s tatins (hmg-coa reductase inhibitors) reduce cd40 expression in

Cardiovascular Research 59 (2003) 755–766www.elsevier.com/ locate/cardiores

S tatins (HMG-CoA reductase inhibitors) reduce CD40 expression inhuman vascular cells

a c a , a*Flore Mulhaupt , Christian M. Matter , Brenda R. Kwak , Graziano Pelli ,a a b c a¨Niels R. Veillard , Fabienne Burger , Pierre Graber , Thomas F. Luscher , Franc¸ois Mach

aCardiology Division, Department of Medicine, University Hospital, Geneva Medical School, Foundation for Medical Research, 64 Avenue Roseraie,1211 Geneva 4, Switzerland

bSerono Pharmaceutical Research Institute, Geneva, SwitzerlandcDivision of Cardiology, Cardiovascular Research, Institute of Physiology and Division of Cardiology, Cardiovascular Center, University Hospital,

Zurich, Switzerland

Received 2 December 2002; accepted 30 May 2003

Abstract

Objective: HMG-CoA reductase inhibitors (statins) possess anti-inflammatory and immunomodulatory properties that are independentof their lipid-lowering action. As the CD40–CD40L signaling pathway is implicated in the modulation of inflammatory responsesbetween vascular cells, involving adhesion molecules, pro-inflammatory cytokines, chemokines, we sought to investigate the potential roleof statins in regulating the expression of CD40.Methods and Results: Using Western blot, flow cytometry and immunohistochemistryanalyses, we observed that four different statins reduced IFN-g-induced CD40 expression in human vascular cells (endothelial cells,smooth muscle cells, macrophages and fibroblasts). This effect was dose-dependent (from 5mM to 80 nM) and reversed by addition ofL-mevalonate. Activation of vascular cells by human recombinant CD40L, as measured by ELISA for IL-6, IL-8 and MCP-1, was stronglyreduced when cells were treated with statins. Immunostaining of human carotid atherosclerotic lesions of patients subjected to statintreatment revealed less CD40 expression on a ‘per vascular cell’ basis compared to control patients. Although many pleiotropic effects ofstatins are mediated by nitric oxide synthase (NOS)- or peroxisome proliferator-activated receptor (PPAR)-dependent signaling pathways,we observed similar statin-induced reduction of CD40 expression using NOS inhibitors or different PPAR ligands.Conclusion: Statinsdecrease CD40 expression and CD40-related activation of vascular cells. These effects are partially reversed by the HMG-CoA reductaseproductL-mevalonate and are mediated by NOS- or PPAR-dependent pathways. Altogether, these findings provide mechanistic insightinto the beneficial effects of statins on atherogenesis. They also provide a scientific rationale for the use of statins as immunomodulatorsafter organ transplantation. 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Keywords: Atherosclerosis; Cytokines; Immunology; Lipid metabolism; Receptors

1 . Introduction that these drugs greatly reduce cardiovascular-relatedmorbidity and mortality in patients with and without

Statins are effective lipid-lowering agents, extensively coronary disease[2,3]. Recently, in vitro and in vivoused in medical practice[1]. They are competitive in- findings have indicated that statins, beside their lipid-hibitors of 3-hydroxy-3-methylglutaryl coenzyme A lowering effects, possess anti-inflammatory properties[4].(HMG-CoA) reductase, an enzyme crucial for cholesterol In addition, we recently demonstrated that these drugssynthesis. Several large clinical trials have demonstrated might also be recognized as a new type of immuno-

suppressor[5].Atherosclerosis is considered as an immuno-inflamma-

*Corresponding author. Tel.:141-22-382-7233; fax:141-22-382-tory disease[6,7] and increasing evidence suggests a7245.

E-mail address: [email protected](B.R.Kwak). Time for primary review 32 days.

0008-6363/03/$ – see front matter 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.doi:10.1016/S0008-6363(03)00515-7

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756 F. Mulhaupt et al. / Cardiovascular Research 59 (2003) 755–766

central role for the CD40–CD40L signaling pathway in the bodies for IL-6, IL-8 and MCP-1 were obtained frompathogenesis of this disease[8,9]. Recently, two reports R&D.L-Mevalonate was purchased from Sigma (St. Louis,demonstrated that hypercholesterolemia, one of the major MO, USA). Human recombinant CD40 ligand (rCD40L)cardiovascular risk factors, increases CD40 and CD40L was generated as described previously[17]. L-NAME wasexpression on platelets and monocytes[10] as well as purchased from Sigma. 15d-PGJ was obtained from2

plasma levels of soluble CD40L[11]. In addition, it has Calbiochem (La Jolla, CA, USA), ETYA from Sigma,been shown that blocking CD40–CD40L interactions WY14643 from Biomol (Plymouth Meeting, PA, USA),significantly prevents the development of atherosclerotic and Troglitazone a gift from Park Davis Pharmaceuticalsplaques and reduces already pre-established lesions (Morris Plains, NJ, USA).[12,13]. Moreover, CD40 signaling has been implicated inother chronic disorders such as rheumatoid arthritis, multi- 2 .2. Cellsple sclerosis and allograft rejection following organ trans-plantation[14]. The investigation conforms with the principles outlined

CD40L (recently renamed CD154) and CD40 are mem- in the Declaration of Helsinki (Cardiovasc Res 1997;35:2-bers of the tumor necrosis factor (TNF) and TNF-receptor 3) as well as theGuide for the Care and Use offamily, respectively. The original function of CD40L in T Laboratory Animals published by the US National Institutecell-dependent humoral immunity involves the activation of Health (NIH publication No. 85-23, revised 1996).and differentiation of B-lymphocytes, the switching of Human vascular ECs and SMCs were isolated fromimmunoglobulin classes as well as the formation of saphenous veins and cultured as described previouslygerminal center and memory cells. More recently, activa- [5,18]. Primary human fibroblasts were isolated from nasaltion of vascular cells such as macrophages (MF), endo- polyps and cultured as previously described[19]. Cellsthelial cells (ECs) and smooth muscle cells (SMCs) via were used at passages two to four for all experiments.CD40 signaling has been shown to induce inflammatory Human monocytes were obtained from peripheral blood ofresponses with expression of adhesion molecules and healthy donors as described previously[20]. They weresecretion of pro-inflammatory cytokines, chemokines, ma- grown in RPMI 1640 medium containing 10% fetal calftrix metalloproteinases and tissue factor[15]. All these serum (FCS) for 10 days. The human Raji cell line wasmolecules are known to be crucial in the pathogenesis of obtained from ATCC (Rockville, MD, USA).atherosclerosis as well as during graft-versus-host disease.

In view of the large clinical beneficial effects of statins 2 .3. Western blot analysison cardiovascular morbidity and mortality, and the emer-gence of numerous non-lipid lowering anti-inflammatory Cells were harvested in ice-cold radio-immuno-precipi-effects of these drugs, we sought to investigate whether tation assay (RIPA) solubilization buffer, and total amountsstatins regulate the expression of CD40 in vascular cells. of protein determined using a bicinchoninic acid quantifi-

cation assay (Pierce, Rockford, IL, USA). Western blottingwas performed as described before[5]. Blots were blocked

2 . Methods in 5% defatted dry milk /phosphate-buffered saline (PBS)/0.1% Tween, incubated for 1 h at room temperature with

2 .1. Reagents primary antibody (1:40; rabbit polyclonal anti-CD40), ormouse monoclonal anti-humanb-actin (1:5000; Pharming-

Human and mouse recombinant IFN-g were obtained en) as control of loading, followed by 1 h incubation withfrom Endogen (Cambridge, MA, USA) and R&D Systems secondary antibody (1:10,000; Jackson ImmunoResearch).(Abingdon, UK), respectively. The statins used in these Quantification was performed using AIDA softwarestudies, i.e. atorvastatin, simvastatin, lovastatin and pravas- (Raytest, Urdorf, Switzerland).tatin were obtained from commercial sources. BecauseECs lack lactonases, to process simvastatin and lovastatin2 .4. Flow cytometryto their active forms, these agents were chemically acti-vated before their use as previously described[16]. Mouse Cells were incubated with FITC-conjugated specificanti-human CD40 monoclonal antibody fluorescein iso- antibody (60 min, 48C) and analyzed in a Becton Dickin-thiocyanate-conjugated (FITC) and mouse anti-human son FACScan flow cytometer as described[5]. At leastHLA-DR monoclonal antibody (FITC) were obtained from 100,000 viable cells were analyzed per condition. DataPharmingen (San Diego, CA, USA). Rabbit anti-human were analyzed using CELLQUEST software (Becton Dic-CD40 polyclonal antibody, anti-rabbit IgG FITC, and HRP kinson).(horseradish peroxidase) goat anti-rabbit IgG were pur-chased from Santa Cruz (Santa Cruz, CA, USA), Jackson2 .5. Cytokine assaysImmunoResearch (West Grove, PA, USA) and VectorLaboratories (Burlingame, CA, USA), respectively. Anti- Release of IL-6, IL-8 and MCP-1 was measured using a

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sandwich-type enzyme-linked immunosorbent assay snap-frozen in liquid nitrogen. There was no significant(ELISA) according to the manufacturer’s instructions difference for age, gender, cardiovascular risk factors and(R&D). Experiments were performed in the presence of other medications between patients treated or not withpolymyxin B (1mg/ml). Optical density was measured at simvastatin (Table 1). Serial cryostat sections (5mm) were450 nm using a Dynatech plate reader (Chantilly, VA, cut, air dried onto microscope slides (Fisher Scientific,USA). The amount of IL-6, IL-8 and MCP-1 detected was Wohlen, Switzerland), and fixed in acetone at220 8C for 5calculated from a standard curve prepared with the recom- min. Sections were pre-incubated with blocking bufferbinant protein. Samples were assayed in duplicate. (PBS/Tween with 8% normal horse serum) and then

incubated successively with an anti-human CD40 antibody2 .6. Fluorescence immunolabeling for 1 h. Thereafter, sections were incubated with

biotinylated secondary antibody (45 min; Vector Laborator-Monocytes/macrophages grown on coverslips were ies) followed by avidin–biotin–alkaline phosphatase com-

rinsed and fixed for 15 min with paraformaldehyde (4%). plex (Vectastain ABC kit). Antibody binding was visual-Coverslips were rinsed and cells incubated successively in ized by alkaline phosphatase substrate (Vector Laborator-0.5 M NH Cl/PBS for 15 min and PBS supplemented with ies). Quantitative analyses for CD40 and CD68 expression4

2% bovine serum albumin (Sigma) for a further 20 min. were performed with a computer-based color image analy-Human macrophages were incubated overnight with pri- sis system[21].mary rabbit polyclonal anti-CD40 (1:50) in 10% normalgoat serum (Sigma)/PBS. After rinsing, human macro- 2 .8. Statistical analysisphages were incubated with secondary antibodies FITC-conjugated (1:800) for 3 h. All steps were performed at Data are presented as mean6S.E.M. Mean valuesroom temperature and between incubation steps cells were between two groups were compared using a two-tailedrinsed with PBS. Finally, coverslips were mounted on Student’st-test, after having performed anF-test forslides in Vectashield (Vector Laboratories). Cells were homogeneity of variances. If data failed to meet require-examined using a Zeiss Axiophot microscope. Replace- ments for use of the parametrict-test, a Mann–Whitneyment of the primary antibody with PBS/10% normal goat U-test was used. Differences were considered statisticallyserum was used to control the specificity of the immuno- significant atP,0.05.labeling of human macrophages.

2 .7. Immunohistochemistry 3 . Results

Surgical specimens of human carotid atheroma were 3 .1. Statins reduce CD40 expression on vascular cellsobtained by protocols approved by the Review Committeeat the University Hospital of Geneva. All atherosclerotic To determine whether statins influence CD40 expres-carotid specimen analyzed were obtained from patients sion, human vascular endothelial cells (ECs) were acti-treated (for at least 3 months) or not with the simvastatin. vated with human recombinant IFN-g (1000 U/ml /24 h)Materials were directly embedded in OCT compound or in the presence or absence of atorvastatin, lovastatin,

pravastatin or simvastatin. As previously described, un-stimulated vascular cells showed little expression of CD40,

T able 1 which was greatly induced by IFN-g [18]. Western blotBaseline characteristics of patients in the simvastatin and control groups analysis (Fig. 1A) revealed that statin treatment reduced

Simvastatin (n518) Control (n514) both, basal CD40 expression as well as IFN-g-inducedCD40. Simvastatin appeared to be the most potent inhib-Age (years) 68 65

Sex (F/M) 8/10 8/6 itor, followed by lovastatin, pravastatin and atorvastatinDiabetes (%) 32 38 (Fig. 1A). Raji cells, known to express a large amount ofSmokers (%) 34 30 CD40, were used as positive control (Fig. 1A, lane 8). TheHypertension (%) 73 68

reduction of CD40 expression by statins was dose-depen-Medication (%)dent (Fig. 1B). The concentration range (0.08 to 10mM)Aspirin 33 28

ACEIs 45 48 of statins used in these experiments was similar to earlierb-Blockers 24 22 reports [5,22], and neither cell survival nor protein syn-Anti-diabetic agents 33 38 thesis was affected at these concentrations (data notDiuretics 32 21

shown). Interestingly, simvastatin treatment significantlyreduced CD40 expression (Fig. 1B) at a concentration ofTotal cholesterol (mmol / l) 5.3 (61.3) 6.4 (61.4)

LDL-c, 3.3 (61.1) 4.1 (61.2) only 80 nM, which corresponds to the effective serumHDL-c 1.3 (60.3) 1.2 (60.3) levels seen with statin treatment in clinical practice[23].Triglycerides 1.9 (61.4) 2.3 (61.4) These effects of statins on CD40 expression were specific

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for the inhibition of the HMG-CoA reductase enzyme, under statin therapy expressed much less CD40 comparedsince addition ofL-mevalonate largely reversed the statin- to control patients (Fig. 4A, B). In addition, we observedinduced inhibition of CD40 expression (Fig. 1A). Similar that individual macrophages within lesions from statin-results were obtained for human vascular SMCs (Fig. 1C). treated patients showed markedly less CD40 expressionThese results were confirmed using flow cytometry analy- compared to individual macrophages in lesions of controlsis (Fig. 1D). Western blot results were supported by patients (Fig. 4C). This reduction was mainly detectedimmunohistochemical analysis on human monocyte– within the shoulder region of the atherosclerotic plaques.macrophages (Fig. 2A–F) and on human fibroblasts (Fig.2G–I), two other cell types present in atherosclerotic 3 .4. Statin reduction of CD40 expression is neitherlesions. Taken together, these experiments demonstratemediated via nitric oxide nor via PPARsthat statin treatment decreased CD40 expression on allhuman vascular cells tested. Due to numerous recent reports demonstrating that

several anti-inflammatory effects of statins could be largely3 .2. Statins inhibit functional CD40 signaling related to their ability to stabilize and increase nitric oxide

synthase (NOS)[25], we investigated whether statinsTo examine functional consequences of statin treatment reduce CD40 expression via NOS pathways. To address

on CD40 expression, we stimulated vascular cells with this question, we stimulated human ECs with recombinanthuman recombinant CD40 ligand (rCD40L) in the presence IFN-g and simvastatin in the presence or absence of theor absence of the statins. As presented inFig. 3 for ELISA NOS inhibitor L-NAME. As shown in Fig. 5 by flowexperiments on ECs, rCD40L induced the secretion of the cytometry analysis, the level of CD40 expression afterpro-inflammatory molecules IL-6, IL-8 and MCP-1, as statin treatment was similarly reduced with or withoutpreviously described[17,24]. When stimulated in the L-NAME treatment.presence of the statins, the synthesis and secretion of all Furthermore, as statins have been recently reported tothese pro-inflammatory proteins were largely reduced. As activate PPARs[26], we investigated whether PPARpreviously described, statins also reduced constitutive ligands could regulate CD40 expression on human vascularcytokine secretion, these effects were however not statisti- cells. We did not detect any effect of either PPAR-a orcally significant. The statin-induced reduction in cytokine/ PPAR-g ligands on CD40 cell surface expression followingchemokine secretion was partially reversed by the addition IFN-g activation (Fig. 6). These findings indicate thatof L-mevalonate (Fig. 3A–C). Specificity of these experi- neither NOS nor PPARs are involved in the reduction ofments was confirmed by blocking CD40–CD40L signaling CD40 expression by statins on vascular cells.with the addition of an anti-CD40L antibody (Fig. 3C).

3 .3. Statin treatment decreases CD40 expression within 4 . Discussionhuman atherosclerotic tissue

Increasing evidence supports the central role of theIn view of our findings that statins reduce CD40 CD40–CD40L signaling pathway in atherosclerosis. Acti-

expression and CD40 signaling in vitro, we decided to vation of vascular cells via CD40–CD40L interactions hasexplore the expression of CD40 on vascular cells within been shown to induce inflammatory responses with expres-human atherosclerotic lesions obtained from patients sion of adhesion molecules, secretion of pro-inflammatorytreated or not with simvastatin (20 mg/day for at least 3 cytokines, matrix metalloproteinases, tissue factor andmonths). Baseline patients characteristics were not statisti- chemokines[8,9,18], molecules considered as crucialcally different and are shown inTable 1.Immunohistoch- players in atherogenesis. Recent in vitro and in vivoemical in situ and Western blot analyses of human carotid findings suggest that statins also have anti-inflammatoryarteries revealed that atherosclerotic lesions from patients properties[24,27].Statins inhibit HMG-CoA reductase that

Fig. 1. Statins decrease IFN-g-induced CD40 expression on human endothelial cells. (A–C) Western blot analysis for CD40. (A) Human vascular ECsunder unstimulated conditions (1); treated with IFNg (1000 U/ml, 24 h) alone (2); or in the presence of atorvastatin (10mM) (3); lovastatin (10mM) (4);pravastatin (20mM) (5); simvastatin (10mM) (6); or simvastatin (10mM) and L-mevalonate (400mM) (7); Raji cells under unstimulated condition aspositive control (8). *P,0.05 (compared to 2); **P,0.05 (compared to 6). Quantification of Western blots is expressed as ratio of CD40/b-actin signalfor each samples. (B) Human vascular ECs under unstimulated conditions (Ctrl), or treated with IFNg (1000 U/ml, 24 h) alone; or in the presence ofsimvastatin (5 to 0.08mM). b-actin is shown as loading control. (C) Human vascular SMCs under unstimulated conditions (1), treated with IFNg (500U/ml, 24 h) alone (2); or in the presence of simvastatin (10mM) (3). b-actin is shown as loading control. (D) Flow cytometry analysis for CD40. Humanvascular ECs treated with IFNg (1000 U/ml, 24 h) alone (green line); or in the presence of simvastatin (5mM) (blue line). Expression of MHC class II forECs stimulated with IFNg (1000 U/ml, 24 h) is shown as positive control (red line). Shown is a histogram representing cell numbers (y-axis) vs. CD40 logfluorescence intensity (x-axis) for 20,000 viable cells, and ECs under unstimulated conditions are shown (solid histograms) as well as isotype controls(dotted lines). For all analysis, similar results were obtained in separate experiments using cells from four different donors.

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Fig. 2. Statins reduce CD40 expression on human macrophages and fibroblasts. Immunohistochemical analysis of CD40 expression (green fluorescence).Macrophages (A–F) and fibroblasts (G–I). Cells were counterstained with Evans Blue (red color). (A) Cells under unstimulated conditions. (B) Cellstreated with IFN-g alone (1000 U/ml, 24 h); or in the presence of simvastatin (5mM) (C); simvastatin (1mM) (D); or pravastatin (20mM) (E). (F) Cellstreated with IFN-g (1000 U/ml, 24 h) and stained with secondary antibody only (negative control). (G) Cells under control conditions; treated with IFN-g

alone (500 U/ml, 24 h) (H); or in the presence of simvastatin (10mM) (I). Scale bar represents 50mm. Similar results were obtained in separateexperiments using cells from three different donors.

transforms HMG-CoA into mevalonate. Indeed, as mevalo- CD40–CD40L in atherogenesis as well as the pleiotropicnate is not only the precursor of cholesterol but also of effects of statins in this disease, we sought to investigate inmany non-steroidal isoprenoids, statins may regulate the vitro and in vivo the effect of statins on CD40 expressionexpression and activities of important molecules in the as well as on related CD40 activation in human vascularpathogenesis of atherosclerosis. Given the importance of cells.

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showed that the reduction of CD40 expression by statins isnot mediated via mevalonate synthesis, but are in agree-ment with three other recent reports[10,29,30].

All four statins used in this study reduce IFN-g-inducedCD40 expression: simvastatin being the strongest, fol-lowed by lovastatin, pravastatin and atorvastatin. Althoughother in vitro studies have also revealed differences inpotency of individual statins, these results should beinterpreted with great care. One possible explanation forthe strong effect of simvastatin on IFN-g-induced CD40expression might be that this specific statin possessesadditional anti-inflammatory effects through inhibitoryactions in other signaling pathways. Alternatively, simplydue to different pharmacokinetic properties simvastatinmay be more effectively processed by cells in culture. It isof importance to note in this respect that in vivo in mousemodels both atorvastatin and simvastatin are effectivetreatments against multiple sclerosis and rheumatoid arth-ritis [30,31].

CD40 signaling plays a critical role in the initiation ofthe immune response by inducing inflammatory responseswith secretion of pro-inflammatory cytokines andchemokines. These chemokines probably attract and directT lymphocytes and macrophages to the atheroma, thussustaining chronic inflammation[32,33]. Indeed, lack ofchemoattractants or their receptors is known to signifi-cantly diminish atherosclerotic lesion progression in mice[34,35]. We found that statin treatment influences func-tional cellular CD40 signaling by reducing the release ofcytokines and chemokines. By secreting these pro-inflam-matory mediators and growth factors, vascular cells canFig. 3. Statins reduce CD40 function on CD40L-activated human vascu-

lar endothelial cells. IL-6 (A), IL-8 (B) and MCP-1 (C) releases measured promote the migration and proliferation of SMCs. Inby ELISA in supernatants of ECs exposed (24 h) to normal media (1); response to inflammatory stimulation, SMCs and MFsimvastatin (10mM) alone (2); activated by rCD40L (5mg/ml) alone secrete MMPs that will subsequently degrade matrix(3); or in the presence of atorvastatin (10mM) (4); lovastatin (10mM)

compounds of the plaque as well as other inflammatory(5); simvastatin (10mM) (6), or simvastatin (10mM) and L-mevalonatemediators that inhibit collagen synthesis. As extracellular(400mM) (7); rCD40L (5mg/ml) preincubated with polyclonal CD40L

antibody (8). Similar results were obtained in independent experiments matrix components are considered crucial determinants ofwith ECs from four different donors. *P,0.05 (compared to 3); **P, fibrous cap integrity and stability, statin-induced reduction0.05 (compared to 6); ***P,0.05 (compared to 3). of CD40 signaling with consequent reduction in secretion

of pro-inflammatory cytokines and chemokines may notPro-inflammatory cytokines expressed within atheroma only exert beneficial actions on progression of atheros-

provide a chemotactic stimulus to adherent leukocytes, clerotic plaque development but on the composition of thedirecting their migration into the intima. Our data show by plaque as well. Indeed, one of the major complications indifferent techniques that statin treatment largely reduces clinical cardiology is plaque rupture with local thrombusCD40 expression both on human ECs and on monocyte– formation, potentially leading to acute coronarymacrophages, thereby potentially reducing EC–monocyte syndromes.interactions. Similar results were obtained on murine Interruption of CD40–CD40L interactions by adminis-macrophages (data not shown). Importantly, we observed a tration of anti-CD40L blocking antibody or gene targetingreduction of CD40 expression at a concentration of 80 nM, has been shown to prevent atherogenesis and limit thewhich is within the range of effective serum concentrations progression of established atherosclerotic lesions in miceseen in clinical practice[23]. In addition, L-mevalonate [12,13]. Both strategies have resulted in a decrease inlargely reversed the statin-induced decreased in CD40 pro-inflammatory factors and an increase in collagenexpression, indicating that indeed the inhibition of HMG- content of atherosclerotic lesions. Recently, it has beenCoA reductase mediates the statin-induced repression of shown that in a short prospective treatment period statinsCD40 on vascular cells. These results are partly in reduced the pro-inflammatory state of human atheromatouscontradiction with the data from Wagner et al.[28], which lesions[36]. Our results showing that patients under statin

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therapy express less CD40-positive cells within atheros- deed, clinical studies have suggested a better outcome afterclerotic plaques compared to control patients without statin cardiac transplantation in patients subjected to statintherapy. In addition, they show that individual macro- therapy[39,40].We recently demonstrated that statins arephages express less CD40, suggesting direct effects of involved in the immune response by inhibiting IFN-g

statins on macrophages in vivo. induced MHC-II[5], and thus might be used as immuno-Most of the anti-inflammatory effects of statins have modulators. In addition, CD40 has been shown to play a

been related to their ability to stabilize and increase NO crucial role in graft-versus-host disease[41,42]. Oursynthase[4,22]. This endogenous vasodilator molecule has current findings indicating that statins decrease CD40by itself anti-inflammatory properties[37]. However, our expression on antigen presenting cells may thereforeexperiments illustrate that the reduction of CD40 expres- provide a new scientific rationale for their use as immuno-sion after statin treatment does not involve the NOS modulators following organ transplantation.signaling pathway. Recently, statins have been reported to In summary, we show that statins decrease CD40activate PPARs[26]. In addition, we recently reported that expression in vascular cells in vitro as well as in vivo. ByPPAR-g-induced repression of major histocompatibility reducing CD40 expression and signaling, statins maycomplex class II (MHC-II), another molecule involved in reduce inflammation and thus stabilize atheroscleroticthe immune response[38]. We did not detect an effect of plaques, features believed to account for the beneficialeither PPAR-a or PPAR-g ligands on CD40 cell surface effects of statins on cardiovascular morbidity and mortali-expression following IFN-g activation, thus excluding also ty. Our findings not only provide an additional explanationthese signaling pathways from mediating statin-induced for previously unknown non-lipid effects of statins inreduction in CD40 expression. atherogenesis, but also suggest their application in further

One of the major problems following organ transplanta- immuno-inflammatory diseases in which CD40 is in-tion is the development of graft-versus-host disease. In- volved, such as multiple sclerosis, systemic lupus erythe-

Fig. 5. Statins effect on CD40 expression is not mediated through NOS. Flow cytometry analysis for CD40 expression. Human vascular ECs treated withIFNg (1000 U/ml, 24 h) alone (green line); or with simvastatin (5mM) (blue line); or in the presence of simvastatin (5mM) and L-NAME (5 mM) (redline). Shown is a histogram representing cell numbers (y-axis) vs. CD40 log fluorescence intensity (x-axis) for 20,000 viable cells, and ECs underunstimulated conditions are shown (solid histograms) as well as isotype controls (dotted lines). Similar results were obtained in independent experimentswith ECs from three different donors.

Fig. 4. Statin therapy decreases CD40 expression on human atherosclerotic lesions. (A) Representative photomicrographs (3100) of CD40 expression (redcolor) within human carotid atherosclerotic lesions in situ. Atherosclerotic lesion in patient treated (1) or not (2) by statin therapy. L, lumen of the vessel.Quantitative analysis of CD40 expression on atherosclerotic lesions obtained from patients treated with statin (1) (n518), or without statin therapy (2)(n514) is shown on the right side of this panel. *P,0.05. (B) Western blot analysis of human carotid atherosclerotic tissues obtained from patients treated(1) or not (2) with statin therapy. Quantitative analysis of CD40 expression in patients treated with statin (1) (n512), or without statin therapy (2)(n510) is shown on the right side of this panel. *P,0.05. (C) Double immunofluorescence stain for macrophages (CD68) in green and CD40 in red inpatient treated (1) or not (2) by statin therapy. *P,0.05. Similar results were obtained in separate experiments using tissues from five different donors.

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Fig. 6. Effects of PPAR ligands on IFN-g-induced CD40 expression on human endothelial cells. Flow cytometry analysis for CD40 expression. Human vascular endothelial cells (ECs) treated withIFN-g (500 U/ml, 24 h) alone (green line), or with the PPAR-g ligands (red lines) 15d-PGJ (10mmol/ l) (A), Troglitazone (50mmol / l) (B); or with the PPAR-a ligands (red lines) ETYA (50mmol / l)2

(C), and WY14643 (50mmol / l) (D). Each panel is a histogram representing cell numbers (y-axis) vs. log fluorescence intensity (x-axis) for 15,000 viable cells, and ECs under unstimulated conditionsare shown (solid histograms) as well as isotype controls (dotted lines). Similar results were obtained in independent experiments with ECs from five different donors.

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is expressed on human vascular endothelial cells, smooth musclematosus, autoimmune diabetes mellitus, chronic arthritiscells, and macrophages: implications for CD40–CD40 ligand sig-and graft-versus-host disease.naling in atherosclerosis. Proc Natl Acad Sci USA 1997;94:1931–1936.

[19] W iszniewski L, Limat A, Saurat JH, Meda P, Salomon D. Differen-A cknowledgements tial expression of connexins during stratification of human keratino-

cytes. J Invest Dermatol 2000;115:278–285.[20] K ol A, Sukhova GK, Lichtman AH, Libby P. Chlamydial heat shockThe authors thank Dr Ludovic Wiszniewski (Department

protein 60 localizes in human atheroma and regulates macrophageof Pediatrics, Geneva University Hospital) for providing us tumor necrosis factor-alpha and matrix metalloproteinase expres-with primary human fibroblasts. This work was supported sion. Circulation 1998;98:300–307.by grants from the Swiss National Research Foundation [21] A ikawa M, Voglic SJ, Sugiyama S et al. Dietary lipid lowering

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[24] D iomede L, Albani D, Sottocorno M et al. In vivo anti-inflammatoryR eferences effect of statins is mediated by nonsterol mevalonate products.

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