Aspirin Inhibits Chlamydia pneumoniae–Induced NuclearFactor-�B Activation, Cytokine Expression, and Bacterial

Development in Human Endothelial CellsAndreas Tiran, Hans-Jürgen Gruber, Wolfgang F. Graier, Andreas H. Wagner,

Ellen B.M. van Leeuwen, Beate Tiran

Objective—Chlamydia pneumoniae has been associated with atherosclerosis. Infection of vascular endothelial cells withC pneumoniae increases the expression of proatherogenic cytokines mediated by nuclear factor (NF)-�B, a transcriptionfactor. 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 strongconcentration-dependent inhibitory effect on chlamydial growth, indicated by the reduction of bacterial inclusions andthe titer of infectious progeny. Involvement of the transcription factor NF-�B was confirmed by electrophoretic mobilityshift assay and by transfection experiments with appropriate decoy oligodeoxynucleotides. Attenuation of theC 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 apoptosisof 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 thedevelopmental cycle of C pneumoniae. Aspirin exerts an anti-chlamydial effect that is due to the inhibition ofC 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 � transcription factor � Chlamydia pneumoniae � nuclear factor-�B

The obligate intracellular bacterial pathogen Chlamydiapneumoniae causes acute and chronic respiratory dis-

ease.1 Recently, there has been increasing evidence thatchronic or recurrent chlamydial infections of vessel wallsmight contribute to atherogenesis and, possibly, to de-creased plaque stability and acute coronary syndromes.2,3

However, the molecular mechanisms by which C pneu-moniae might initiate or promote plaque development arepoorly understood. C pneumoniae is known to infect andreplicate in cell types found within the atheroscleroticlesion, including endothelial cells (ECs), smooth musclecells, and macrophages.4 – 6 Infection of these cells resultsin the increased expression of proinflammatory cytokinesand adhesion molecules.7–11 The activation of nuclearfactor (NF)-�B in the host cell obviously plays a crucialrole in this bacteria-induced target cell activation.11,12

NF-�B contains a family of cellular transcription factorsthat get activated in response to a number of stimuli,including bacterial lipopolysaccharide, phorbol esters, in-

terleukin (IL)-1, and tumor necrosis factor-�.13 NF-�Bdimers, most commonly composed of the RelA (p65) andNF-�B1 (p50) or NF-�B2 (p52) subunits, are sequesteredin an inactive cytoplasmic complex by binding to itsinhibitory subunit, I�B. Phosphorylation by I�B kinase, acellular kinase complex, on the stimulation and subsequentdegradation of I�B leads to translocation of NF-�B to thenucleus, where it binds to specific DNA sequences andregulates the transcription of specific genes.14 Transcrip-tion factors of the NF-�B/Rel family are critical for theinducible expression of multiple genes involved in inflam-matory responses and apoptosis.15

The present study was designed to elucidate whether theactivation of NF-�B represents a crucial step in thechlamydial growth cycle. The effect of aspirin and itsmetabolite sodium salicylate (both known inhibitors ofNF-�B)16,17 on C pneumoniae–induced NF-�B activation,cytokine expression, and bacterial growth cycle was as-sessed in human ECs.

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 theDepartment 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-mailandreas.tiran@uni-graz.at

© 2002 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000022695.22369.BE

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MethodsMaterialsAspirin (acetylsalicylic acid), sodium salicylate, and pyrrolidinedithiocarbamate (PDTC) were obtained from Sigma Chemical Co.Bay-117082 (BAY), an inhibitor of I�B-� phosphorylation, wasfrom Alexis.18

Cell Culture and Bacterial StrainGeneration and characterization of the EC line EVLC2, a cell linederived from human umbilical vein ECs by immortalization withsimian 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 fromATCC. EVLC2, ECV-304, and Hep-2 cells were maintained inRPMI 1640 medium, medium 199, and MEM-� medium, respec-tively (GIBCO-BRL). All media were supplemented with 2 mmol/LL-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 concen-tration of 50 �g/mL. Cycloheximide (1 �g/mL) was added only forthe propagation of bacteria in Hep-2 cells. Normal human umbilicalvein ECs (HUVECs) were obtained from Technoclone and main-tained in EC growth medium supplemented with EC growth supple-ment (Technoclone). Only cells up to the fourth passage were usedfor 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 mono-layers, as described previously.20

Growth and Staining of C pneumoniaeCells were grown to near confluence in 12-well tissue culture plates.One-day-old monolayers were infected with C pneumoniae bycentrifugation (1 hour, 10°C, 2400g) in a reduced volume of therespective cell-specific growth medium. The titer of inocula usedwas 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, theinocula were replaced by growth media containing the various testdrugs at different concentrations. In a series of experiments, apossible effect of pretreatment on the outcome parameters investi-gated or an irreversible effect of the studied drugs was excluded (datanot shown). Media were replaced every 24 hours. Positive controlinfections without added drug and negative controls with mock-infected cells were always run in parallel. Cultures were incubatedfor 72 hours at 35°C in a humidified atmosphere containing 5% CO2.Chlamydial inclusions were visualized by staining methanol-fixedmonolayers with a genus-specific fluorescein-labeled monoclonalantibody (PathoDX, DPC). The effect of treatment on inclusion sizewas quantitatively assessed by morphometry with the use of Optimas6.51 image analysis software. The number of viable cells in themonolayers was determined routinely by trypan blue dye exclusionafter detachment of the monolayers from the culture plates withtrypsin/EDTA to ensure that recorded results were not due tochanges in cell number after infection.

Determination of Inclusion Count and of Titer ofInfectious ProgenyThe effect of inhibition of NF-�B on chlamydial growth was definedin terms of the reduction of visible inclusion bodies and in terms ofthe decrease in infectious progeny titers. The reduction of inclusionswas expressed as percent inhibition, calculated as follows: [(inclu-sions of control)�(inclusions of treated sample)]/(inclusions ofcontrol)�100.21 Inclusion counts represent average numbers ofinclusion bodies per field at a magnification of �400 determinedfrom 10 fields per well from triplicate samples. To assess theincrease or decrease in infectious progeny titers, infected ECs andtheir growth media were sonicated 72 hours after infection (hpi).This suspension was diluted 4-fold with fresh medium and inocu-lated in triplicate on indicator HEp2 cells. Inclusions were visualized

and counted 72 hours later, as described above. Titers were ex-pressed as IFU per milliliter.5

Deconvolution MicroscopyThe effect of aspirin and other NF-�B inhibitors on the morphologyof chlamydial inclusions was studied by high-resolution imageanalysis with the use of deconvolution microscopy after staining witha genus-specific fluorescein-labeled monoclonal antibody. Briefly, at72 hpi, C pneumoniae–infected cells were washed twice with PBS(pH 7.4) and fixed in methanol for 10 minutes. The cells were thenincubated 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 includeda liquid-cooled CCD camera (Quantix) on an inverted microscope(Nikon Eclipse TE300) with a CFI Plan Fluor �100 oil immersionobjective (numerical aperture 1.3, 0.068 �m/pixel, Nikon) in asso-ciation with the Image Pro 3.0 software (Media Cybernetics).

Electrophoretic Mobility Shift AssayFor electrophoretic mobility shift assay (EMSA), cells were har-vested at 6 hpi. Preparation of nuclear extracts, labeling, and bindingreaction were carried out as described previously.22 The double-stranded gel shift oligonucleotides for NF-kB, NF-kBmut andactivator protein (AP)-1, and the appropriate gel supershift antibod-ies were obtained from Santa Cruz Biotechnology. EMSAs wererepeated 3 times, and representative gels are shown.

Decoy ODN TechniqueDouble-stranded decoy oligodeoxynucleotides (ODNs) were preparedfrom the complementary single-stranded phosphorothioate-bondedODNs (Eurogentec) by melting at 95°C for 5 minutes, followed by acool-down phase of 3 hours at ambient temperature.22 The efficiency ofthe 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 finalconcentration of 10 �mol/L to the growth medium after completionof the infection of EVLC2 cells with C pneumoniae. The sequence ofthe single-stranded ODN contained either an NF-�B binding site(5�-AGTTGAGGGGACTTTCCCAGGC-3�) or a mutated NF-�Bbinding site (5�-AGTTGAGGTGAGTTTCACAGGC-3�). Under-lined letters denote phosphorothioate-bonded bases. Transfection ofthe decoy ODN was achieved without using any cationic lipid orliposomal complex.

Determination of IL-6 and IL-8 in SupernatantTo measure the cytokine secretion from individual monolayers, thesupernatant medium was removed at 48 hpi and stored at �70°Cuntil tested. IL-6 and IL-8 were determined by enzyme immunoassaywith signal detection by chemiluminescence, as described by themanufacturer (Quantiglo Immunoassay, R&D Systems).

ApoptosisInduction of apoptosis was shown by fluorescent staining of nuclearDNA with fluorescent bisbenzimide dye Hoechst 33342 (Calbio-chem) at 12, 24, and 48 hpi with C pneumoniae. The cells grown onglass coverslips in 12-well plates were washed twice with HBSS andincubated with fixation buffer (5% formaldehyde in 145 mmol/LNaCl and 10 mmol/L HEPES-KOH, pH 7.5) for 20 minutes at roomtemperature. Then H33342 was added at a final concentration of 10�g/mL for an incubation period of 20 minutes. Thereafter, the bufferwas replaced by 50% glycerol in HBSS. Nuclear staining intensityand morphology were evaluated optically. As a control experimentfor the test performance, EVLC2 cells were incubated for 6 to 8hours with 2 �g/mL cycloheximide and 40 ng/mL tumor necrosisfactor (Boehringer Biochemicals). This treatment induced morpho-logical changes typical of apoptosis in a high percentage of ECs (datanot shown). Nuclear morphology was assessed with the use of afluorescence microscope. At least 300 nuclei per sample werecounted in each of 4 independent experiments.

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Statistical AnalysisStatistical analysis was performed on a personal computer with use ofthe SPSS software package (version 10.0). All values are expressed asmean�SEM; IC50 values represent mean and 95% CIs. The Studentunpaired t test was used to determine differences between the means.For comparison between multiple drug concentrations, data were ana-lyzed by ANOVA and the Dunnett post hoc test. All tests were 2-sided,and values of P�0.05 were considered significant.

ResultsEffect of Aspirin on Chlamydial GrowthFigure 1 (top) shows infection of human EVLC2 cells at 72 hpiby conventional fluorescence microscopy. Large intracellularinclusion bodies are visible in the control infection; they con-tained actively replicating C pneumoniae. Treatment with a lowconcentration of aspirin (0.5 mmol/L) exerted a significant effecton the morphology of intracellular inclusion bodies. Inclusionswere smaller (median [25% to 75% percentile] of inclusion areawas 32 [16 to 49] �m2 versus 35 [19 to 53] �m2 in controls,P�0.017), sometimes irregularly shaped, and often of dullappearance. Sodium salicylate, PDTC, and BAY had similareffects (data not shown). At higher concentrations, the effect onthe 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 visiblein the control infection, whereas treatment with high concentra-tions of aspirin (2.5 mmol/L), salicylic acid (2.5 mmol/L), orPDTC (25 �mol/L) resulted in small, barely stained, disinte-grated inclusions. All these drugs showed a clear concentration-dependent inhibitory effect on chlamydial growth (P�0.001).The number of inclusions and the titer in infectious progeny at72 hpi were reduced significantly, as shown in Figure 2A and2B. Inclusions were reduced, with IC50 values of 1.27 (0.88 to1.83) mmol/L, 0.82 (0.71 to 0.96) mmol/L, and 25.3 (19.3 to33.6) �mol/L for aspirin, salicylic acid, and PDTC, respectively.The titer of infectious progeny was inhibited with similarpotency. This was not due to a direct toxic effect of the drugs onthe host cells because the percentage of dead cells in ourexperiments did not differ between treated and untreated sam-ples (Figure 2A). Essentially the same results were obtained inHUVECs (please see online Figure I, available at http://atvb.a-hajournals.org) and ECV-304 cells (data not shown).

Growth-Inhibiting Effects of Aspirin Are Specificto NF-�B InhibitionThe effect of the drugs on transcription factor activation wasinvestigated by EMSA. Results are shown in Figure 3. NF-�B

Figure 1. Effect of aspirin, salicylic acid, and PDTCon the morphology of C pneumoniae inclusions inhuman ECs. EVLC2 cells infected with C pneu-moniae (multiplicity of infection [MOI] 1) weretreated with the indicated concentrations of aspi-rin, salicylic acid, or PDTC or were left untreated(control). At 72 hours after infection, monolayerswere stained with a genus-specific FITC-labeledantibody and counterstained with Evans blue. Thecells were examined at �400 magnification undera conventional fluorescence microscope (top) orby high-resolution image analysis (bottom) with theuse of deconvolution microscopy (see Methods).

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activity was increased at 6 hours after C pneumoniae infection.Only weak NF-�B DNA binding activity was seen in mock-infected cells. Competitive inhibition by unlabeled NF-�B oligo-nucleotide, but not by an AP-1 DNA binding site, and themissing binding to the mutated NF-�B site suggest specificity ofthe interaction. Supershift experiments with p65 and p50 anti-bodies showed that these components of the NF-�B family wereactivated by C pneumoniae. All investigated drugs reducedactivation of NF-�B in infected cells to a level comparable tothat of mock-infected control cells. To further support thefinding that NF-�B may contribute to the life cycle of chlamyd-ial infection, the decoy ODN technique was used. Transfectionof the infected ECs with the NF-�B–specific decoy ODNinhibited chlamydial growth by 50�2.6% (P�0.001), whereasthe mutated decoy ODN had no effect on chlamydial growth(1�2.3% inhibition, P�NS). These data represent mean�SDvalues of 3 independent experiments.

Aspirin Treatment Prevents C pneumoniae–Induced IL ExpressionTo confirm the ability of aspirin in the functional inhibition ofNF-�B activation in infected ECs, we next investigated theeffect of aspirin on the expression of IL-6 and IL-8, with bothunder the transcriptional control of NF-�B.

It has been shown previously that infection of ECs with Cpneumoniae induces secretion of IL-6 and IL-8, that maximalcytokine induction occurs by 48 hpi, and that it is sustained forthe entire 72-hour study period.11 Figure 4 shows IL-6 and IL-8concentrations in the supernatant of EC cultures at 48 hpi. Cpneumoniae infection induced IL-6 and IL-8 concentrationsnearly 3- and 6-fold, respectively. Treatment of cell cultures withaspirin or sodium salicylate reduced IL-6 and IL-8 levels in adose-dependent manner.

Inhibition of Chlamydial Growth Is Not Causedby ApoptosisTo investigate whether the inhibition of chlamydial growthwas indeed related to reduced bacterial proliferation orwhether the cells were driven to apoptosis, chromatin con-densation was assessed by staining with the nuclear dyeHoechst 33342 in ECs with and without treatment with thevarious investigated drugs. The results for the highest drugconcentrations at 12, 24, and 48 hpi are shown in the onlineTable (which can be accessed at http://atvb.ahajournals.org).The percentage of cells with nuclear changes indicative ofapoptosis ranged between 6% and 15%, with a tendencytoward higher rates at later time points. Compared with theuntreated control cells, in the treated cells no significantlyincreased rates of apoptotic cells were seen after treatmentwith aspirin, salicylic acid, BAY, or decoy ODN. In contrast,treatment with PDTC efficiently induced apoptosis in up to60% of cells at the highest concentration of 50 �mol/L onlybut not at concentrations �25 �mol/L. These data suggestthat the described concentration-dependent effect of aspirinand the other inhibitors of NF-�B is not caused by apoptosisof the host cells.

Figure 2. Inhibition of chlamydial growth and decrease of infec-tious progeny titers by aspirin (ASA), sodium salicylate (SSA),PDTC, and BAY in EVLC2 cells. A, EVLC2 cells were stained at72 hpi with a genus-specific FITC-labeled antibody. Inclusionswere counted, and percent inhibition was calculated asdescribed in Methods. Mean�SEM values represent data of 5independent experiments. B, EVLC2 cells were sonicated at 72hpi, and growth titers were determined subsequently in HEp2indicator cells to assess production of infectious progeny.Results were expressed as IFU per milliliter and representmean�SEM of 3 independent experiments. *P�0.05 and**P�0.001 vs control by Dunnett test.

Figure 3. Effect of ASA, SSA, PDTC, and BAY on C pneumoniae–induced NF-�B activation in EVLC2 cells at 6 hpi, analyzed byelectromobility shift assay. Subunit composition of the NF-�B bind-ing 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.

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DiscussionThe present study demonstrates that aspirin and its metabolitesalicylic acid counteract the intracellular development andmaturation of chlamydial inclusions indicated by the reduc-tion of bacterial inclusions and the titer of infectious progeny.The clear concentration-response relationship indicates ayet-unknown effect of these substances on chlamydialgrowth. The inhibitory effect of aspirin/salicylic acid onchlamydial development was accompanied by morphologicalchanges of the inclusions that became smaller and lessuniformly shaped. Our data suggest that the intracellulargrowth of C pneumoniae is mediated by an activation of thetranscription factor NF-�B. These findings apply not only toa single cell line but also to various endothelial and epithelialcell lines and to cultured HUVECs. It is tempting to speculatethat the bacterium has evolved to benefit from cellularmechanism of gene activation to promote its own replication.

Our data suggest that the effect of aspirin/salicylic acid onchlamydial growth is caused, at least in part, by its capacity toinhibit NF-�B.17 However, aspirin is a drug with many knownmechanisms of action.23 First of all, aspirin is known to irreversiblyinhibit cyclooxygenase (COX) by acetylation. Our findings thatsodium salicylate, which lacks the respective acetyl group, mimicsthe inhibitory properties of aspirin on chlamydial growth indicatethat the observed effect is not mediated by COX inhibition.Recently, it has been proposed that salicylates exert part of theirantiinflammatory action by the suppression of COX gene transcrip-tion.24 However, because PDTC and BAY, structurally unrelatedinhibitors of NF-�B,18,25 had anti-chlamydial effects similar to those

of the salicylates, it seems unlikely that the inhibitory effect ofsalicylates on chlamydial growth is mediated primarily by thesuppression of COX gene transcription.

Because most of the chemical inhibitors lack exclusive specific-ity for NF-�B inhibition and also affect the activity of othertranscription factors and signaling cascades,26 we used a highlyspecific decoy ODN technique22 to verify that NF-�B is causallyinvolved in the observed inhibition of chlamydial growth. Indeed,reduction of chlamydial inclusions by 50% occurred after transfec-tion of ECs with the consensus decoy ODN but not when thecorresponding mutant decoy ODN was used. These data stronglysuggest that activation of NF-�B constitutes an essential part of thehost-pathogen relationship, with important implications for chla-mydial growth. However, we cannot completely exclude the pos-sibility that the effect of aspirin on chlamydial growth also involvesother cellular signal transduction pathways. Besides its direct inhib-itory effect on NF-�B, aspirin is known to be a scavenger of reactiveoxygen species, which are involved as intracellular signal transduc-tion messengers in the regulation of many transcription factors.27

Therefore, part of the effects of aspirin that we have observed in thisinvestigation might be due to this activity. Moreover, salicylatesmight also exert an additional direct toxic effect on the chlamydialdevelopmental cycle that has not yet been recognized. Furtherstudies are necessary to elucidate these issues.

In the next step, we investigated whether treatment of ECswith aspirin resulted in a decreased expression of genes undercontrol of NF-�B to prove efficient NF-�B inhibition at thefunctional level. For this purpose, we determined the secretion ofIL-6 and IL-8 into the supernatant medium. Both have beenshown previously to be under the regulatory control of NF-�B28,29 and to be upregulated after the infection of ECs with Cpneumoniae.9–11 As with chlamydial growth, treatment withaspirin/salicylic acid resulted in a similar concentration-dependent inhibition of the secretion of IL-6 and IL-8 (Figure 4).We interpret this finding as another strong indication supportingthe hypothesis that the growth inhibition of C pneumoniae byaspirin is caused, at least partly, by inhibition of NF-�B.

Recent studies have broadened the role of NF-�B from that of aregulator of immune response and inflammatory responses to that ofa regulator of apoptosis and cell growth.15 Therefore, we wonderedwhether the growth-inhibitory effect of aspirin reflected apoptoticcell death of the host cell rather than attenuation of chlamydialproliferation. This idea was supported by a recent study by Wahl etal,30 who showed that activation of NF-�B in C pneumoniae–infected macrophages is associated with protection of these cellsagainst apoptosis. When investigating the effect of treatment withaspirin and the other drugs on cell viability or the occurrence of cellswith nuclear changes characteristic of apoptosis, we did not find anyindication of a toxic or proapoptotic effect of these drugs (Figure 2and online Table). The only exception was PDTC at �25 �mol/L,concentrations for which signs of induction of apoptosis weredetected in a high percentage of cells, whereas the same cells werestill classified as viable in the dye exclusion test. PDTC is known tobe an unspecific inhibitor of NF-�B but a potent scavenger ofreactive oxygen species with a high potential to induce apopto-sis.25,31 Therefore, we conclude that the described growth-inhibitingeffect of aspirin and the other inhibitors of NF-�B was not due toincreased rates of necrosis or apoptosis of the host cells. This isconsistent with other studies concerning the antiapoptotic activity

Figure 4. Effect of aspirin and salicylic acid on C pneumoniae–induced IL-6 and IL-8 expression of EVLC2. The cell superna-tant was removed at 48 hpi. IL-6 and IL-8 were determined byenzyme immunoassay. Mean�SEM values represent data oftriplicates. The experiment was repeated once.

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conferred to the host cell on infection with C pneumoniae, resultingin the conclusion that no NF-�B–dependent cellular factors wereinvolved in the protection against apoptosis.32–34

The presented results show that aspirin at concentrations aslow 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 arecommonly achieved by therapeutic doses of aspirin in humans.23

In coronary heart disease, aspirin has proven to reduce morbidityand mortality in primary prevention and secondary intervention.In addition to its antithrombotic effects, other mechanisms maycontribute to these clinical benefits of aspirin.23 Therefore, it istempting to speculate that mechanisms such as described in thepresent investigation play a role.

Taken together, these data strongly suggest that C pneu-moniae has developed a strategy to exploit host cell biology topromote its own development. Our findings suggest that activa-tion of NF-�B plays a pivotal role in the developmental cycle ofC pneumoniae and that aspirin and other inhibitors of NF-�Bactivation might have previously unrecognized anti-chlamydialeffects. The identification of NF-�B as a critical transcriptionfactor in C pneumoniae infection may provide insights intospecific therapeutic strategies for the treatment of diseasesassociated with this organism.

AcknowledgmentsThis study was supported by grants from the Austrian Science Funds(P13401-Med, SFB 714). We thank Dr Oleksiy Tsybrovskyy for theperformance of morphometric measurements and Nicole Friedl andElisabeth Winter for their excellent technical assistance.

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Leeuwen and Beate TiranAndreas Tiran, Hans-Jürgen Gruber, Wolfgang F. Graier, Andreas H. Wagner, Ellen B.M. van

Expression, and Bacterial Development in Human Endothelial CellsB Activation, CytokineκInduced Nuclear Factor-−Chlamydia pneumoniaeAspirin Inhibits

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 2002 American Heart Association, Inc. All rights reserved.

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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.

A

0

25

50

75

100

ASA (mmol/L) - 0.1 0.5 2.5 5.0 - - - - - - - -SSA (mmol/L) - - - - - 0.1 0.5 2.5 5.0 - - - -PDTC (µmol/L) - - - - - - - - - 5.0 12 25 50% dead cells 6 5 7 6 7 9 3 5 11 7 9 3 11

Inhi

bitio

n of

chl

amyd

ial

grow

th(%

) *

***

*

*

B

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50

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ASA (mmol/L) - 0.1 0.5 2.5 5.0 - - - - - - - -SSA (mmol/L) - - - - - 0.1 0.5 2.5 5.0 - - - -PDTC (µmol/L) - - - - - - - - - 5.0 12 25 50

IFU/

ml

(x 1

04 )

* **

*

* *

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 FITC-labeled 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.