Vascular Biology
Aspirin Inhibits Chlamydia pneumoniae–Induced Nuclear
Factor-␬B Activation, Cytokine Expression, and Bacterial
Development in Human Endothelial Cells
Andreas Tiran, Hans-Jürgen Gruber, Wolfgang F. Graier, Andreas H. Wagner,
Ellen B.M. van Leeuwen, Beate Tiran
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Objective—Chlamydia pneumoniae has been associated with atherosclerosis. Infection of vascular endothelial cells with
C pneumoniae increases the expression of proatherogenic cytokines mediated by nuclear factor (NF)-␬B, a transcription
factor. The present study was designed to test the effect of aspirin on C pneumoniae–induced NF-␬B activation,
interleukin expression, and bacterial development in cultured human endothelial cells.
Methods and Results—Aspirin, its metabolite salicylic acid, and 2 other unrelated NF-␬B inhibitors showed a strong
concentration-dependent inhibitory effect on chlamydial growth, indicated by the reduction of bacterial inclusions and
the titer of infectious progeny. Involvement of the transcription factor NF-␬B was confirmed by electrophoretic mobility
shift assay and by transfection experiments with appropriate decoy oligodeoxynucleotides. Attenuation of the
C pneumoniae–induced activation of NF-␬B by aspirin also reduced the secretion of interleukin-6 and interleukin-8,
indicating efficient inhibition of NF-␬B gene expression. Reduction of chlamydial growth was not caused by apoptosis
of the host cell, as determined by monitoring characteristic chromatin condensation.
Conclusions—These data provide evidence that NF-␬B–mediated gene activation represents a crucial step in the
developmental cycle of C pneumoniae. Aspirin exerts an anti-chlamydial effect that is due to the inhibition of
C pneumoniae–induced NF-␬B activation, which might account for some of the cardioprotective activity of aspirin.
(Arterioscler Thromb Vasc Biol. 2002;22:1075-1080.)
Key Words: aspirin 䡲 transcription factor 䡲 Chlamydia pneumoniae 䡲 nuclear factor-␬B
terleukin (IL)-1, and tumor necrosis factor-␣.13 NF-␬B
dimers, most commonly composed of the RelA (p65) and
NF-␬B1 (p50) or NF-␬B2 (p52) subunits, are sequestered
in an inactive cytoplasmic complex by binding to its
inhibitory subunit, I␬B. Phosphorylation by I␬B kinase, a
cellular kinase complex, on the stimulation and subsequent
degradation of I␬B leads to translocation of NF-␬B to the
nucleus, where it binds to specific DNA sequences and
regulates the transcription of specific genes.14 Transcription factors of the NF-␬B/Rel family are critical for the
inducible expression of multiple genes involved in inflammatory responses and apoptosis.15
The present study was designed to elucidate whether the
activation of NF-␬B represents a crucial step in the
chlamydial growth cycle. The effect of aspirin and its
metabolite sodium salicylate (both known inhibitors of
NF-␬B)16,17 on C pneumoniae–induced NF-␬B activation,
cytokine expression, and bacterial growth cycle was assessed in human ECs.
T
he obligate intracellular bacterial pathogen Chlamydia
pneumoniae causes acute and chronic respiratory disease.1 Recently, there has been increasing evidence that
chronic or recurrent chlamydial infections of vessel walls
might contribute to atherogenesis and, possibly, to decreased plaque stability and acute coronary syndromes.2,3
However, the molecular mechanisms by which C pneumoniae might initiate or promote plaque development are
poorly understood. C pneumoniae is known to infect and
replicate in cell types found within the atherosclerotic
lesion, including endothelial cells (ECs), smooth muscle
cells, and macrophages.4 – 6 Infection of these cells results
in the increased expression of proinflammatory cytokines
and adhesion molecules.7–11 The activation of nuclear
factor (NF)-␬B in the host cell obviously plays a crucial
role in this bacteria-induced target cell activation.11,12
NF-␬B contains a family of cellular transcription factors
that get activated in response to a number of stimuli,
including bacterial lipopolysaccharide, phorbol esters, in-
Received February 17, 2002; revision accepted April 22, 2002.
From the Department of Laboratory Medicine (A.T., H-J.G., B.T) and the Institute of Medical Biochemistry and Medical Molecular Biology (W.F.G.),
University of Graz, Graz, Austria; the Department of Cardiovascular Physiology (A.H.W.), University of Goettingen, Goettingen, Germany; and the
Department of Haematology (E.B.M.v.L.), University of Groningen, Groningen, the Netherlands.
Correspondence to Andreas Tiran, Department of Laboratory Medicine, University of Graz, Auenbruggerplatz 15, A-8036 Graz, Austria. E-mail
[email protected]
© 2002 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
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DOI: 10.1161/01.ATV.0000022695.22369.BE
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Methods
Materials
Aspirin (acetylsalicylic acid), sodium salicylate, and pyrrolidine
dithiocarbamate (PDTC) were obtained from Sigma Chemical Co.
Bay-117082 (BAY), an inhibitor of I␬B-␣ phosphorylation, was
from Alexis.18
Cell Culture and Bacterial Strain
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Generation and characterization of the EC line EVLC2, a cell line
derived from human umbilical vein ECs by immortalization with
simian virus 40 large T antigen, is described elsewhere.19 ECV-304
(American Type Culture Collection [ATCC] CRL 1998) and HEp-2
(ATCC CCL 23) cells, 2 epithelial cell lines, were obtained from
ATCC. EVLC2, ECV-304, and Hep-2 cells were maintained in
RPMI 1640 medium, medium 199, and MEM-␣ medium, respectively (GIBCO-BRL). All media were supplemented with 2 mmol/L
L-glutamine, 10% FCS (PAA Laboratories), 1 mg/mL vancomycin
(ICN), and 1 mg/mL streptomycin sulfate (Sigma). For EVLC2 cells,
EC growth supplement (Technoclone) was added to a final concentration of 50 ␮g/mL. Cycloheximide (1 ␮g/mL) was added only for
the propagation of bacteria in Hep-2 cells. Normal human umbilical
vein ECs (HUVECs) were obtained from Technoclone and maintained in EC growth medium supplemented with EC growth supplement (Technoclone). Only cells up to the fourth passage were used
for experiments.
C pneumoniae strain 2023, a kind gift from Dr J. Ossewaarde
(National Institute of Public Health and the Environment, Bilthoven,
the Netherlands) was propagated and titrated in HEp-2 cell monolayers, as described previously.20
Growth and Staining of C pneumoniae
Cells were grown to near confluence in 12-well tissue culture plates.
One-day-old monolayers were infected with C pneumoniae by
centrifugation (1 hour, 10°C, 2400g) in a reduced volume of the
respective cell-specific growth medium. The titer of inocula used
was in the range of 1 to 5 inclusion-forming units (IFU) per cell,
resulting in a multiplicity of infection of 0.7 to 1. After infection, the
inocula were replaced by growth media containing the various test
drugs at different concentrations. In a series of experiments, a
possible effect of pretreatment on the outcome parameters investigated or an irreversible effect of the studied drugs was excluded (data
not shown). Media were replaced every 24 hours. Positive control
infections without added drug and negative controls with mockinfected cells were always run in parallel. Cultures were incubated
for 72 hours at 35°C in a humidified atmosphere containing 5% CO2.
Chlamydial inclusions were visualized by staining methanol-fixed
monolayers with a genus-specific fluorescein-labeled monoclonal
antibody (PathoDX, DPC). The effect of treatment on inclusion size
was quantitatively assessed by morphometry with the use of Optimas
6.51 image analysis software. The number of viable cells in the
monolayers was determined routinely by trypan blue dye exclusion
after detachment of the monolayers from the culture plates with
trypsin/EDTA to ensure that recorded results were not due to
changes in cell number after infection.
Determination of Inclusion Count and of Titer of
Infectious Progeny
The effect of inhibition of NF-␬B on chlamydial growth was defined
in terms of the reduction of visible inclusion bodies and in terms of
the decrease in infectious progeny titers. The reduction of inclusions
was expressed as percent inhibition, calculated as follows: [(inclusions of control)⫺(inclusions of treated sample)]/(inclusions of
control)⫻100.21 Inclusion counts represent average numbers of
inclusion bodies per field at a magnification of ⫻400 determined
from 10 fields per well from triplicate samples. To assess the
increase or decrease in infectious progeny titers, infected ECs and
their growth media were sonicated 72 hours after infection (hpi).
This suspension was diluted 4-fold with fresh medium and inoculated in triplicate on indicator HEp2 cells. Inclusions were visualized
and counted 72 hours later, as described above. Titers were expressed as IFU per milliliter.5
Deconvolution Microscopy
The effect of aspirin and other NF-␬B inhibitors on the morphology
of chlamydial inclusions was studied by high-resolution image
analysis with the use of deconvolution microscopy after staining with
a genus-specific fluorescein-labeled monoclonal antibody. Briefly, at
72 hpi, C pneumoniae–infected cells were washed twice with PBS
(pH 7.4) and fixed in methanol for 10 minutes. The cells were then
incubated for 30 minutes with the monoclonal antibody (PathoDX,
DPC) at a final dilution of 1:200 and counterstained with Evans blue.
The instrumental setup of the deconvolution microscope included
a liquid-cooled CCD camera (Quantix) on an inverted microscope
(Nikon Eclipse TE300) with a CFI Plan Fluor ⫻100 oil immersion
objective (numerical aperture 1.3, 0.068 ␮m/pixel, Nikon) in association with the Image Pro 3.0 software (Media Cybernetics).
Electrophoretic Mobility Shift Assay
For electrophoretic mobility shift assay (EMSA), cells were harvested at 6 hpi. Preparation of nuclear extracts, labeling, and binding
reaction were carried out as described previously.22 The doublestranded gel shift oligonucleotides for NF-kB, NF-kBmut and
activator protein (AP)-1, and the appropriate gel supershift antibodies were obtained from Santa Cruz Biotechnology. EMSAs were
repeated 3 times, and representative gels are shown.
Decoy ODN Technique
Double-stranded decoy oligodeoxynucleotides (ODNs) were prepared
from the complementary single-stranded phosphorothioate-bonded
ODNs (Eurogentec) by melting at 95°C for 5 minutes, followed by a
cool-down phase of 3 hours at ambient temperature.22 The efficiency of
the hybridization was checked on 2.5% agarose gels containing 0.1%
ethidium bromide and usually found to exceed 85%.
The double-stranded decoy ODN was added at an optimized final
concentration of 10 ␮mol/L to the growth medium after completion
of the infection of EVLC2 cells with C pneumoniae. The sequence of
the single-stranded ODN contained either an NF-␬B binding site
(5⬘-AGTTGAGGGGACTTTCCCAGGC-3⬘) or a mutated NF-␬B
binding site (5⬘-AGTTGAGGTGAGTTTCACAGGC-3⬘). Underlined letters denote phosphorothioate-bonded bases. Transfection of
the decoy ODN was achieved without using any cationic lipid or
liposomal complex.
Determination of IL-6 and IL-8 in Supernatant
To measure the cytokine secretion from individual monolayers, the
supernatant medium was removed at 48 hpi and stored at ⫺70°C
until tested. IL-6 and IL-8 were determined by enzyme immunoassay
with signal detection by chemiluminescence, as described by the
manufacturer (Quantiglo Immunoassay, R&D Systems).
Apoptosis
Induction of apoptosis was shown by fluorescent staining of nuclear
DNA with fluorescent bisbenzimide dye Hoechst 33342 (Calbiochem) at 12, 24, and 48 hpi with C pneumoniae. The cells grown on
glass coverslips in 12-well plates were washed twice with HBSS and
incubated with fixation buffer (5% formaldehyde in 145 mmol/L
NaCl and 10 mmol/L HEPES-KOH, pH 7.5) for 20 minutes at room
temperature. Then H33342 was added at a final concentration of 10
␮g/mL for an incubation period of 20 minutes. Thereafter, the buffer
was replaced by 50% glycerol in HBSS. Nuclear staining intensity
and morphology were evaluated optically. As a control experiment
for the test performance, EVLC2 cells were incubated for 6 to 8
hours with 2 ␮g/mL cycloheximide and 40 ng/mL tumor necrosis
factor (Boehringer Biochemicals). This treatment induced morphological changes typical of apoptosis in a high percentage of ECs (data
not shown). Nuclear morphology was assessed with the use of a
fluorescence microscope. At least 300 nuclei per sample were
counted in each of 4 independent experiments.
Tiran et al
Effects of Aspirin on Chlamydia pneumoniae
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Figure 1. Effect of aspirin, salicylic acid, and PDTC
on the morphology of C pneumoniae inclusions in
human ECs. EVLC2 cells infected with C pneumoniae (multiplicity of infection [MOI] 1) were
treated with the indicated concentrations of aspirin, salicylic acid, or PDTC or were left untreated
(control). At 72 hours after infection, monolayers
were stained with a genus-specific FITC-labeled
antibody and counterstained with Evans blue. The
cells were examined at ⫻400 magnification under
a conventional fluorescence microscope (top) or
by high-resolution image analysis (bottom) with the
use of deconvolution microscopy (see Methods).
Statistical Analysis
Statistical analysis was performed on a personal computer with use of
the SPSS software package (version 10.0). All values are expressed as
mean⫾SEM; IC50 values represent mean and 95% CIs. The Student
unpaired t test was used to determine differences between the means.
For comparison between multiple drug concentrations, data were analyzed by ANOVA and the Dunnett post hoc test. All tests were 2-sided,
and values of P⬍0.05 were considered significant.
Results
Effect of Aspirin on Chlamydial Growth
Figure 1 (top) shows infection of human EVLC2 cells at 72 hpi
by conventional fluorescence microscopy. Large intracellular
inclusion bodies are visible in the control infection; they contained actively replicating C pneumoniae. Treatment with a low
concentration of aspirin (0.5 mmol/L) exerted a significant effect
on the morphology of intracellular inclusion bodies. Inclusions
were smaller (median [25% to 75% percentile] of inclusion area
was 32 [16 to 49] ␮m2 versus 35 [19 to 53] ␮m2 in controls,
P⫽0.017), sometimes irregularly shaped, and often of dull
appearance. Sodium salicylate, PDTC, and BAY had similar
effects (data not shown). At higher concentrations, the effect on
the morphology of the inclusions became more distinct. Figure 1
(bottom) shows high-resolution images of C pneumoniae–in-
fected ECs at 72 hpi. A well-developed inclusion body is visible
in the control infection, whereas treatment with high concentrations of aspirin (2.5 mmol/L), salicylic acid (2.5 mmol/L), or
PDTC (25 ␮mol/L) resulted in small, barely stained, disintegrated inclusions. All these drugs showed a clear concentrationdependent inhibitory effect on chlamydial growth (P⬍0.001).
The number of inclusions and the titer in infectious progeny at
72 hpi were reduced significantly, as shown in Figure 2A and
2B. Inclusions were reduced, with IC50 values of 1.27 (0.88 to
1.83) mmol/L, 0.82 (0.71 to 0.96) mmol/L, and 25.3 (19.3 to
33.6) ␮mol/L for aspirin, salicylic acid, and PDTC, respectively.
The titer of infectious progeny was inhibited with similar
potency. This was not due to a direct toxic effect of the drugs on
the host cells because the percentage of dead cells in our
experiments did not differ between treated and untreated samples (Figure 2A). Essentially the same results were obtained in
HUVECs (please see online Figure I, available at http://atvb.ahajournals.org) and ECV-304 cells (data not shown).
Growth-Inhibiting Effects of Aspirin Are Specific
to NF-␬B Inhibition
The effect of the drugs on transcription factor activation was
investigated by EMSA. Results are shown in Figure 3. NF-␬B
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Figure 3. Effect of ASA, SSA, PDTC, and BAY on C pneumoniae–
induced NF-␬B activation in EVLC2 cells at 6 hpi, analyzed by
electromobility shift assay. Subunit composition of the NF-␬B binding activity by supershift (SS) with antibodies against p65 and p50,
by competition with AP-1 and unlabeled NF-␬B oligonucleotide,
and by binding to mutated NF-␬B binding site is demonstrated.
Aspirin Treatment Prevents C pneumoniae–
Induced IL Expression
Figure 2. Inhibition of chlamydial growth and decrease of infectious progeny titers by aspirin (ASA), sodium salicylate (SSA),
PDTC, and BAY in EVLC2 cells. A, EVLC2 cells were stained at
72 hpi with a genus-specific FITC-labeled antibody. Inclusions
were counted, and percent inhibition was calculated as
described in Methods. Mean⫾SEM values represent data of 5
independent experiments. B, EVLC2 cells were sonicated at 72
hpi, and growth titers were determined subsequently in HEp2
indicator cells to assess production of infectious progeny.
Results were expressed as IFU per milliliter and represent
mean⫾SEM of 3 independent experiments. *P⬍0.05 and
**P⬍0.001 vs control by Dunnett test.
activity was increased at 6 hours after C pneumoniae infection.
Only weak NF-␬B DNA binding activity was seen in mockinfected cells. Competitive inhibition by unlabeled NF-␬B oligonucleotide, but not by an AP-1 DNA binding site, and the
missing binding to the mutated NF-␬B site suggest specificity of
the interaction. Supershift experiments with p65 and p50 antibodies showed that these components of the NF-␬B family were
activated by C pneumoniae. All investigated drugs reduced
activation of NF-␬B in infected cells to a level comparable to
that of mock-infected control cells. To further support the
finding that NF-␬B may contribute to the life cycle of chlamydial infection, the decoy ODN technique was used. Transfection
of the infected ECs with the NF-␬B–specific decoy ODN
inhibited chlamydial growth by 50⫾2.6% (P⬍0.001), whereas
the mutated decoy ODN had no effect on chlamydial growth
(1⫾2.3% inhibition, P⫽NS). These data represent mean⫾SD
values of 3 independent experiments.
To confirm the ability of aspirin in the functional inhibition of
NF-␬B activation in infected ECs, we next investigated the
effect of aspirin on the expression of IL-6 and IL-8, with both
under the transcriptional control of NF-␬B.
It has been shown previously that infection of ECs with C
pneumoniae induces secretion of IL-6 and IL-8, that maximal
cytokine induction occurs by 48 hpi, and that it is sustained for
the entire 72-hour study period.11 Figure 4 shows IL-6 and IL-8
concentrations in the supernatant of EC cultures at 48 hpi. C
pneumoniae infection induced IL-6 and IL-8 concentrations
nearly 3- and 6-fold, respectively. Treatment of cell cultures with
aspirin or sodium salicylate reduced IL-6 and IL-8 levels in a
dose-dependent manner.
Inhibition of Chlamydial Growth Is Not Caused
by Apoptosis
To investigate whether the inhibition of chlamydial growth
was indeed related to reduced bacterial proliferation or
whether the cells were driven to apoptosis, chromatin condensation was assessed by staining with the nuclear dye
Hoechst 33342 in ECs with and without treatment with the
various investigated drugs. The results for the highest drug
concentrations at 12, 24, and 48 hpi are shown in the online
Table (which can be accessed at http://atvb.ahajournals.org).
The percentage of cells with nuclear changes indicative of
apoptosis ranged between 6% and 15%, with a tendency
toward higher rates at later time points. Compared with the
untreated control cells, in the treated cells no significantly
increased rates of apoptotic cells were seen after treatment
with aspirin, salicylic acid, BAY, or decoy ODN. In contrast,
treatment with PDTC efficiently induced apoptosis in up to
60% of cells at the highest concentration of 50 ␮mol/L only
but not at concentrations ⱕ25 ␮mol/L. These data suggest
that the described concentration-dependent effect of aspirin
and the other inhibitors of NF-␬B is not caused by apoptosis
of the host cells.
Tiran et al
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Figure 4. Effect of aspirin and salicylic acid on C pneumoniae–
induced IL-6 and IL-8 expression of EVLC2. The cell supernatant was removed at 48 hpi. IL-6 and IL-8 were determined by
enzyme immunoassay. Mean⫾SEM values represent data of
triplicates. The experiment was repeated once.
Discussion
The present study demonstrates that aspirin and its metabolite
salicylic acid counteract the intracellular development and
maturation of chlamydial inclusions indicated by the reduction of bacterial inclusions and the titer of infectious progeny.
The clear concentration-response relationship indicates a
yet-unknown effect of these substances on chlamydial
growth. The inhibitory effect of aspirin/salicylic acid on
chlamydial development was accompanied by morphological
changes of the inclusions that became smaller and less
uniformly shaped. Our data suggest that the intracellular
growth of C pneumoniae is mediated by an activation of the
transcription factor NF-␬B. These findings apply not only to
a single cell line but also to various endothelial and epithelial
cell lines and to cultured HUVECs. It is tempting to speculate
that the bacterium has evolved to benefit from cellular
mechanism of gene activation to promote its own replication.
Our data suggest that the effect of aspirin/salicylic acid on
chlamydial growth is caused, at least in part, by its capacity to
inhibit NF-␬B.17 However, aspirin is a drug with many known
mechanisms of action.23 First of all, aspirin is known to irreversibly
inhibit cyclooxygenase (COX) by acetylation. Our findings that
sodium salicylate, which lacks the respective acetyl group, mimics
the inhibitory properties of aspirin on chlamydial growth indicate
that the observed effect is not mediated by COX inhibition.
Recently, it has been proposed that salicylates exert part of their
antiinflammatory action by the suppression of COX gene transcription.24 However, because PDTC and BAY, structurally unrelated
inhibitors of NF-␬B,18,25 had anti-chlamydial effects similar to those
Effects of Aspirin on Chlamydia pneumoniae
1079
of the salicylates, it seems unlikely that the inhibitory effect of
salicylates on chlamydial growth is mediated primarily by the
suppression of COX gene transcription.
Because most of the chemical inhibitors lack exclusive specificity for NF-␬B inhibition and also affect the activity of other
transcription factors and signaling cascades,26 we used a highly
specific decoy ODN technique22 to verify that NF-␬B is causally
involved in the observed inhibition of chlamydial growth. Indeed,
reduction of chlamydial inclusions by 50% occurred after transfection of ECs with the consensus decoy ODN but not when the
corresponding mutant decoy ODN was used. These data strongly
suggest that activation of NF-␬B constitutes an essential part of the
host-pathogen relationship, with important implications for chlamydial growth. However, we cannot completely exclude the possibility that the effect of aspirin on chlamydial growth also involves
other cellular signal transduction pathways. Besides its direct inhibitory effect on NF-␬B, aspirin is known to be a scavenger of reactive
oxygen species, which are involved as intracellular signal transduction messengers in the regulation of many transcription factors.27
Therefore, part of the effects of aspirin that we have observed in this
investigation might be due to this activity. Moreover, salicylates
might also exert an additional direct toxic effect on the chlamydial
developmental cycle that has not yet been recognized. Further
studies are necessary to elucidate these issues.
In the next step, we investigated whether treatment of ECs
with aspirin resulted in a decreased expression of genes under
control of NF-␬B to prove efficient NF-␬B inhibition at the
functional level. For this purpose, we determined the secretion of
IL-6 and IL-8 into the supernatant medium. Both have been
shown previously to be under the regulatory control of NF␬B28,29 and to be upregulated after the infection of ECs with C
pneumoniae.9 –11 As with chlamydial growth, treatment with
aspirin/salicylic acid resulted in a similar concentrationdependent inhibition of the secretion of IL-6 and IL-8 (Figure 4).
We interpret this finding as another strong indication supporting
the hypothesis that the growth inhibition of C pneumoniae by
aspirin is caused, at least partly, by inhibition of NF-␬B.
Recent studies have broadened the role of NF-␬B from that of a
regulator of immune response and inflammatory responses to that of
a regulator of apoptosis and cell growth.15 Therefore, we wondered
whether the growth-inhibitory effect of aspirin reflected apoptotic
cell death of the host cell rather than attenuation of chlamydial
proliferation. This idea was supported by a recent study by Wahl et
al,30 who showed that activation of NF-␬B in C pneumoniae–
infected macrophages is associated with protection of these cells
against apoptosis. When investigating the effect of treatment with
aspirin and the other drugs on cell viability or the occurrence of cells
with nuclear changes characteristic of apoptosis, we did not find any
indication of a toxic or proapoptotic effect of these drugs (Figure 2
and online Table). The only exception was PDTC at ⬎25 ␮mol/L,
concentrations for which signs of induction of apoptosis were
detected in a high percentage of cells, whereas the same cells were
still classified as viable in the dye exclusion test. PDTC is known to
be an unspecific inhibitor of NF-␬B but a potent scavenger of
reactive oxygen species with a high potential to induce apoptosis.25,31 Therefore, we conclude that the described growth-inhibiting
effect of aspirin and the other inhibitors of NF-␬B was not due to
increased rates of necrosis or apoptosis of the host cells. This is
consistent with other studies concerning the antiapoptotic activity
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conferred to the host cell on infection with C pneumoniae, resulting
in the conclusion that no NF-␬B–dependent cellular factors were
involved in the protection against apoptosis.32–34
The presented results show that aspirin at concentrations as
low as 10⫺4 mol/L markedly decreased chlamydial growth.
These findings may be of pharmacological relevance in vivo,
inasmuch as serum concentrations of 10⫺4 to 10⫺5 mol/L are
commonly achieved by therapeutic doses of aspirin in humans.23
In coronary heart disease, aspirin has proven to reduce morbidity
and mortality in primary prevention and secondary intervention.
In addition to its antithrombotic effects, other mechanisms may
contribute to these clinical benefits of aspirin.23 Therefore, it is
tempting to speculate that mechanisms such as described in the
present investigation play a role.
Taken together, these data strongly suggest that C pneumoniae has developed a strategy to exploit host cell biology to
promote its own development. Our findings suggest that activation of NF-␬B plays a pivotal role in the developmental cycle of
C pneumoniae and that aspirin and other inhibitors of NF-␬B
activation might have previously unrecognized anti-chlamydial
effects. The identification of NF-␬B as a critical transcription
factor in C pneumoniae infection may provide insights into
specific therapeutic strategies for the treatment of diseases
associated with this organism.
Acknowledgments
This study was supported by grants from the Austrian Science Funds
(P13401-Med, SFB 714). We thank Dr Oleksiy Tsybrovskyy for the
performance of morphometric measurements and Nicole Friedl and
Elisabeth Winter for their excellent technical assistance.
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Aspirin Inhibits Chlamydia pneumoniae−Induced Nuclear Factor-κB Activation, Cytokine
Expression, and Bacterial Development in Human Endothelial Cells
Andreas Tiran, Hans-Jürgen Gruber, Wolfgang F. Graier, Andreas H. Wagner, Ellen B.M. van
Leeuwen and Beate Tiran
Arterioscler Thromb Vasc Biol. 2002;22:1075-1080; originally published online May 23, 2002;
doi: 10.1161/01.ATV.0000022695.22369.BE
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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TABLE. Effect of treatment of C. pneumoniae infected endothelial cells with
various inhibitors of NF-κB on the percentage of apoptotic cells*
% cells (SD) with apoptotic nuclear morphology
Inhibitor
12 hpi
24 hpi
48 hpi
untreated
8 (4.5)
8 (0.9)
12 (3.5)
Aspirin (5 mmol/L)
8 (2.7)
8 (2.6)
13 (1.6)
Salicylic acid (5 mmol/L)
7 (4.8)
7 (2.2)
10 (1.9)
Bay 117082 (4 µmol/L)
8 (3.7)
8 (1.2)
13 (1.5)
PDTC (25 µmol/L)
9 (4.2)
12 (1.3)
15 (3.0)
PDTC (50 µmol/L)
14 (1.9)†
48 (1.9)‡
60 (5.7)‡
NF-κB decoy ODN
8 (2.9)
9 (1.6)
15 (1.9)
NF-κBmut decoy ODN
6 (1.8)
8 (1.7)
15 (3.0)
*C. pneumoniae infected EVCL2 cells were incubated in medium containing the
various inhibitors or were left untreated as a control. At the indicated time points,
cells were stained with Hoechst 33342 and nuclear morphology was assessed by
fluorescence microscopy. At least 300 nuclei per sample were counted. Mean
percentages represent data from 4 independent experiments. † P<0.05, ‡ P<0.001 by
Student’s t test as compared to the untreated control.
Inhibition of chlamydial
growth(%)
A
*
100
75
* *
*
*
*
50
25
0
ASA (mmol/L)
SSA (mmol/L)
PDTC (µmol/L)
- 0.1 0.5 2.5 5.0 - - - - - - - - - - - - 0.1 0.5 2.5 5.0 - - - - - - - - - - - - 5.0 12 25 50
% dead cells
6
5
7
6
7
9
3
5
11
7
9
3
11
B
IFU/ml (x 10 4)
75
50
25
0
ASA (mmol/L)
SSA (mmol/L)
PDTC (µmol/L)
*
*
* *
* *
- 0.1 0.5 2.5 5.0 - - - - - - - - - - - - 0.1 0.5 2.5 5.0 - - - - - - - - - - - - 5.0 12 25 50
Figure I. Inhibition of chlamydial growth and decrease of infectious progeny titers by aspirin (ASA), sodium
salicylate (SSA), and PDTC in HUVECs. A, HUVECs were stained at 72 hpi with a genus-specific FITClabeled antibody. Inclusions were counted and percent inhibition was calculated as given in “Methods”.
Means + SEM represent data of 2 independent experiments. B, HUVECs were sonicated at 72 hpi and
growth titers were determined subsequently in HEp2 indicator cells to assess production of infectious
progeny. Results were expressed as inclusion forming units (IFU) per milliliter and represent means + SEM
of 2 independent experiments. *P<0.05, by Dunnett’s test vs control.