induced by the application of hydrogen
peroxide (1 mM). The activity of NF-
B could be monitored by expression of membrane CD25 by flow cytometric analysis. Untreated cells showed nearly equal expression of CD25. TNF
increased CD25 expression evenly in the CHO/control, CHO/TLR2, and CHO/TLR4 lines after 24 h of treatment. However, application of hydrogen peroxide consistently increased NF-
B activity in the CHO/TLR2 cells to a
greater extent than in the CHO/control and CHO/TLR4 cell lines (Fig.
2a). In addition, hydrogen
peroxide increased nuclear translocation of NF-
B in the CHO/TLR2
line, but was ineffective in the CHO/control line after 2 h of
treatment, as measured by EMSA. There was no difference in the amount
of nuclear NF-
B for TNF
-treated cells (Fig. 2b).
Moreover, a 1-h preincubation with a TLR2 blocking antibody but not
control IgG (data not shown) completely abrogated the nuclear
translocation of NF-
B. The binding of Oct-1, a transcription factor
that is constitutively expressed, was unchanged by the treatment (Fig.
2c). CHO cells showed a high basal level of nuclear AP-1 by
EMSA without treatment, and was unchanged by preincubation with the
TLR2 antibody or treatment with hydrogen peroxide (data not shown).
These results suggest that TLR2 enhances hydrogen peroxide-induced
activation of NF-
B.
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Fig. 2.
TLR2 is involved in hydrogen peroxide-induced
NF- B activation in CHO fibroblast.
a, TLR2-overexpressing CHO cells showed an increase in
NF-
B activity, as measured by CD25 expression, when compared with
control and TLR4-overexpressing cells. No difference could be detected
in unstimulated or TNF
-stimulated cells. All experiments were done
in triplicate. b, electrophoretic mobility shift assays
could detect an increase in nuclear translocation of NF-
B after
stimulation with H2O2 (1 mM) in
TLR2-overexpressing CHO fibroblasts when compared with the control cell
line. c, NF-
B translocation could be suppressed by
pretreatment with a TLR2 blocking antibody, as measured by EMSA,
whereas DNA binding of the constitutively expressed transcription
factor Oct-1 was unchanged.
B after
Oxidative Stress in Neonatal Rat Cardiac Myocytes--
To test whether
TLR2 participates in the response to oxidative stress in cardiac
myocytes, NRVM were preincubated with the blocking TLR2 antibody for
1 h and then treated with hydrogen peroxide for another 2 h.
As for the CHO cells, the TLR2 blocking antibody could completely
abrogate the nuclear translocation of NF-
B (Fig.
3a), whereas preincubation
with control IgG was without effect. NF-
B activation by TNF
was
unchanged by treatment with the TLR2 blocking antibody. Similar to
NF-
B, AP-1 induction by hydrogen peroxide could be blocked by the
TLR2 blocking antibody, but not AP-1 induction by TNF-
. The
constitutively expressed Oct-1 was unchanged by the treatments.
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Fig. 3.
TLR2 is involved in hydrogen peroxide-induced
NF- B activation in neonatal rat ventricular
myocytes. a, pretreatment with a TLR2 blocking antibody
inhibited nuclear translocation of NF-
B and AP-1 after a 2-h
treatment with hydrogen peroxide (left panels) but had no
influence on the constitutively expressed transcription factor Oct-1 or
on nuclear translocation of NF-
B after treatment with TNF
(middle panels). A nonimmune IgG antibody (NI
IgG) did not affect hydrogen peroxide-induced NF-
B
translocation (right panel). b, hydrogen
peroxide-induced phosphorylation of p38 MAP kinase, shown in the
top panel using an antibody specific for phosphorylated p38
MAPK, is unaffected by the anti-TLR2 blocking antibody. The lower
panel shows equal amounts of p38 MAPK in each lane. All
experiments were done in triplicate.
B activation can elicit anti-apoptotic and
pro-apoptotic pathways in various cell types. Whether NF-
B promotes
or inhibits apoptosis seems dependent on the type of cell and the type
of inducer (27). Under the conditions of ischemic injury and oxidative stress, NF-
B appears to have an anti-apoptotic role in cardiac myocytes (28). To investigate the role of TLR2 in myocyte survival after oxidative stress, NRVM were preincubated with a blocking TLR2
antibody for 1 h and then treated with 0.5 mM hydrogen
peroxide. The fraction of myocytes with DNA fragmentation, measured as
the percentage of cells in sub-G1 phase, ranged from 6.9 to
10.9% under basal, serum-free conditions over the 6-h incubation.
Hydrogen peroxide increased this number to 12 ± 4.6% at 30 min,
to a maximum of 33.2 ± 5.9% at 6 h. Preincubation with the
TLR2 antibody further increased hydrogen peroxide-induced DNA
fragmentation at all time points examined, to a maximum of 40.5 ± 4.9% at 6 h. A nonimmune IgG was without effect (Fig.
4a) on the response to
hydrogen peroxide.
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Fig. 4.
TLR2 has an anti-apoptotic/cytotoxic
function. a, cells were treated with hydrogen
peroxide (0.5 mM) for the indicated times in the absence or
presence of TLR2 antibody or matched nonimmune antibody
(NI). 10,000 cells per experimental group were analyzed for
DNA fragmentation by quantitating the fraction of cells in
sub-G1 phase by FACS. Pretreatment with the TLR2 antibody
further increased hydrogen peroxide-induced DNA fragmentation.
NI denotes a nonimmune rabbit IgG. Data were pooled from
three to six separate experiments and are the mean sub-G1
populations. b, annexin V binding to NRVM following exposure
to hydrogen peroxide (0.5 mM) in the presence or absence of
TLR2 antibody or matched nonimmune antibody (NI IgG).
Results are representative of two experiments with similar results;
10,000 cells/experimental group were analyzed by FACS.
-TLR2 Ab, 65%), and at 2 h
(H2O2, 35% versus
H2O2+
-TLR2 Ab, 46%). Pretreatment with a
nonimmune IgG was without effect. Thus, blockade of TLR2 inhibits
hydrogen peroxide-induced NF-
B and AP-1 activity but enhances
hydrogen peroxide-induced DNA fragmentation and PS externalization in
NRVM, suggesting an anti-apoptotic function of TLR2 in cardiac myocytes
after oxidative stress.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and IL-6, occurs in human and experimental heart failure,
seemingly irrespective of initiating events and without evidence of
infection (33). Although there is a growing consensus that one or more
of these proteins contribute to maladaptive cardiac remodeling, the
proximal events that trigger and sustain their expression are not well
understood. However, each of these cytokines is now known to be related
to innate immunity, and their expression may be initiated through
activation of specific innate immunity receptors. We have previously
shown that TLR4 is expressed in cardiac myocytes and that
overexpression of dominant positive TLR4 activates NF-
B. In normal
murine and human myocardium, TLR4 expression was diffuse and
predominantly expressed in cardiac myocytes. However, in both
"remodeling" murine and human myocardium remote from sites of
ischemic injury and in heart tissue from patients with idiopathic
congestive heart failure, focal areas of intense TLR4 staining in
myocytes could be detected (17). Therefore, pattern recognition
receptors, as for example TLRs, may be important to trigger activation
of the innate immune system in congestive heart failure and may have an
important pathogenic role.
B after stimulation with hydrogen peroxide. First, hydrogen
peroxide increased NF-
B activity and nuclear translocation in CHO
cells that overexpress TLR2 but not in CHO cells that lack a functional TLR2 or that overexpress TLR4. Second, an anti-TLR2 antibody, but not a
nonimmune IgG, abrogated hydrogen peroxide-induced activation of
NF-
B and AP-1 in TLR2-overexpressing CHO cells and NRVM. Several experiments discount any nonspecific effect of the TLR2 antibody: 1) a
single immunoreactive band was present in NRVM that corresponded to a
band in CHO/TLR2 cell lysates, 2) TNF
-induced NF-
B and AP-1
activity was unaffected by the antibody, 3) DNA binding of the
constitutive transcription factor OCT-1 was unaffected, and 4) hydrogen
peroxide-induced phosphorylation of p38 MAPK was similarly unaffected
by the TLR2 antibody. Thus, from these criteria, we suggest that
hydrogen peroxide triggers NF-
B and AP-1 activation through a
mechanism that involves TLR2.
B and has been
thought to be crucial for NF-
B activation in the cell (reviewed in
Ref. 21). However, the recently characterized IL-1 and TNF pathways
activate NF-
B independent of ROS, and the activation of NF-
B by
ROS seems to be distinct from IL-1 and TNF. Moreover, different cell
types respond to H2O2 in different ways. The
kinetics for NF-
B activation by H2O2 are
slow and may therefore be characteristic for a secondary response of
NF-
B to agents that induce stress. An exact pathway for NF-
B
activation by ROS has not been characterized yet, but may involve
direct phosphorylation of tyrosine at position 42 on I
B
(reviewed
in Refs. 21, 34, 35). The current results suggest an alternative
pathway, whereby H2O2 activates a surface receptor, TLR2, which in turn triggers the signaling events that degrade I
B and activate NF-
B. The IL1R-like pathway utilized by
TLR2 to activate NF-
B has recently been reported (36).
B activation and is reminiscent of the "alarm" or "danger" signals that activate
innate immunity as outlined by Matzinger (19). In support of this, a
recent report demonstrated that endogenous heat shock protein 60 (HSP60) may function as a ligand for TLR4 (18). HSPs are known to
induce NF-
B (38). Of note, we could
demonstrate2 the release of
HSP60 in the supernatant of cardiac myocytes treated with
H2O2, thus verifying that
H2O2 can elicit release of a potential TLR
ligand in NRVM. Further experiments are necessary and currently underway to address this hypothesis.
B occurs
following activation of TLR2 (36). NF-
B then directs transcription
of a variety of genes, including those related to apoptosis (27). A
pro-apoptotic role of TLR2 has been proposed in a monocytic cell line
(39, 40). However, the function of NF-
B in cell death is highly
dependent on the type of cell as well as the type of inducer (27).
Blockade of TLR2 increased the rate of hydrogen peroxide-induced
apoptosis and injury in NRVM, based on two independent measures of
cytotoxicity and apoptosis, hypodiploidy and externalization of
phosphatidylserine. Therefore, TLR2 may have an anti-apoptotic function
in the heart. In heart failure as well as after myocardial infarction,
necrosis and apoptosis have a major pathogenetic role in the
development of ventricular dysfunction (41, 42). Considering that we
have seen increased levels of TLR expression specifically in the border
zones between viable and damaged myocardium after infarction
(17),2 one could speculate that activation of TLRs play a
role in the protection of ischemic but viable myocardium.
B and AP-1 in NRVM. We suggest
that activation of TLR2 is triggered by hydrogen peroxide-generated endogenous signals that may increase survival of cardiac myocytes after
oxidative stress.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. T. Golenbock for the gift of the CHO cell lines, and Dr. Min-Ying Pu for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft and by National Institutes of Health Grants HL52320 and HL36141.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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Cardiovascular
Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA
02115. Tel.: 617-732-5876; Fax: 617-732-5132; E-mail:
tbourcier@rics.bwh.harvard.edu.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M009160200
2 S. Frantz, R. A. Kelly, and T. Bourcier, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: TLR, toll-like receptor; IL-1, interleukin-1; JNK, c-Jun NH2-terminal kinase; LPS, lipopolysaccharide; AP-1, activator protein-1; TNF, tumor necrosis factor; CHO, Chinese hamster ovary; NRVM, neonatal rat ventricular myocyte; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair(s); PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorting; EMSA, electrophoretic mobility shift assay; MAPK, mitogen-activated protein kinase; PS, phosphatidylserine; Ab, antibody; PRR, pattern recognition receptor; ROS, reactive oxygen species; HSP, heat shock protein.
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