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Differential effects of statins on relevant functions of human monocyte-derived dendritic cells Atilla Yilmaz,*,1 Christine Reiss,* Alexander Weng,* Iwona Cicha,* Christian Stumpf,* Alexander Steinkasserer,† Werner G. Daniel,* and Christoph D. Garlichs* *Medical Clinic II and †Department of Dermatology, University of Erlangen-Nuremberg, Germany

Abstract: Statins were shown to possess immunomodulating properties, but the mechanisms of statin effects on the immune system are poorly understood. We analyzed the influence of statins on professional antigen-presenting dendritic cells (DC). Immature DC were cultivated from monocytes of healthy donors. DC maturation was induced by lipopolysaccharide (LPS; 1 ␮g/mL). Unstimulated and LPS-stimulated DC were treated with simvastatin or atorvastatin (0.1–1 ␮M). The expression of CD40, CD83, CD86, and human leukocyte antigen-DR on unstimulated and LPS-stimulated DC was reduced significantly by statins, and the expression of Toll-like receptor 2 (TLR2) and TLR4 on LPS-stimulated DC was enhanced temporarily. Statins caused a significant reduction of endocytosis of fluorescein isothiocyanate-dextran by DC. Statins significantly inhibited the basal secretion of interleukin (IL)-6, IL-8, IL-12, and tumor necrosis factor ␣ from unstimulated DC, and their release from LPS-stimulated DC was enhanced. In mixed leukocyte reaction, preincubation of LPS-stimulated DC with statins significantly suppressed their clustering with T cells and their ability to induce T cell proliferation, CD71, and CD25 up-regulation on T cells and the secretion of interferon-␥ and IL-2 from T cells. In conclusion, this study showed that statins suppressed endocytosis, basal secretion of proinflammatory cytokines, and the ability of DC to induce T cell proliferation, activation, and T helper cell type 1 differentiation. However, statin preincubation of LPS-stimulated DC caused a further increase in their secretion of proinflammatory cytokines. J. Leukoc. Biol. 79: 529 –538; 2006. Key Words: atherosclerosis 䡠 inflammation 䡠 immunity

INTRODUCTION Statins, inhibitors of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase, are widely used in clinical practice as cholesterol-lowering agents. In addition, they reduce morbidity and mortality of patients with atherosclerotic diseases [1]. In a former clinical study, improved survival and reduced rejection episodes were observed in pravastatin-treated patients after heart transplantation, and therefore, it was assumed that statins 0741-5400/06/0079-529 © Society for Leukocyte Biology

might possess immunomodulating properties [2]. Subsequently, Kwak et al. [3] reported that statins inhibit major histocompatibility complex (MHC) expression on interferon-␥ (IFN-␥)stimulated human macrophages and endothelial cells, leading to a reduced ability of these cells to stimulate T cells. In further studies, beneficial effects of statins related to immunomodulation were reported in various animal models of immunological disorders such as experimental autoimmune encephalomyelitis [4, 5], sepsis [6], or cardiac allograft vasculopathy [7]. Recently, it was shown that in the presence of statins, the priming of murine naı¨ve T cells with spleen antigen-presenting cells (APC) caused a suppression of T helper cell type 1 (Th1) differentiation [8]. Furthermore, a down-regulation of human leukocyte antigen (HLA)-DR and CD38 was observed in healthy persons treated with atorvastatin [9]. Dendritic cells (DC) are ubiquitously distributed, professional APC with the unique ability to initiate a primary immune response by the activation of naı¨ve T cells [10]. To acquire the ability to stimulate T cells, DC must undergo a maturation process with the up-regulation of surface MHC II and costimulatory molecules. The presence of DC was described in atherosclerotic plaques, suggesting an association of DC with atherogenesis [11, 12]. In this regard, it was shown that DC maturation is induced by several atherogenic stimuli such as oxidized low-density lipoprotein (LDL)-cholesterol [13], lysophosphatidylcholine [14], nicotine [15], and angiotensin II [16]. Moreover, we recently demonstrated that the emergence of DC in atherosclerotic plaques, particularly in colocalization with T cells, is associated with plaque complications and ischemic syndromes, indicating a contribution of DC to plaque destabilization by the induction of inflammation [17]. It is interesting that in that study, fewer mature DC and a more stable plaque morphology were observed in atherosclerotic plaques of patients under current statin medication, so that we assumed that the migration of DC into atheroma might be reduced by statins. Indeed, it was recently shown that statin incubation decreases DC adhesion and transmigration over an endothelial monolayer [18]. Another explanation for the decrease of DC in atherosclerotic plaques of statin-treated patients might be an enhanced migration out of the atheroma. In

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Correspondence: Medical Clinic II, University of Erlangen-Nuremberg, Ulmenweg 18, 91054 Erlangen, Germany. E-mail: [email protected] Received February 2, 2005; revised August 20, 2005; accepted October 26, 2005; doi: 10.1189/jlb.0205064.

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this context, a recent study showed that dyslipidemia caused a reduction of the migration of DC from atherosclerotic plaques into the regional lymph nodes, leading to their local increase [19]. Therefore, we hypothesized that statins influence not only DC migration but also other important DC functions. In a recent study, we showed that the statin preincubation of human monocyte-derived DC, stimulated with cytokines, caused a significant suppression of their maturation [20]. Similar findings about the suppressive effect of statins on the maturation of murine bone marrow-derived DC were recently reported by Sun and Fernandes [21]. The aim of our present study was to analyze the influence of statins on several important functions of human monocyte-derived DC such as endocytosis and cytokine secretion and particularly, their ability to induce T cell proliferation, activation, and differentiation.

MATERIALS AND METHODS Cell culture medium and reagents DC were cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 ␮g/mL streptomycin (all from Biochrom AG, Berlin, Germany), 10 mM Hepes (Sigma-Aldrich, Taufkirchen, Germany), and 1% heat-inactivated (56°C, 30 min) human serum. Merck (Darmstadt, Germany) and Pfizer (Karlsruhe, Germany) provided simvastatin and atorvastatin, respectively. Mevalonate (MVA) was obtained from Sigma-Aldrich. Simvastatin was activated before use to an open acid form as described previously [22].

Generation of DC Monocyte-derived DC were generated according to an established method with minor modifications [23]. Briefly, peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation with Biocoll (Biochrom AG) from leukapheresis products from healthy subjects, fulfilling the guidelines for blood donors (Department of Transfusion Medicine, Erlangen, Germany). Monocytes were isolated from PBMC by their adherence to plastic. Immature DC were differentiated from monocytes by incubation with 800 U/mL granulocyte macrophage-colony stimulating factor (Novartis, Munich, Germany) and 125 U/mL interleukin-4 (IL-4; Strathmann Biotec AG, Hamburg, Germany) for 6 days. The differentiation of DC was monitored by the up-regulation of CD1a expression and down-regulation of CD14 expression. Immature DC were incubated with simvastatin or atorvastatin (0.1–1 ␮M), sometimes subsequently stimulated with 1 ␮g/mL lipopolysaccharide (LPS; Sigma-Aldrich) or 1 ␮g/mL CD154 (R&D Systems, Wiesbaden, Germany) up to 24 h. Statin incubation of DC was performed for 48 h, as our former study demonstrated this duration was most effective [20]. In some experiments, the cells were coincubated with MVA (100 ␮M) simultaneously with statin treatment.

Cell viability DC viability was determined microscopically by trypan blue exclusion. To detect any toxic effects of statins, annexin V and propidium iodide (PI) staining was analyzed by flow cytometry using the apoptosis detection kit (Becton Dickinson, Heidelberg, Germany) according to the manufacturer’s instructions.

Flow cytometric analysis [fluorescein-activated cell sorter (FACS)] DC were incubated with the mouse anti-human antibodies CD1a-fluorescein isothiocyanate (FITC), CD14-FITC, CD40-FITC, CD83-FITC, CD86-FITC, HLA-DR-phycoerythrin (PE; all from Caltag, Hamburg, Germany), Toll-like receptor 2 (TLR2)-FITC (Serotec, Du¨sseldorf, Germany), TLR3-PE (eBioscience, San Diego, CA), and TLR4-FITC (Serotec) for 30 min at 4°C. Cells were analyzed by FACSCalibur using CellQuest software of Becton Dickinson. Appropriate isotype-matched immunoglobulins (Caltag) were used as negative

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controls. The geo-mean or median was used to describe the mean fluorescence intensity (MFI) for at least 5000 cells per sample.

Endocytosis of FITC-dextran Endocytotic capacity of DC was determined by their uptake of FITC-dextran according to a method described previously [24]. Briefly, 0.2 ⫻ 106 DC were incubated with 1 mg/mL FITC-dextran (Molecular Probes, Leiden, Netherlands) for 1 h at 37°C and at 4°C (as negative control). After washing twice with FACS buffer (Becton Dickinson), cellular uptake of FITC-dextran was analyzed by FACS.

Cytokine analysis Cytokines were analyzed by FACS using the cytometric bead array (CBA) kit (Becton Dickinson), according to the manufacturer’s instructions. The cytokine concentration (pg/mL) was determined with calibration curves separately established using the CBA analysis software (Becton Dickinson). IL-6, IL-8, IL-12, and tumor necrosis factor ␣ (TNF-␣) were measured in the supernatant of DC cultures with the CBA inflammation kit of Becton Dickinson.

Analysis of T cell proliferation, T cell activation, and Th1 differentiation in a mixed leukocyte reaction (MLR) The proliferation of T cells induced by DC was analyzed in an allogeneic MLR using the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) as described previously [25]. The ability of DC to induce T cell activation was assessed by the expression of certain activation markers, CD71 (early) and CD25 (late), on T cells. Briefly, allogeneic peripheral blood lymphocytes (PBL), obtained from buffy coats from healthy donors (Department of Transfusion Medicine), were labeled with 10 ␮M CFSE for 10 min at 37°C. DC were mixed with 200,000 allogeneic PBL at DC/PBL ratios of 1:180, 1:60, 1:20, and 1:6. After 6 days of coculture, cells were harvested, and CFSE staining was analyzed by FACS on T cells, identified with a FITC-stained antibody against CD3-Tricolor (Caltag). In each experiment, the intensity of CFSE fluorescence (CFSE-MFI) of T cells, which were not coincubated with DC (unstimulated control), was set as 100%, and the CFSE-MFI of DC-stimulated T cells was related to this value as percentage. As CFSE-MFI of T cells is generally reduced with each cell division by half, the reduction in CFSE-MFI of CD3-positive cells correlated directly with the level of T cell proliferation. The expression of T cell activation markers was analyzed by FACS on CD3-positive cells using the mouse anti-human antibodies: CD71-PE and CD25-PE (both from Caltag). Concentrations of IL-2 and IFN-␥ were determined in the supernatant of DC-T cell cocultures after 6 days using the Th1/Th2 CBA kit of Becton Dickinson.

Statistical analysis All results for continuous variables are expressed as mean ⫾ SEM. Statistical analyses were performed by Mann-Whitney Rank Sum test. P values of 0.05 or less were considered statistically significant.

RESULTS Effect of statins on the expression of costimulatory molecules and TLRs on DC The expression of CD40, CD83, CD86, and HLA-DR on statinincubated, LPS-unstimulated DC (S⫹/L– DC) and statin-preincubated, LPS-stimulated DC (S⫹/L⫹ DC) was compared with statin-untreated, LPS-unstimulated DC (S–/L– DC, immature control) and statin-untreated, LPS-stimulated DC (S–/L⫹ DC, mature control). Regarding unstimulated DC, statin incubation led to a significant dose-dependent reduction of the basal expression of CD40, CD83, CD86, and HLA-DR on S⫹/L– DC compared http://www.jleukbio.org

with S–/L– DC (Fig. 1, A and B; CD86 and HLA-DR shown as an example). As expected, upon LPS stimulation, CD40, CD83, CD86, and HLA-DR were strongly up-regulated on S–/L⫹ DC. Similar to our former results [20], preincubation with statins before LPS stimulation caused a significant, dose-dependent suppression of the up-regulation of CD40, CD83, CD86, and HLA-DR on S⫹/L⫹ DC (Fig. 1, C and D; CD86 and HLA-DR). Coincubation with MVA significantly reversed the statin-induced suppression of CD40, CD83, CD86, and HLA-DR expression on S⫹/L– DC and S⫹/L⫹ DC. However, incubation with MVA without simultaneous statin incubation did not significantly alter CD40, CD83, CD86, and HLA-DR expression (data not shown). Comparing simvastatin and atorvastatin, no significant differences in their efficiency to suppress the up-regulation of CD40, CD83, CD86, and HLA-DR on S⫹/L⫹ DC were observed (Fig. 1, E and F). Statin incubation of unstimulated DC did not significantly alter the expression of TLR2, TLR3, or TLR4 (data not shown). In contrast, statin preincubation (1 ␮M, 48 h), prior to LPS stimulation, caused a significant increase in TLR2 and TLR4 expression on S⫹/L⫹ DC compared with S-/L⫹ DC (Fig. 2, A and C), which was reversed specifically by MVA. Comparing different time-points during LPS stimulation (3 h, 6 h, and 24 h), it was shown that the highest increase of TLR2 and TLR4 expression on S⫹/L⫹ DC occurred at 6 h of LPS stimulation. Afterwards, at 24 h, TLR2 and TLR4 expression on S⫹/L⫹ DC decreased remarkably, so that their statin-induced increase seems to be temporary. In contrast, although TLR3 was highly expressed on DC in our experiments, no significant changes of TLR3 were observed by statins (Fig. 2B).

Suppression of the endocytotic capacity of DC by statins The effect of statins on the capacity of DC to take up FITCdextran was analyzed. Statin incubation of S⫹/L– DC led to a significant reduction of their endocytotic capacity compared with S–/L– DC (Fig. 3, A and B). As expected, upon LPS stimulation, endocytotic capacity of S–/L⫹ DC was reduced remarkably compared with S–/L– DC. Statin preincubation of LPS-stimulated DC led to a further significant decrease in their endocytotic capacity compared with S–/L⫹ DC (Fig. 3C). The inhibitory statin effect on endocytosis was equipotent for simvastatin and atorvastatin and reversible by MVA.

Exclusion of a statin-induced cell death of DC To exclude the possibility that the suppression of costimulatory molecules or endocytotic capacity of DC might be a result of cell death, cell viability and apoptosis were monitored. By trypan blue exclusion, no higher percentage of dead cells was observed for statin-treated DC than for statin-untreated DC (data not shown). In addition, staining with PI and annexin V was performed to detect any toxic effects of statins more sensitively. Neither simvastatin nor atorvastatin incubation of DC significantly altered annexin V or PI expression (data not shown), proving that statins did not induce cell death in our experiments.

Effect of statins on the secretion of proinflammatory cytokines from DC The influence of statins on the secretion of proinflammatory cytokines by unstimulated and LPS-stimulated DC was investigated. As shown in Figure 4A (IL-6 and IL-8 shown as an example), statin incubation of unstimulated DC led to a significant reduction of their basal secretion of proinflammatory cytokines (percent reduction of S⫹/L– DC compared with S–/L– DC: IL-6, 35%, P⫽0.04; IL-8, 20%, P⫽0.05; IL-12, 53%, P⫽0.02; and TNF-␣, 69%, P⫽0.04). As expected, LPS stimulation caused a strong increase in the secretion of proinflammatory cytokines from DC. It was surprising that, and in contrast to the effect of statins on unstimulated DC, statin preincubation (1 ␮M, 48 h) of LPS-stimulated DC led to a further significant increase in their secretion of proinflammatory cytokines (percent increase of S⫹/L⫹ DC compared with S–/L⫹ DC: IL-6, 260%, P⫽0.03; IL-8, 257%, P⫽0.008; IL-12, 173%, P⫽0.05; and TNF-␣, 328%, P⫽0.02; Fig. 4B). This increase was independent of the type of statin used (simvastatin or atorvastatin) and could be reversed by MVA. To clarify the question if statins themselves induce the secretion of proinflammatory cytokines without a further stimulus, IL-6 and IL-8 concentrations were analyzed in the cell culture supernatant after a statin incubation and during the following LPS stimulation. In Figure 4C, the concentrations of IL-6 and IL-8 in the culture medium of S–/L⫹ DC, S⫹/L⫹ DC, and S⫹/L⫹ DC and MVA are shown at different time-points: after the 48-h statin incubation with statins before LPS stimulation (0) and during the following LPS stimulation at 8 h, 16 h, and 24 h. The bar graph shows that statin incubation itself did not cause a higher secretion of IL-6 and IL-8 from S⫹/L⫹ DC compared with S–/L⫹ DC (Fig. 4C; 0 h). In contrast, at 16 and 24 h, during LPS stimulation, significantly higher IL-6 and IL-8 concentrations were detected for S⫹/L⫹ DC than for S–/L⫹ DC. This finding indicates that the increase in the secretion of proinflammatory cytokines from statin-preincubated, LPS-stimulated DC is not caused by statins themselves but seems to be an amplification of the LPS effect by statins. Therefore, statin-preincubated DC were stimulated with CD154 to investigate whether the statin-enhanced release of proinflammatory cytokines by stimulated DC is dependent on LPS or whether it can also be induced by an alternative maturation stimulus. Analogous to LPS stimulation, preincubation of CD154-stimulated DC with statins caused an enhanced release of proinflammatory cytokines compared with statin-untreated, CD154-stimulated DC (data not shown), implying that the statin effect is independent of the kind of DC stimulation.

Suppression of the ability of DC to induce T cell proliferation, T cell activation, and Th1 differentiation by statins The most relevant function of DC is to activate T cells against certain antigens, thereby inducing an antigen-specific immune response. Therefore, we investigated the effect of statins on the ability of LPS-stimulated DC to induce T cell proliferation, T cell activation, and Th1 differentiation. As expected, compared with S–/L– DC, the use of S–/L⫹ DC in MLR led to a visible Yilmaz et al. Dendritic cell functions and statins

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Fig. 2. Statin effect on the expression of TLRs on DC (FACS). Expression of TLR2 (A), TLR3 (B), and TLR4 (C; MFI) on S–/L⫹ DC, S⫹/L⫹ DC, and S⫹/L⫹ DC ⫹ MVA after 48 h statin incubation at 3 h, 6 h, and 24 h of subsequent LPS stimulation. S–/L⫹ versus S⫹/L⫹ and S⫹/L⫹ versus S⫹/L⫹ ⫹ MVA, n ⫽ 5; *, P ⱕ 0.05.

increase in DC-T cell clustering (Fig. 5A) and a higher level of T cell proliferation (Fig. 5B) and T cell activation (Fig. 5C). Statin preincubation of LPS-stimulated DC caused a remarkable decrease in their clustering with T cells, reversed by MVA (Fig. 5A). Compared with S–/L⫹ DC, simvastatin or atorvastatin preincubation significantly reduced the ability of S⫹/L⫹ DC to induce T cell proliferation in MLR, even below the level of S–/L– DC (Fig. 5B). In addition, statins significantly suppressed the up-regulation of T cell activation markers, CD71 (early) and CD25 (late), induced by S⫹/L⫹ DC, below the level of S–/L– DC (Fig. 5C). As expected, a remarkably higher secretion of IFN-␥ and IL-2 from T cells was observed using S–/L⫹ DC rather than S–/L– DC in MLR (Fig. 5D). Statin preincubation of LPS-stimulated DC caused a significant suppression of their ability to induce IFN-␥ or IL-2 secretion from T cells in MLR.

DISCUSSION In recent studies, it was shown that statins possess immunomodulating effects, which can influence various immunological disorders, such as graft rejection, after cardiac transplantation [2, 7], experimental encephalomyelitis [4, 5], and sepsis [6]. However, the exact molecular mechanisms by which statins modulate the immune response had not been elucidated completely. Kwak et al. [3] described that statins inhibit the up-regulation of MHC II on nonprofessional APC such as

endothelial cells or macrophages. In addition, we recently showed that statins suppress the maturation of human monocyte-derived DC [20]. In our present study, we extended our investigations about the effect of statins on DC to further relevant DC functions such as endocytosis, cytokine secretion, and T cell stimulation. In contrast to our former study, a longer incubation time of 48 h enabled us to use smaller statin concentrations (0.1–1 ␮M). In addition, we analyzed the effect of statins on unstimulated (immature) as well as stimulated (maturing) DC. Instead of cytokines, we used the strong proinflammatory LPS for DC maturation, enabling us to analyze the effect of statins on the secretion of proinflammatory cytokines from DC. In concordance with our former study [20], statins suppressed the upregulation of costimulatory molecules (CD40, CD86), CD83, as well as of HLA-DR on LPS-stimulated DC. In addition, we now showed that even the basal expression of costimulatory molecules, CD83, and HLA-DR on unstimulated DC was downregulated by statins. TLRs, which recognize pathogen-associated molecular patterns, were proven to be crucial for the recognition of microbes by the innate immune system and thus, bridging the innate and acquired immune system [26]. Depending on the subtype of DC, a different pattern of TLR is expressed on myeloid DC (TLR1 to -3, -5 to -8, and -10), on plasmacytoid DC (TLR1, -6, -7, -9, and -10), and on monocyte-derived DC (TLR1 to -6 and -8) [26]. This variety of TLR expression allows DC to respond

4 Fig. 1. Statin effect on the expression of costimulatory molecules and HLA-DR on DC (FACS). (A, B) Histographic presentation and FACS graphs of CD86 and HLA-DR expression (MFI) on simvastatin (Simva)-treated (0.1–1 ␮M, 48 h), unstimulated DC (S⫹/L–) compared with statin-untreated, unstimulated DC (S–/L–) and statin (1 ␮M, 48 h) and MVA (100 ␮M)-coincubated, unstimulated DC (S⫹/L–⫹MVA). Statistically compared were S–/L– versus S⫹/L– and S⫹/L– versus S⫹/L– ⫹ MVA, n ⫽ 11; *, P ⱕ 0.05, and **, P ⱕ 0.005. (C, D) Expression of CD86 and HLA-DR (MFI) on statin-treated, LPS-stimulated DC (S⫹/L⫹) compared with statin-untreated, LPS-stimulated DC (S–/L⫹ DC) and statin (1 ␮M, 48 h) and MVA (100 ␮M)-coincubated, LPS-stimulated DC (S⫹/L⫹ DC⫹MVA). S–/L⫹ versus S⫹/L⫹ and S⫹/L⫹ versus S⫹/L⫹ ⫹ MVA, n ⫽ 13. (E, F) Comparison between the efficiency of simvastatin and atorvastatin (Atorva; each 1 ␮M, 48 h) to suppress CD86 or HLA-DR up-regulation on S⫹/L⫹ DC. S–/L⫹ versus S⫹/L⫹, n ⫽ 5.

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Fig. 3. Effects of statins on the endocytotic capacity of DC. (A, B) FACS example and histographic presentation of FITC-dextran uptake at 37°C by S⫹/L– DC compared with S–/L– DC and S⫹/L– DC ⫹ MVA at 4°C as negative control. Statistically compared were S–/L– versus S⫹/L– and S⫹/L– versus S⫹/L– ⫹ MVA, n ⫽ 5; *, P ⱕ 0.05. (C) FITC-dextran uptake of S⫹/L⫹ DC compared with S–/L⫹ DC and S⫹/L⫹ DC ⫹ MVA. S–/L⫹ versus S⫹/L⫹ and S⫹/L⫹ versus S⫹/L⫹ ⫹ MVA, n ⫽ 9.

differentially to microbial ligands [26]. Despite these differences in TLR patterns, signals through TLR generally result in the activation and maturation of all DC subtypes, leading to a nuclear factor-␬B-dependent up-regulation of costimulatory molecules, secretion of cytokines and chemokines, and an increase in their ability to stimulate T cells [26]. In human atherosclerotic plaques, the expression of TLR1, -2, and -4 was described recently [27]. In addition, the functional association of TLR4 with atherosclerosis was shown in apolipoprotein E knockout mice as well as in humans [28, 29]. In our study, we analyzed the effect of statins on the expression of TLR2, -3, and -4 on human monocyte-derived DC. Statin incubation of unstimulated DC did not significantly alter the expression of TLR2, -3, or -4. It was surprising that preincubation of LPSstimulated DC with statins caused a significant, temporary increase in the expression of TLR2 and -4 with a peak at 6 h. In contrast, TLR3 expression on LPS-stimulated DC was not affected significantly by statins. This divergent finding might be explained by major differences between TLR3 and TLR-2 or -4. First, TLR2 and -4 molecules are expressed mainly on the surface of DC, and TLR3 is located mainly in the intracellular endosomal compartments of DC [26] so that an up-regulation of TLR3 by statins might occur delayed. Second, the expression of TLR is generally up-regulated in the presence of their ligands. Thus, in contrast to TLR2 and -4, which respond to bacterial products, the expression of TLR3 is not up-regulated 534

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by LPS but in response to viral double-stranded RNA of polyinosinic-polycytidylic acid [30]. Third, in a previous study, a differential regulation of TLR3 and -4 expression was observed in the intestinal mucosa of patients with inflammatory bowel diseases [31]. However, analyses of the time kinetics of TLR2 and -4 expression on statin-treated DC showed that their increase peaked at 6 h of LPS stimulation and subsequently decreased. As the increase of TLR2 and -4 expression on LPS-stimulated DC by statins seems to be a temporary phenomenon, its relevance might therefore be limited in vivo. The uptake of molecules is a crucial function of immature DC and the prerequisite for their subsequent intracellular processing and presentation of the corresponding antigenic fragments on DC [24]. Antigen uptake simultaneous to DC maturation, induced by certain stimuli such as LPS, CD154, or proinflammatory cytokines, leads to a specific immune response against the antigen by presentation on DC to T cells. Otherwise, the uptake of self-antigens, e.g., from apoptotic cells, without a simultaneous DC maturation mediates tolerance by presenting them to T cells. Immature DC possess a high capacity of endocytosis, which is reduced remarkably during DC maturation, and the expression of costimulatory molecules and MHC II is up-regulated. In our study, the uptake of FITC-dextran by statin-incubated DC was analyzed. The endocytotic capacity of unstimulated as well as LPSstimulated DC was reduced significantly by statins. As the http://www.jleukbio.org

Fig. 4. Effects of statins on the secretion of proinflammatory cytokines from DC. (A) Levels (pg/mL) of IL-6 and IL-8, released by S⫹/L– DC, S–/L– DC, and S⫹/L– DC ⫹ MVA in the cell culture supernatant. Statistically compared were S–/L– versus S⫹/L– and S⫹/L– versus S⫹/L– ⫹ MVA, n ⫽ 7; *, P ⱕ 0.05. (B) Levels (pg/mL) of IL-6 and IL-8, released by S⫹/L⫹ DC, S–/L⫹ DC, and S⫹/L⫹ DC ⫹ MVA in the cell culture supernatant. S–/L⫹ versus S⫹/L⫹ and S⫹/L⫹ versus S⫹/L⫹ ⫹ MVA, n ⫽ 9. (C) Analyses of the time kinetics of IL-6 and IL-8 release (pg/mL, logarithmic presentation) from DC after simvastatin incubation (1 ␮M, 48 h) and before (0 h) and during LPS stimulation (8, 16, and 24 h). S–/L⫹ versus S⫹/L⫹ and S⫹/L⫹ versus S⫹/L⫹ ⫹ MVA, n ⫽ 4.

expression of costimulatory molecules and HLA-DR was reduced simultaneously by statins, DC maturation could not be the reason for the statin-induced reduction of endocytotic capacity. A similar finding of a discrepancy of endocytosis and maturation of DC was already described in a former study [32], indicating that changes of the endocytotic capacity can occur independently of DC maturation status. Thus, our results suggest that statins significantly suppress the endocytosis of antigens into DC. With regard to other cells, it was shown that

statins suppress LDL-cholesterol uptake into macrophages or smooth muscle cells [33, 34]. The local cytokine milieu plays a key role in the progression of atherosclerosis and emergence of acute ischemic complications. It was proven that proinflammatory cytokines such as IFN-␥, IL-6, IL-12, and TNF-␣ promote plaque progression and destabilization [35]. Therefore, we analyzed the effect of statins on the secretion of proinflammatory cytokines from DC. Regarding immature DC, statin incubation over 48 h caused a Yilmaz et al. Dendritic cell functions and statins

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Fig. 5. Suppression of the ability of LPS-stimulated DC to induce T cell proliferation, T cell activation, and Th1 differentiation by statins. (A) Characteristic pictures showing DC-T cell clustering in MLR cultures of S–/L– DC, S–/L⫹ DC, S⫹/L⫹ DC (simvastatin: 1 ␮M, 48 h), and S⫹/L⫹ DC ⫹ MVA (light microscopy: original magnification, 40⫻, unstained). (B) T cell proliferation, induced by allogeneic DC in MLR, correlated with the logarithmic reduction of incorporated CFSE fluorescence of CD3-labeled cells. Left: Histographic presentation of the CFSE-MFI of CD3-labeled T cells, stimulated with S–/L– DC, S–/L⫹ DC, S⫹/L⫹ DC, and S⫹/L⫹ DC ⫹ MVA, as a percentage of the CFSE-MFI value of unstimulated control T cells; DC/PBL ratios of 1:180, 1:60, 1:20, and 1:6. Statistically compared were S–/L⫹ versus S⫹/L⫹ and S⫹/L⫹ versus S⫹/L⫹ ⫹ MVA, n ⫽ 6; *, P ⱕ 0.05; **, P ⱕ 0.005. Right: FACS examples of CFSE staining (x-axis) of CD3-stained (y-axis) T cells, cocultured with S–/L– DC, S–/L⫹ DC, S⫹/L⫹ DC, and S⫹/L⫹ DC ⫹ MVA at a DC/PBL ratio of 1:20. (C) Expression of the activation markers CD71 (early) and CD25 (late) on T cells (MFI), stimulated with S–/L– DC, S–/L⫹ DC, S⫹/L⫹ DC, and S⫹/L⫹ DC ⫹ MVA, at a DC/PBL ratio of 1:20. S–/L⫹ versus S⫹/L⫹ and S⫹/L⫹ versus S⫹/L⫹ DC ⫹ MVA, n ⫽ 4. (D) Levels (pg/mL) of Th1-associated cytokines, IFN-␥ and IL-2, released from T cells, stimulated with S–/L– DC, S–/L⫹ DC, S⫹/L⫹ DC, and S⫹/L⫹ DC ⫹ MVA at a DC/PBL ratio of 1:20, into the MLR culture medium. S–/L⫹ versus S⫹/L⫹ and S⫹/L⫹ versus S⫹/L⫹ DC ⫹ MVA, n ⫽ 6.

significant, slight reduction of their basal secretion of several strong proinflammatory cytokines (IL-6, IL-8, IL-12, and TNF). In contrast, statin preincubation of CD154- or LPS-stimulated DC led to a further, significant increase in their IL-6, IL-8, IL-12, and TNF-␣ secretion. We showed that the increased production of proinflammatory cytokines was not induced by statins themselves but seems to be an amplification of the effect 536

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of different maturation stimuli. A similar finding was described for LPS-stimulated murine bone marrow-derived DC by Sun and Fernandes [21], who observed a significant increase in the secretion of TNF-␣, IL-6, and IL-12 by a lovastatin preincubation. Moreover, in recent studies, it was shown that statins enhance the secretion of proinflammatory cytokines from LPSstimulated monocytes or macrophages [36 –38]. However, the http://www.jleukbio.org

observed increase in the secretion of proinflammatory cytokines by statins was not yet confirmed in clinical studies. In contrast, a decrease in IL-6, IL-8, and monocyte chemoattractant protein-1 in the blood of patients with hypercholesterolemia under a statin treatment was described recently [39]. In addition, a decrease in proinflammatory cytokines by statins was observed in the case of intravenous application of LPS into healthy subjects [40, 41]. In small clinical studies, it was further shown that statin therapy reduced the incidence or mortality of severe sepsis [42, 43]. However, large, clinical, randomized trials are still needed to decide if the application of statins is feasible in patients with bacteremia or sepsis. T cells, present in atherosclerotic plaques, control local immunity through cytokine release or CD154-mediated activation of other immune-competent cells [35]. The progression of atherosclerosis is mainly associated with a Th1-immune response [35]. LPS is a strong, proinflammatory stimulus enabling APC to induce T cell stimulation and Th1 differentiation. Thus, we analyzed whether a statin preincubation of DC prior to LPS stimulation can effectively suppress the LPSinduced increase in their ability to induce T cell proliferation, activation, and Th1 differentiation. In our study, we showed that the ability of LPS-stimulated DC to induce T cell proliferation was reduced significantly by statin preincubation. Furthermore, statin preincubation significantly suppressed the ability of LPS-stimulated DC to induce the up-regulation of certain functionally important T cell activation markers: CD71 and CD25. CD71, up-regulated in the first 12 h after T cell activation, is a serum iron-transport protein, which interacts with the T cell receptor (TCR), and thus, is important for the efficient formation of cell contacts of T cells with APC [44, 45]. CD25, an IL-2 receptor ␣-chain, is required for the clonal expansion of T cells through stimulation of the TCR/CD3 complex during the induction of an antigen-specific immune response. CD25 is up-regulated after 48 h and thus, is considered to be a hallmark as late activation/memory marker expressed on T cells [44, 45]. The influence of statins on DCinduced Th1 differentiation was investigated by analysis of the release of Th1-associated cytokines (IFN-␥ and IL-2) from T cells in MLR. We showed that statin preincubation of LPSstimulated DC significantly reduced their ability to induce IFN-␥- and IL-2-secreting T cells. These results are likely to explain the beneficial effects of statins observed in animal models with Th1-associated disorders such as autoimmune encephalitis [4, 5]. As it is known that a Th1-immune response to certain antigens, e.g., oxidized LDL-cholesterol, accelerates atherogenesis [35], the suppressive effect of statins on the ability of DC to induce T cell proliferation, activation, and Th1 differentiation can be considered as another beneficial property of these substances in the treatment of atherosclerosis. For all statin effects described, no significant differences between simvastatin or atorvastatin were observed, and all effects were reversed by MVA, indicating an inhibition of the HMG-CoA reductase as an underlying mechanism. As the used statin concentrations were in a therapeutic range (0.1–5 ␮M) [46], an in vivo relevance of the described effects can be assumed. In conclusion, in the present study, we showed that statins suppress endocytotic capacity of immature DC, necessary for

the uptake of foreign antigens, and the ability of matured DC to activate T cells, necessary for the induction of immune responses. Regarding cytokine secretion, divergent results were obtained. On the one hand, statins suppressed the basal secretion of proinflammatory cytokines from immature DC. Conversely, statins enhanced the secretion of proinflammatory cytokines from LPS-stimulated DC. The results of our study elucidate an underlying molecular mechanism of immunomodulation by statins, suggesting their benefit in the treatment of Th1-driven immunological diseases. However, statin-induced amplification of proinflammatory cytokine secretion from stimulated DC needs to be investigated further.

ACKNOWLEDGMENTS This work was supported by the Interdisciplinary Center for Clinical Research of the University of Erlangen-Nuremberg, German Heart Foundation, and Pfizer Global Research and Development. We are grateful to Mrs. Sabine Kesting, Mrs. Doris Flick, and Ms. Katja Schubert for technical assistance and to Ms. Mona Woyciewski for her helpful corrections of this work. A. Y. and C. R. contributed equally to this paper.

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