The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 180, 6988 -6996
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, X.
Right arrow Articles by Markovic-Plese, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, X.
Right arrow Articles by Markovic-Plese, S.

Simvastatin Inhibits IL-17 Secretion by Targeting Multiple IL-17-Regulatory Cytokines and by Inhibiting the Expression of IL-17 Transcription Factor RORC in CD4+ Lymphocytes

Xin Zhang*, Jianping Jin{dagger}, Xueyan Peng§, Vinod S. Ramgolam* and Silva Markovic-Plese1,*,{ddagger}

* Department of Neurology, {dagger} Center for Bioinformatics, and {ddagger} Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599; and § Department of Neurology, Yale University School of Medicine, New Haven, CT 06510


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Statins, extensively used as cholesterol-lowering agents, have recently been identified as immunomodulatory agents. This study investigated the statins’ mechanisms that target the autoimmune response in humans, and evaluated their therapeutic potential in multiple sclerosis. Our results demonstrated statin-mediated increases in suppressor of cytokine secretion (SOCS) 3 and suppressor of cytokine secretion 7, which negatively regulate the STAT/JAK signal transduction pathway and IL-6 and IL-23 gene expression in monocytes. Simvastatin also induced IFN-{gamma}, IL-4, and IL-27 production in monocytes, which together inhibited IL-17 transcription and secretion in CD4+ T cells. IL-17-producing CD4+ cells, referred to as Th17 cells, have recently been found to play a central role in the development of autoimmune diseases. Furthermore, simvastatin directly inhibited the expression of retinoic acid-related orphan nuclear hormone receptor C, a transcription factor that controls IL-17 production in CD4+ T cells. This effect was reversed by mevalonic acid, a downstream metabolite of 3-hydroxy-3-methylglutaryl CoA reductase, confirming that simvastatin’s specific effect is through the inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase. These results provide evidence for the novel immunomodulatory mechanisms of statins, which selectively target the regulation of cytokine transcription involved in the development of the human autoimmune response. Based on the described immunomodulatory mechanisms, good safety profile and oral bioavailability, statins represent a promising therapeutic approach for multiple sclerosis and other chronic inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple sclerosis (MS)2 is a chronic progressive inflammatory CNS disease with a chronic inflammatory response directed against the myelin-derived proteins (1, 2). MS is one of the best-characterized neurological diseases, with effective therapies targeting specific steps in the disease development (3). Available therapies for relapsing remitting (RR) MS target peripheral autoreactive T cell activation (glatiramer acetate, GA), immunocompetent cell migration into the CNS (IFN β), and natalizumab), or suppress inflammatory response (GA, mitoxantrone) (4). Despite effective suppression of the inflammatory response, disease often progresses, which reflects the complexity of the immune dysregulation and neurodegenerative processes that are only partially blocked by targeting individual steps in the disease pathogenesis. In addition, close to 30% of patients are considered nonresponders, as they experience a breakthrough disease activity while on therapy. Therefore, new therapies and combination thereof are actively sought with even more enthusiasm, which is supported by the more comprehensive understanding of the disease pathogenesis.

Statins are inhibitors of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, an early and rate-limiting step in cholesterol biosynthesis. Statins are effective cholesterol-lowering agents, which have been extensively used for primary and secondary prevention of cardiovascular disease. Recent studies have demonstrated statins’ immunomodulatory effects, which are only partially mediated by the lowering of cholesterol levels (5). Kwak et al. (6) first reported a dose-dependent inhibition of IFN-{gamma}-induced MHC class II expression by human monocytes, macrophages, and endothelial cells. Studies of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, have indicated that statins induce a shift from the production of proinflammatory (Th1) cytokines (IL-2, IL-12, IFN-{gamma}, and TNF-{alpha}) to the production of Th2 cytokines (IL-4, IL-5, and IL-10) (7). The multiple immunomodulatory effects of statins resulted in the inhibition of CNS lesion formation, and the delayed onset and ameliorated severity of EAE (7, 8). However, statins’ specific targets in human autoimmune response are not well defined.

Our study reports that simvastatin induces suppressor of cytokine secretion (SOCS) 3 and 7 expression in monocytes, which inhibit transcription of IL-6 and IL-23, cytokines that play an important role in the development of the autoimmune response in MS. The recently identified SOCS protein family (CIS, SOCS1, 2, 3, 4, 5, 6, and 7) inhibits cytokine signaling via a negative feedback loop and prevents excessive cellular responses to cytokines. SOCS3, one of the best characterized members of the SOCS family, is induced in a multitude of cell types by IL-2, IL-3, IL-6, IL-12, IL-23, IFN{alpha}/β/{gamma}, IL-27, IL-4, IL-10, IL-1, TGF-β, TNF-{alpha}, GM-CSF, LPS, growth hormone, prolactin, and insulin. The pattern of SOCS3 expression is dependent on the cytokine induction, but also on the cell type in which it is induced. SOCS3 blocks JAK1 and JAK2, and phosphorylation-dependent activation of STAT3 in response to IL-6. It targets the IL-6 receptor signaling complex gp130 for proteosomal degradation. Similarly, SOCS3 inhibits the STAT3 transduction pathways for IL-23 (9). Therefore, SOCS3 induction by statins may have an inhibitory effect on multiple inflammatory cytokine signal transduction pathways that mediate the autoimmune response.

Th17 is a recently identified subset of CD4+ T cells that is critical for the development of autoimmune diseases. Th17 cells produce IL-17A (also known as IL-17), IL-17F, TNF-{alpha}, GM-CSF, and IL-6, in response to TGFβ and IL-6 in mice. The expansion of this T cell subset is mediated by IL-23 and inhibited by IL-27 (10), and its relation to Th1 and Th2 cell lineages is currently investigated. Recent studies have revealed that great differences exist between mice and human Th17 cell lineage differentiation. Several groups have reported that TGFβ does not stimulate, but instead inhibits the human Th17 differentiation (11, 12, 13). As reported by Chen et al. (13), IL-23 induces Th17 differentiation in humans, while it has been established that it does not induce Th17 differentiation in naive animal cells due to the lack of IL23R expression. In humans, IL-23 up-regulates its own receptor on T cells and increases retinoic acid-related orphan nuclear hormone receptor C (RORC) and IL-17 expression (13). IL-21 has recently been identified in animal systems as a cytokine that induces ROR{gamma}t expression and IL-17 production through the induction of IL23R expression. IL-21 is secreted by Th17 cells and in an autocrine fashion may further enhance IL-17 secretion via induction of STAT3 phosphorylation (14).

IL-17 mRNA is elevated in the active MS brain lesions, as reported by Lock et al. (15) and detected in our unpublished results (Montes et al., submitted for publication). IL-17 gene expression is significantly increased in mononuclear cells derived from the blood and from cerebrospinal fluid of MS patients in comparison to healthy controls (HCs), and numbers of IL-17-expressing blood mononuclear cells are higher during MS exacerbations than in clinically silent periods (16). Because IL-17 has been detected in the target organs of patients with other autoimmune diseases, including rheumatoid arthritis, psoriasis and autoimmune uveitis, it is conceivable that it plays a role in human autoimmune processes (17) and represents a selective target for the treatment of autoimmune diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Study subjects

Thirty-four patients with confirmed diagnoses of RR MS and 10 age-, sex-, and race-matched HCs were enrolled in the study upon signing an institutional review board-approved informed consent form. The inclusion criteria for the MS patients consisted of a confirmed diagnosis of RR MS according to McDonald’s diagnostic criteria (18), age 18–55, and an extended disability status score of 1.5–5.5. The exclusion criteria were history of hyperlipidemia treated with statins and immunomodulatory or immunosuppresive therapy at the time of blood sample collection. The treatment-free period was at least 6 wk for IV methylprednisolone, 3 mo for IFN-β and GA, and 6 mo for azathioprine, methotrexate, mitoxantrone, cyclophosphamide, and other immunosuppressive medications.

Gene arrays

PBMCs were separated using Lymphocyte Separation Medium (BioWhittaker) from blood samples of 10 patients with RR MS. Five x 106 cells per condition were stimulated with plate immobilized {alpha}CD3 (1 µg/ml) and {alpha}CD28 (5 µg/ml) mAb (BD Pharmingen) in the absence or presence of simvastatin (1 µM) (Calbiochem) for 24 h. Total RNA was isolated by RNeasy kit (Quiagen). cDNA was prepared by reverse transcription and hybridized to a positively charged membrane containing the arrayed DNA. Gene expression of 102 inflammatory genes was measured using GEA Th1–2–3 expression system (SuperArray). Arrays contained cDNA probes including control sequences (blank, Puc-18, β-actin, GAPDH, Cyclophillin-1) in four replicates. Gene expression was measured using Gene Pix Pro 5.0 software (Axon Instruments). The background-subtracted signals were used to calculate the expression level for each gene. The median expression value of all genes on the membrane was used for the normalization of each individual gene on the membrane. Each value was normalized again against the median of that gene in all membranes. The genes with <1.2-fold change between untreated and simvastatin-treated cultures and genes with raw expression levels below 600 in 5 out of 20 membranes were filtered out from statistical analysis, which was performed by a paired t test comparing each of the normalized values of the simvastatin-treated samples to those of the nontreated samples. Gene tree clustering was conducted using Gene Spring software (Agilent Technologies).

Cell cultures

CD14+ monocytes were isolated from MS patients (n = 10) and age-, sex-, and race-matched HCs (n = 10) by EasySep Negative Selection Monocyte Enrichment Kit (StemCell Technologies) and CD4+ T cells by MACS CD4+ T Cell Isolation Kit (Miltenyi Biotec). Cell purity was confirmed by flow cytometry (>95%). Five x 106 monocytes per condition were pretreated with simvastatin (10 µM) for 24 h in serum-free media (Opti MEM, Life Technologies), and then stimulated with LPS (1 µg/ml) (Sigma-Aldrich) for 2 h before RNA extraction and for 48 h before supernatants collection. Five x 106 CD4+ T cells per condition were pretreated as above, and then stimulated with plate immobilized {alpha}CD3 (1 µg/ml) and {alpha}CD28 (5 µg/ml) mAb for 4 h before RNA extraction and for 48 h before supernatant collection. In some experiments, 5 x 106 CD4+ purified T cells per condition from the MS patients were pretreated in the presence or absence of mevalonic acid (Sigma-Aldrich) (100 µM) for 24 h before stimulation.

CD45RA cells were separated by MACS CD45RA+ T Cell Isolation Kit (Miltenyi Biotec) and cultured for 24 h in the absence or presence of simvastatin and indicated cytokines (IL-6, IL-23, IFN-{gamma}, IL-4, IL-27 at 50 ng/ml) or cytokine blocking Abs (at concentration 10 µg/ml) for 24 h. Cells were then stimulated with plate immobilized {alpha}CD3 and {alpha}CD28 mAb. After 5 days, IL-2 (20 U/ml) was added and the cultures were expanded for an additional 7 days. At day 12, cells were harvested for quantitative RT-PCR (qRT-PCR), while supernatants were frozen for the measurement of secreted cytokines by ELISA.

qRT-PCR

Total RNA was isolated using a RNeasy kit, and cDNA was synthesized using a High Capacity cDNA Archive Kit (Applied Biosystems). The primers for SOCS1, SOCS3, SOCS5, SOCS7, IL-23A, IL-6, IFN-{gamma}, IL-27, IL-4, IL-17A, IL-21, and RORC were purchased from Applied Biosystems and gene expression was measured by qRT-PCR using Taqman Gene Expression Assays (Applied Biosystems). Results are expressed for each subject as relative gene expression normalized for the 18S mRNA expression.

ELISA

Supernatants from the cell cultures were collected and stored at –80°C until the cytokine measurements by ELISA. IL-17 was measured using a Human IL-17A ELISA Constuction Kit (Antigenix America), IL-27 using an ELISA kit purchased from R&D Systems. IL-2, IFN-{gamma}, IL-4, IL-6, IL-10, IL-12p40, IL-12p70 (BD Pharmingen), and IL-23 (eBiosciences) were measured in duplicate by ELISA following the manufacturer’s recommendation. Results are expressed for each subject as cytokine concentration in pg/ml.

Western blotting

Human negatively selected CD14+ monocytes were plated at 3 x 106 cells per condition in the absence or presence of simvastatin (10 µM) for 24 h in serum-free media, and then stimulated with LPS (1 µg/ml) for 2 h before protein extraction. Proteins from cell lysates were denatured in SDS, resolved by 7.5% SDS-PAGE for STAT detection and by 12.5% SDS-PAGE for SOCS3 detection, and transferred on polyvinylidene difluoride membranes prewetted by 100% methanol. Nonspecific binding was blocked for 30 min in the blocking buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 2% FishGelatin, 1% OVA in double distilled water) and incubated overnight with the mouse anti-human Ab for SOCS3, Tubulin (Santa Cruz Biotechnology), and STAT3 (Cell Signaling Technology), and the rabbit anti-human Ab for STAT1, pSTAT1, and pSTAT3 (Cell Signaling Technology). Blots were washed with TBS Tween 20, and then incubated with IRDye 680 conjugated anti-rabbit IgG or anti-mouse IgG secondary Ab (Li-Cor Biosciences) for 1 h. The blots were washed again and protein bands detected with Odyssey Infrared Imaging System (Li-Cor Biosciences). The intensity of the visualized bands was quantified by Odyssey 2.0 software (Li-Cor Biosciences).

Statistics

A linear regression model in the R package was used to investigate the relationship between SOCS3/SOCS7 and IL-6/IL-23 gene expression. A negative coefficient with p ≤ 0.05 was considered to indicate a change in gene expression.

The statistical analysis for the qRT-PCR and ELISA results was performed by Wilcoxon matched pairs test analysis (GraphPad InStat), followed by a Benjamini and Hochberg correction for multiple comparisons. Adjusted p values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Simvastatin changes the expression of multiple inflammatory genes in activated PBMCs, most significantly SOCS3 and SOCS7

Our results, and the studies of others, indicate that statins have pleiotropic immunomodulatory effects that include inhibition of Ag presentation, T cell activation and differentiation, and mononuclear cell recruitment into the CNS (19). To simultaneously capture simvastatin-induced changes in the expression of multiple genes involved in the human inflammatory response, and to establish simvastatin’s operative mechanism of action targeting PBMCs in patients with RR MS, we used Th1–2–3 cDNA arrays (SuperArrays). The arrays contained 102 specific cytokines, chemokines, and their receptors; activation-, costimulatory-, and adhesion-molecules; transcription factors; apoptotic molecules; and negative and positive control probes. Because our previous results (19) and a study by Neuhaus et al. (20) reported statins’ anti-proliferative effect only in activated PBMCs, we tested simvastatin’s effect on immune response gene expression in PBMCs stimulated with plate immobilized {alpha}CD3 and {alpha}CD28 mAbs for 24 h. PBMCs were derived from 10 RR MS patients.

Simvastatin significantly increased the expression of SOCS3 and SOCS7 mRNA (Fig. 1). The expression of SOCS3 increased 17-fold, and the expression of SOCS7 increased 11-fold in simvastatin-treated cultures, in comparison to the untreated PBMCs. Simvastatin significantly decreased the expression of the CD25 (12.6-fold), consistent with our reported results on the simvastatin-induced decrease in CD25 surface expression in CD4+ and CD8+ lymphocytes as measured by flow cytometry (19). The costimulatory molecule CTLA-4 decreased 8.5-fold and CD27 decreased 2.3-fold in the simvastatin-treated PBMCs compared with the untreated samples. IL-4R expression decreased 4.7-fold. These changes likely reflect decreased cell activation.


Figure 1
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 1. Simvastatin induces changes in the expression of multiple genes. The gene tree presents an average result for all genes with at least a >2-fold change in the simvastatin-treated vs untreated samples. Inflammatory gene expression profiling was performed using a GEA Th1–2–3 expression system. Arrays contained 102 cDNA probes for immune response genes, including control sequences. The PBMCs were derived from 10 patients with RR MS. Gene expression was measured in 5 x 106 cells stimulated with plate-immobilized {alpha}CD3 and {alpha}CD28 mAbs in the absence or presence of simvastatin (1 µM) in serum-free media for 24 h. Statistical analysis was performed by a paired t test comparing each of the normalized values of the simvastatin-treated samples to those of the nontreated samples. Asterisk denotes genes with statistically different expression in simvastatin-treated vs untreated samples.

 
qRT-PCR experiments confirmed the significant up-regulation of SOCS3 (p = 0.014) and SOCS7 (p = 0.002) in the PBMCs derived from 10 RR MS patients, data not shown. Our qRT-PCR results confirm the gene array results and provide more accurate quantitative measurements; however, they do not reveal which cell subsets contribute to the gene expression changes induced by simvastatin in PBMCs.

Simvastatin induces an increase in SOCS3 and SOCS7 mRNA expression, which inhibits IL-23 and IL-6 gene expression in monocytes derived from MS patients and HCs

To identify which cell subsets are selectively targeted by simvastatin, we performed qRT-PCR for multiple members of the SOCS family (SOCS1, 3, 5, 7) and cytokine genes (IFN-{gamma}, IL-27, IL-6, IL-23, and IL-4) in separated CD14+ monocytes and CD3+ (CD4+ and CD8+) lymphocytes. The simvastatin-mediated increase in SOCS3 and SOCS7 gene expression is present in monocytes derived from MS patients (p = 0.006 and p = 0.006, respectively, Fig. 2A), and not in CD3+ T cells (data not shown). LPS-stimulated monocytes derived from age-, sex-, and race-matched HCs also exhibited a significant, but less prominent, increase in SOCS3 expression (p = 0.006 in MS patients; p = 0.0118 in HCs). Up-regulation of SOCS7 in HCs was not statistically significant. Study of simvastatin’s effect on cytokine gene expression in monocytes revealed a significant decrease in IL-23 expression in the MS patients (p = 0.006), while the decrease in the HCs did not reach statistical significance (Fig. 2A). IL-6, another cytokine negatively regulated by SOCS3, exhibited decreased expression in simvastatin-treated cultures in both the MS patients and HCs, but the differences were not significant. Measurement of monocyte-secreted IL-6 in simvastatin-treated cultures revealed a significantly decreased IL-6 secretion in both MS patients (p = 0.003) and the HCs (p = 0.012), data not shown.


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 2. Simvastatin induces an increase in SOCS3 and SOCS7 gene expression in monocytes. A, Simvastatin increases SOCS3 and SOCS7 gene expression, and inhibits IL-23A and IL-6 transcription in monocytes. Five x 106 purified monocytes derived from 10 RR MS patients and 10 matched HCs were cultured in the absence or presence of simvastatin (10 µM) for 24 h in serum-free media, and then stimulated with LPS (1 µg/ml) for 2 h. SOCS3, SOCS7, IL-6, and IL-23A gene expression was measured by qRT-PCR. The results are expressed as relative gene expression, normalized for 18S mRNA expression. B, Simvastatin increases SOCS3 protein expression and inhibits STAT1 and STAT3 phosphorylation in monocytes. Three x 106 negatively selected CD14+ monocytes were cultured in the absence or presence of simvastatin (10 µM) for 24 h in serum-free media, and then stimulated with LPS for 2 h before protein extraction. SOCS3 expression and STAT1 and STAT3 phosphorylation were measured by Western blotting. The results show a representative blot from three similar experiments.

 
A linear regression analysis was used to examine the relationship of statin-induced increase in SOCS3 and SOCS7 gene expression with the decreased gene expression of IL-23 and IL-6 in the same cultures. Up-regulation of SOCS3 had a significant interaction with down-regulation of IL-6 and IL-23 in RR MS patients (SOCS3-IL-6: p = 0.004; SOCS3-IL-23: p = 0.007) while the interaction of SOCS7 gene expression with the IL-6 and IL-23 cytokine gene expression was less prominent (SOCS7-IL-6: p = 0.035; SOCS7-IL-23: p = ns). In contrast, the interaction of SOCS3 and SOCS7 gene expression with down-regulation of cytokine gene expression in HCs was not statistically significant.

To examine the mechanisms by which a statin-induced increase in SOCS3 expression affects cytokine transcription in monocytes, we determined the effect of SOCS3 induction on the phosphorylation of the STAT1 and STAT3 proteins. Simvastatin-induced SOCS3 expression in monocytes inhibited phosphorylation of STAT3, a key transcription factor involved in the regulation of IL-6 and IL-23 production, as well as the phosphorylation of STAT1 (Fig. 2B). The statins’ inhibition of STAT3 and STAT1 phosphorylation was reversed by mevalonate, a downstream metabolite of HMG-CoA reductase, confirming the simvastatin’s specific effect through the inhibition of HMG-CoA reductase (Fig. 2B).

Simvastatin induces an increase in IFN-{gamma}, IL-4, and IL-27 production in monocytes derived from MS patients and HCs

Further studies on monocytes have identified a significant simvastatin-induced increase in IFN-{gamma} gene expression in MS patients (4.3-fold) and HCs (10.2-fold), Fig. 3A. Similar changes were detected in IFN-{gamma} protein secretion: simvastatin increased IFN-{gamma} secretion in MS patients (6.5-fold) and 1.7-fold in HCs (Fig. 3B). In MS patients, IL-27, a member of the IL-12 family that is induced by IFN-{gamma}, also displayed significantly increased gene expression (1.6-fold, p = 0.025) in monocytes upon treatment with simvastatin, while the difference was not statistically significant in HCs. IL-27 secretion was significantly higher in simvastatin-treated monocytes derived from MS patients (p = 0.004), while the difference in HCs was not statistically significant, Fig. 3B. IL-4 gene expression was increased by simvastatin in the monocytes derived from both MS patients (2.3-fold) and HCs (1.6-fold). Similar increase in secreted IL-4 cytokine level was detected in MS patients (2.0-fold, p = 0.004) while the increase in HCs was not significant (Fig. 3B). It is of note that the gene expression of SOCS1 and SOCS5, which regulate IFN-{gamma} and IL-4 transcription respectively, was not significantly changed by the treatment of monocytes with simvastatin (data not shown).


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 3. Simvastatin increases IFN-{gamma}, IL-4, and IL-27 production in monocytes. A, Five x 106 purified monocytes derived from 10 RR MS patients and 10 matched HCs were cultured in the absence or presence of simvastatin (10 µM) for 24 h in serum-free media, and then stimulated with LPS for 2 h before RNA extraction. IFN-{gamma}, IL-4, and IL-27 gene expression was measured by qRT-PCR and the results presented as a relative expression to the 18S mRA. B, After 48 h of monocyte stimulation with LPS in the absence or presence of simvastatin, IFN-{gamma}, IL-4, and IL-27 were measured in the supernatants by ELISA. The results are expressed as cytokine concentration in pg/ml.

 
The inhibitiory effect on IL-17 expression by CD4+ cells is mediated by the cytokine pattern changes induced by simvastatin treatment of monocytes

Simvastatin inhibited IL-6 and IL-23 and induced IFN-{gamma}, IL-4, and IL-27 gene expression. Because the former cytokines were reported to induce IL-17 production in CD4+ cells and the latter to inhibit IL-17 production (10), we predicted that the net effect of statin-induced changes in the cytokine production by monocytes would be the inhibition of IL-17 production in CD4+ cells. We confirmed that culturing of CD45RA+ cells in the presence of IL-6 and IL-23 increased, and the addition of IFN-{gamma}, IL-4, and IL-27 decreased the IL-17 secretion. In contrast, mAb blocking of IL-6 and IL-23 inhibited, while mAb blocking of IFN-{gamma}, IL-4, and IL-27 induced IL-17 secretion in CD45RA+ cells (Fig. 4A). Consistent results were obtained for RORC and IL-17 gene expression in the same samples tested by qRT-PCR (data not shown).


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 4. Simvastatin’s effects on cytokine production in monocytes inhibit IL-17 secretion in CD4 cells. A, IL-17A secretion by {alpha}CD3 plus {alpha}CD28 mAb-stimulated CD4RA+ cells was induced by IL-6 and IL-23, while the addition of IFN-{gamma}, IL-4, and IL-27 inhibited RORC and IL-17 gene expression. Two x 106 negatively selected CD45RA+ naive cells were cultured in serum-free media with the indicated cytokines or blocking Abs for 24 h, and then stimulated with plate-immobilized {alpha}CD3 and {alpha}CD28 mAb. After 5 days, IL-2 (20 U/ml) was added and the cultures were expanded for an additional 7 days. At day 12, the supernatants were collected and the secreted IL-17 was measured by ELISA. Representative results from two similar experiments performed in duplicate are shown. B, Supernatants from simvastatin-treated monocytes inhibited RORC and IL-17A gene expression in CD4+ cells. Five x 106 purified monocytes derived from three RR MS patients were pretreated with simvastatin (10 µM) for 24 h in serum-free media, and then stimulated with LPS for 24 h. The supernatants from the monocyte cultures were transferred to the negatively separated allogenic CD4+ cells derived from RR MS patients. After 24-h coincubation, the CD4+ cells were stimulated with plate-immobilized {alpha}CD3 and {alpha}CD28 mAbs for 4 h before RNA extraction. RORC and IL-17A gene expression was measured by qRT-PCR. The results represent one out of three similar experiments, performed in triplicate.

 
To directly test the prediction that simvastatin-induced changes in monocyte cytokine secretion suppress IL-17 secretion in CD4 cells, the supernatants from the simvastatin-treated monocytes were transferred to the activated allogenic CD4+ T cells. Supernatants from the simvastatin-treated monocytes inhibited the expression of transcription factor RORC and IL-17 in CD4+ T cells in comparison to the CD4+ T cells cultured with the supernatants of untreated monocytes (Fig. 4B).

Simvastatin directly inhibits gene expression and secretion of IL-17 by CD4+ lymphocytes in MS patients and HCs

The main finding of our study is that simvastatin significantly inhibited RORC and IL-17A gene expression in the activated CD4+ cells derived from MS patients (–2.4 and –5.5-fold, respectively) and HCs (–2.7, and –2.2-fold, respectively). Simvastatin inhibited IL-17A protein secretion in CD4+ T cells from MS patients and HCs (–3.3-fold and –2.5-fold, respectively), Fig. 5A. In addition, simvastatin inhibited IL-21 gene expression in the same samples from MS patients (–2.6-fold, p = 0.003) and from HCs (–2.4-fold, p = 0.003), indicating that it may also inhibit the autocrine IL-21-mediated induction of IL-17 transcription in CD4 cells (data not shown).


Figure 5
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 5. Simvastatin inhibits RORC and IL-17 gene expression and secretion of IL-17A by CD4+ T cells in RR MS patients and HCs. A, Five x 106 negatively selected CD4+ T cells were pretreated with simvastatin (10 µM) for 24 h in serum-free media, and then stimulated with plate-immobilized {alpha}CD3 and {alpha}CD28 mAb. After 4 h of stimulation, total RNA was extracted and RORC and IL-17A gene expression was measured by qRT-PCR. Results are expressed as relative gene expression normalized for 18S mRNA expression. After 48 h of stimulation, IL-17A protein secretion was measured in supernatants by ELISA. The results are expressed as cytokine concentration in pg/ml. B, 5 x 106 negatively selected CD45RA+ naive cells were cultured in serum-free media in the absence or presence of simvastatin and indicated cytokines or blocking Abs for 24 h, and then stimulated with plate immobilized {alpha}CD3 and {alpha}CD28 mAb. After 5 days, IL-2 (20 U/ml) was added and the cultures were expanded for an additional 7 days. At day 12, supernatants were collected and IL-17 secretion was measured by ELISA. The figure shows representative results from three similar experiments performed in triplicate.

 
We also confirmed in long-term cultures that simvastatin inhibits IL-17 secretion in naive CD45RA+ cells differentiated in the presence of Th17 polarizing cytokines (IL-6 and IL-23). Simvastatin’s effect was reversed by addition of anti-IFN-{gamma}, IL-4, and IL-27 mAb (Fig. 5B). Similar results were obtained for RORC and IL-17 gene expression in the same samples tested by qRT-PCR (data not shown).

Simvastatin-mediated inhibition of RORC and IL-17A gene expression is reversed by mevalonic acid

Both the simvastatin-mediated decrease in IL-17A and RORC expression were reversed by mevalonic acid, indicating that simvastatin’s effect is mediated by the inhibition of HMG-CoA reductase (Fig. 6).


Figure 6
View larger version (10K):
[in this window]
[in a new window]

  		FIGURE 6. Simvastatin-mediated inhibition of RORC and IL-17A gene expression is reversed by mevalonic acid. Five x 106 negatively selected CD4+ T cells derived from four RR MS patients were pretreated with simvastatin (10 µM) in the absence or presence of mevalonic acid (100 µM) for 24 h in serum-free media, and then stimulated with plate-immobilized {alpha}CD3 and {alpha}CD28 mAb. After 4-h stimulation, total RNA was extracted and IL-17A and RORC expression was measured by qRT-PCR. The results represent one out of four similar experiments performed in triplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our data identified that treatment with simvastatin increases SOCS3 and SOCS7 expression in PBMCs and monocytes from RR MS patients and HCs (Figs. 1 and 2). Individual SOCS proteins regulate transcription of multiple cytokines, as well as the expression of MHC class II and costimulatory molecules, and represent an attractive therapeutic target in inflammatory diseases (21). The cell-penetrating SOCS3 protein has been successfully used as a treatment for animal model of inflammatory liver disease. The inhibition of MHC class II expression in CD11b+ splenocytes in cell-penetrating SOCS3-treated mice was mediated by the inhibition of STAT1 phosphorylation that interacts with the type IV CIITA promoter for the expression of MHC class II molecules (22). In an animal model of rheumatoid arthritis, intra-articular injection of recombinant SOCS3 reduced joint inflammation and decreased STAT3 phosphorylation and IL-6 production (23).

Therefore, the induction of SOCS3 expression with statins may be therapeutically effective in MS, as supported by studies of the animal model of MS. Li et al. (21) have reported that SOCS3-transduced dendritic cells (DCs) express a decreased level of MHC class II and CD86, have decreased production of IL-12 and IL-23, and were toleragenic, biasing T cell differentiation toward the Th2 phenotype in myelin oligodendrocite-specific T cells. Moreover, transfer of the SOCS3-transfected DCs to naive mice prevented the EAE development. Consistent with our results, Huang et al. (24) have demonstrated that statins induced SOCS3 in stimulated mice macrophage cell line, which was reversed by the mevalonic acid.

Our study demonstrated that simvastatin-induced SOCS3 expression in monocytes inhibited phosphorylation of STAT3 and STAT1 (Fig. 2B). Based on these results, we propose that changes in IL-6 and IL-23 are related to the SOCS3-mediated inhibition of STAT3 phosphorylation. Findings of an increased expression of IFN-{gamma}, IL-4, and IL-27 in simvastatin-treated monocytes will need to be elucidated, because SOCS1 and SOCS5, which regulate transcription of those cytokines are not significantly inhibited in monocytes upon statin treatment. SOCS1 and SOCS3 have significant sequence homology and both predominantly inhibit STAT1 and STAT3 phosphorylation, however, they are often expressed in a reciprocal fashion (9).

Our data (Fig. 3) and a study by Neuhaus et al. (20) and Kiesier et al. (25) reported an increase in IFN-{gamma} production by statins in the activated PBMCs and monocytes derived from MS patients. IFN-{gamma} has a dual role in the autoimmune inflammatory response: it is a proinflammatory Th1 cytokine, but it also plays a role in controlling autoimmune response by inducing apoptosis of autoreactive T cells in EAE (26). More recently, our understanding of the autoimmune response was significantly altered by reports on the Th17 cell lineage, whose development is suppressed by IFN-{gamma}, IL-12, and IL-27 (27). These studies explained previously contradictory findings that STAT1, IL-12RB, IFN-{gamma}-, and IFN-{gamma} receptor-deficient mice develop a severe form of EAE.

We confirmed that inhibition of IL-6 and IL-23 and culturing with IFN-{gamma}, IL-4, and IL-27 inhibited RORC and IL-17 gene expression, as well as IL-17 secretion by CD4 cells. These cytokine changes are consistent with the simvastatin-induced changes in cytokine secretion by monocytes (Fig. 4A). When supernatants from the simvastatin-treated monocytes were used to treat activated allogenic CD4+ cells, they significantly inhibited RORC transcription factor and IL-17A gene expression in CD4+ cells in comparison to the CD4+ cells cultured with the supernatants of untreated monocytes (Fig. 4B). This finding confirmed that statin-induced changes in IL-17-regulatory cytokine production by monocytes indeed suppress IL-17 secretion in CD4 cells.

In addition to this effect of simvastatin on monocytes’ secreted cytokines that regulate IL-17 production by CD4+ cells, we examined whether statins directly affect the IL-17 production by CD4+ T cells. Our results indicated that simvastatin significantly inhibits RORC and IL-17 gene expression, as well as IL-17 secretion in CD4+ cells derived from both RR MS patients and HCs (28) (Fig. 5A). Our previous results indicated that statins also inhibit T-bet and do not significantly change GATA3 expression in the PBMCs derived from MS patients (19). However, at this point it is not clear how RORC, T-bet, and GATA3 cross-regulate each other in the human system, and whether a subset of IL-17-producing CD4+ cells also produce IFN-{gamma} in MS patients, as reported in patients with Crohn’s disease (29) and autoimmune scleritis (17).

In conclusion, our study demonstrated that statin-induced changes in cytokine transcription in monocytes inhibited the production of IL-6 and IL-23, and induced IFN-{gamma}, IL-4, and IL-27, which together suppressed IL-17 secretion in the CD4+ cells derived from both MS patients and HCs. In addition, simvastatin directly suppressed RORC and IL-17 gene expression, as well as IL-17 secretion from the CD4 T cells (30). Our results characterized the novel anti-inflammatory mechanisms of statins, which selectively target the regulation of cytokine transcription and the development of the human autoimmune response. Based on the described immunomodulatory mechanisms, good safety profile, and oral bioavailability, statins represent a promising therapeutic approach for MS and other chronic inflammatory diseases.


   Acknowledgments
 
We thank Dr. Rick Meeker for helpful discussion and Danielle Speer and Leslie Ingram for help with blood samples collection.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Silva Markovic-Plese, 6109 Neuroscience Research Building, 103 Mason Farm Road, Chapel Hill, NC 27599. E-mail address: markovics{at}neurology.unc.edu Back

2 Abbreviations used in this paper: MS, multiple sclerosis; RR, relapsing remitting; GA, glatiramer acetate; EAE, experimental autoimmune encephalomyelitis; SOCS, suppressor of cytokine secretion; HC, healthy control; qRT-PCR, quantitative real time PCR; DC, dendritic cell; RORC, retinoic acid-related orphan nuclear hormone receptor C; HMG, 3-hydroxy-3-methylglutaryl. Back

Received for publication September 28, 2007. Accepted for publication March 10, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sospedra, M., R. Martin. 2005. Immunology of multiple sclerosis. Annu. Rev. Immunol. 23: 683-747. [Medline]
  2. Bielekova, B., B. Goodwin, N. Richert, I. Cortese, T. Kondo, G. Afshar, B. Gran, J. Eaton, J. Antel, J. A. Frank, et al 2000. Encephalitogenic potential of the myelin basic protein peptide (amino acids 83–99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand. Nat. Med. 6: 1167-1175. [Medline]
  3. Calabresi, P. A.. 2002. Considerations in the treatment of relapsing-remitting multiple sclerosis. Neurology 58: S10-S22. [Abstract/Free Full Text]
  4. Hafler, D. A.. 2004. Multiple sclerosis. J. Clin. Invest. 113: 788-794. [Medline]
  5. Zamvil, S. S., L. Steinman. 2002. Cholesterol-lowering statins possess anti-inflammatory activity that might be useful for treatment of MS. Neurology 59: 970-971. [Free Full Text]
  6. Kwak, B., F. Mulhaupt, S. Myit, F. Mach. 2000. Statins as a newly recognized type of immunomodulator. Nat. Med. 6: 1399-1402. [Medline]
  7. Youssef, S., O. Stuve, J. C. Patarroyo, P. J. Ruiz, J. L. Radosevich, E. M. Hur, M. Bravo, D. J. Mitchell, R. A. Sobel, L. Steinman, S. S. Zamvil. 2002. The HMG-CoA reductase inhibitor, atorvastatin, promotes a Th2 bias and reverses paralysis in central nervous system autoimmune disease. Nature 420: 78-84. [Medline]
  8. Nath, N., S. Giri, R. Prasad, A. K. Singh, I. Singh. 2004. Potential targets of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor for multiple sclerosis therapy. J. Immunol. 172: 1273-1286. [Abstract/Free Full Text]
  9. Elliott, J., J. A. Johnston. 2004. SOCS: role in inflammation, allergy and homeostasis. Trends Immunol. 25: 434-440. [Medline]
  10. Iwakura, Y., H. Ishigame. 2006. The IL-23/IL-17 axis in inflammation. J. Clin. Invest. 116: 1218-1222. [Medline]
  11. Acosta-Rodriguez, E. V., G. Napolitani, A. Lanzavecchia, F. Sallusto. 2007. Interleukins 1β and 6 but not transforming growth factor-β are essential for the differentiation of interleukin 17-producing human T helper cells. Nat. Immunol. 8: 942-949. [Medline]
  12. Evans, H. G., T. Suddason, I. Jackson, L. S. Taams, G. M. Lord. 2007. Optimal induction of T helper 17 cells in humans requires T cell receptor ligation in the context of Toll-like receptor-activated monocytes. Proc. Natl. Acad. Sci. USA 104: 17034-17039. [Abstract/Free Full Text]
  13. Chen, Z., C. M. Tato, L. Muul, A. Laurence, J. J. O'Shea. 2007. Distinct regulation of interleukin-17 in human T helper lymphocytes. Arthritis Rheum. 56: 2936-2946. [Medline]
  14. Wei, L., A. Laurence, K. M. Elias, J. J. O'Shea. 2007. IL-21 is produced by Th17 cells and drives IL-17 production in a STAT3-dependent manner. J. Biol. Chem. 282: 34605-34610. [Abstract/Free Full Text]
  15. Lock, C., G. Hermans, R. Pedotti, A. Brendolan, E. Schadt, H. Garren, A. Langer-Gould, S. Strober, B. Cannella, J. Allard, et al 2002. Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat. Med. 8: 500-508. [Medline]
  16. Matusevicius, D., P. Kivisakk, B. He, N. Kostulas, V. Ozenci, S. Fredrikson, H. Link. 1999. Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis. Mult. Scler. 5: 101-104. [Abstract/Free Full Text]
  17. Amadi-Obi, A., C. R. Yu, X. Liu, R. M. Mahdi, G. L. Clarke, R. B. Nussenblatt, I. Gery, Y. S. Lee, C. E. Egwuagu. 2007. T(H)17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1. Nat. Med. 13: 711-718. [Medline]
  18. McDonald, W. I., A. Compston, G. Edan, D. Goodkin, H. P. Hartung, F. D. Lublin, H. F. McFarland, D. W. Paty, C. H. Polman, S. C. Reingold, et al 2001. Recommended diagnostic criteria for multiple sclerosis: guidelines from the international panel on the diagnosis of multiple sclerosis. Ann. Neurol. 50: 121-127. [Medline]
  19. Peng, X., J. Jin, S. Giri, M. Montes, D. Sujkowski, Y. Tang, J. Smrtka, T. Vollmer, I. Singh, S. Markovic-Plese. 2006. Immunomodulatory effects of 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors, potential therapy for relapsing remitting multiple sclerosis. J. Neuroimmunol. 178: 130-139. [Medline]
  20. Neuhaus, O., S. Strasser-Fuchs, F. Fazekas, B. C. Kieseier, G. Niederwieser, H. P. Hartung, J. J. Archelos. 2002. Statins as immunomodulators: comparison with interferon-β 1b in MS. Neurology 59: 990-997. [Abstract/Free Full Text]
  21. Li, Y., N. Chu, A. Rostami, G. X. Zhang. 2006. Dendritic cells transduced with SOCS-3 exhibit a tolerogenic/DC2 phenotype that directs type 2 Th cell differentiation in vitro and in vivo. J. Immunol. 177: 1679-1688. [Abstract/Free Full Text]
  22. Jo, D., D. Liu, S. Yao, R. D. Collins, J. Hawiger. 2005. Intracellular protein therapy with SOCS3 inhibits inflammation and apoptosis. Nat. Med. 11: 892-908. [Medline]
  23. Wong, P. K., P. J. Egan, B. A. Croker, K. O'Donnell, N. A. Sims, S. Drake, H. Kiu, E. J. McManus, W. S. Alexander, A. W. Roberts, I. P. Wicks. 2006. SOCS-3 negatively regulates innate and adaptive immune mechanisms in acute IL-1-dependent inflammatory arthritis. J. Clin. Invest. 116: 1571-1581. [Medline]
  24. Huang, K. C., C. W. Chen, J. C. Chen, W. W. Lin. 2003. Statins induce suppressor of cytokine signaling-3 in macrophages. FEBS Lett. 555: 385-389. [Medline]
  25. Kieseier, B. C., J. J. Archelos, H. P. Hartung. 2004. Different effects of simvastatin and interferon β on the proteolytic activity of matrix metalloproteinases. Arch. Neurol. 61: 929-932. [Abstract/Free Full Text]
  26. Furlan, R., E. Brambilla, F. Ruffini, P. L. Poliani, A. Bergami, P. C. Marconi, D. M. Franciotta, G. Penna, G. Comi, L. Adorini, G. Martino. 2001. Intrathecal delivery of IFN-{gamma} protects C57BL/6 mice from chronic-progressive experimental autoimmune encephalomyelitis by increasing apoptosis of central nervous system-infiltrating lymphocytes. J. Immunol. 167: 1821-1829. [Abstract/Free Full Text]
  27. Kastelein, R. A., C. A. Hunter, D. J. Cua. 2007. Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation. Annu. Rev. Immunol. 25: 221-242. [Medline]
  28. Acosta-Rodriguez, E. V., L. Rivino, J. Geginat, D. Jarrossay, M. Gattorno, A. Lanzavecchia, F. Sallusto, G. Napolitani. 2007. Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat. Immunol. 8: 639-646. [Medline]
  29. Annunziato, F., L. Cosmi, V. Santarlasci, L. Maggi, F. Liotta, B. Mazzinghi, E. Parente, L. Fili, S. Ferri, F. Frosali, et al 2007. Phenotypic and functional features of human Th17 cells. J. Exp. Med. 204: 1849-1861. [Abstract/Free Full Text]
  30. Ivanov, I. I., B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille, D. J. Cua, D. R. Littman. 2006. The orphan nuclear receptor ROR{gamma}t directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126: 1121-1133. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
V. S. Ramgolam, Y. Sha, J. Jin, X. Zhang, and S. Markovic-Plese
IFN-{beta} Inhibits Human Th17 Cell Differentiation
J. Immunol., October 15, 2009; 183(8): 5418 - 5427.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
S.-i. Kagami, T. Owada, H. Kanari, Y. Saito, A. Suto, K. Ikeda, K. Hirose, N. Watanabe, I. Iwamoto, and H. Nakajima
Protein geranylgeranylation regulates the balance between Th17 cells and Foxp3+ regulatory T cells
Int. Immunol., June 1, 2009; 21(6): 679 - 689.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
P. J. Barnes and B. R. Celli
Systemic manifestations and comorbidities of COPD
Eur. Respir. J., May 1, 2009; 33(5): 1165 - 1185.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
X. Zhang, J. Jin, Y. Tang, D. Speer, D. Sujkowska, and S. Markovic-Plese
IFN-{beta}1a Inhibits the Secretion of Th17-Polarizing Cytokines in Human Dendritic Cells via TLR7 Up-Regulation
J. Immunol., March 15, 2009; 182(6): 3928 - 3936.
[Abstract] [Full Text] [PDF]

Home page
 ThoraxHome page
M Imamura, K Okunishi, H Ohtsu, K Nakagome, H Harada, R Tanaka, K Yamamoto, and M Dohi
Pravastatin attenuates allergic airway inflammation by suppressing antigen sensitisation, interleukin 17 production and antigen presentation in the lung
Thorax, January 1, 2009; 64(1): 44 - 49.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
P. J. Barnes
Future Treatments for Chronic Obstructive Pulmonary Disease and Its Comorbidities
Proceedings of the ATS, December 1, 2008; 5(8): 857 - 864.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Qin, S. A. Niyongere, S. J. Lee, B. J. Baker, and E. N. Benveniste
Expression and Functional Significance of SOCS-1 and SOCS-3 in Astrocytes
J. Immunol., September 1, 2008; 181(5): 3167 - 3176.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, X.
Right arrow Articles by Markovic-Plese, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, X.
Right arrow Articles by Markovic-Plese, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS