- Email: [email protected]
Statins as immunomodulators
Transplant Immunology 9 (2002) 197–200
Statins as immunomodulators Francois Mach* ¸ Cardiology Division, Department of Medicine, University Hospital Geneva, Foundation for Medical Research, 64 Avenue Roseraie, 1211 Geneva 4, Switzerland
Abstract HMG-CoA reductase inhibitors, or statins, are effective lipid lowering agents, extensively used in medical practice. Statins have never been shown to be involved in the immune response, although few clinical reports have suggested a better outcome of cardiac transplantation in patients under pravastatin therapy. Major histocompatibility complex class II (MHC-II) molecules are directly involved in the activation of T lymphocytes and in the control of the immune response. Whereas only a limited number of specialized cell types express MHC-II constitutively, numerous other cells become MHC-II positive upon induction by interferon gamma (IFN-g). We and others recently demonstrated that statins act as direct inhibitors of induction of MHC-II expression by IFN-g and thus as repressors of MHC-II-mediated T cell activation. This effect was observed in several cell types, including primary human endothelial cells and macrophages. Interestingly, this inhibition is specific for inducible MHC-II expression and does not concern either constitutive expression of MHC-II or expression of MHC-I. In repressing induction of MHC-II, and subsequent T lymphocyte activation, statins therefore behave as a novel type of immunomodulator. This unexpected effect provides a scientific rationale for suggesting the use of statins as novel immunosuppressors, not only in organ transplantation but in numerous other pathologies as well. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Statins (HMG-CoA reductase inhibitors); Immunology; MHC-II; Transplantation
A major challenge of contemporary medicine is to break the traditional compartmentalization that frequently separates different fields. The same holds between medical practice and basic biochemical mechanism. Unexpected linkages between different areas of medicine are indeed of particular interest. An unsuspected ‘bridge’ across cardiology practice and molecular immunology, as presented here, is a good example of such a linkage. In the last decades, substantial progress has been made in understanding the relationship between lipid disorders and prevention of cardiac ischemic disease. The identification of new therapeutic targets and new lipidmodifying agents expend treatment options. 3hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase inhibitors, the so-called statins, atorvastatin, cerivastatin, fluvastatin, pravavstatin, lovastatin and simvastatin, can induce relatively large reductions in plasma cholesterol levels and are established drugs for the treatment of hypercholesterolemia w1x. Clinical trials have demonstrated that statins can induce regression of *Corresponding author. Tel.: q41-22-382-7234; fax: q41-22-3475979. E-mail address: [email protected] (F. Mach).
vascular atherosclerosis as well as reduction of cardiovascular-related morbidity and mortality in patients with and without coronary artery disease w1–4x. The clinical beneficial results of HMG-CoA reductase inhibitors are usually assumed to result from their ability to reduce cholesterol synthesis w5x. However, because mevalonate, the product of the enzyme reaction, is the precursor not only of cholesterol but also of many nonsteroidal isoprenoid compounds, inhibition of HMGCoA reductase may result in pleiotropic effects w6–10x. Indeed, the mevalonate pathway yields a series of isoprenoids that are vital for diverse cellular functions w7x. These isoprenoids include isopentenyl adenosine, present in some types of transfer of RNA; dolichols required for glycoprotein synthesis; and polyisoprenoid side chains of ubiquinone and heme A, involved in electron transport. Several proteins have also been identified that are port-translationally modified by the covalent attachment of mevalonate-derived isoprenoid groups, either farnesyl or geranylgeranyl pyrophosphosphate w11x. These proteins must be prenylated as a prerequisite for membrane associate, which is required for their function. Members of this family are involved
0966-3274/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 6 6 - 3 2 7 4 Ž 0 2 . 0 0 0 3 0 - 8
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F. Mach / Transplant Immunology 9 (2002) 197–200
in a number of cellular process including cell signaling, cell differentiation and proliferation, myelination, cytoskeleton dynamics and endocytoticyexocytotic transport w6x. Hence, statins trough the inhibition of HMG-CoA reductase, could affect several processes that may help to explain their non-lipid-related pharmacological properties. Indeed, several recent in vitro and in vivo experiments demonstrated that HMG-CoA reductase inhibitors have anti-atherosclerotic effects non-related to lipid. Statins can directly up-regulating endothelial NOS (nitric oxide synthase) expression in vitro under cholesterol-clamped conditions w12–14x. This upregulation of eNOS expression by statins occurs predominantly at post-transcriptional level and is prevented by its isoprenoid derivatives, mevalonate and geranylgeranyl,w12,14x. From other in vitro studies, it has been demonstrated that statins decrease the secretion of the pro-inflammatory cytokines IL-6 and IL-8 but not TNF-a from activated macrophages w15,16x, inhibit chemokines release such as MCP-1 and IP-10 by endothelial cells w17x, inhibit adhesion molecules expression such as CD11 on monocytes w18x or ICAM-1 on endothelial cells w19x. On SMCs, statins inhibit proliferation and migration, possibly via apoptosis w20x. Indeed, statins have been reported to induce apoptosis in cultured SMCs and myocytes w21x. A proapoptotic effect of HMG-CoA reductase inhibitors was also demonstrated in vivo w22x. Statins may have direct effect related to atherosclerotic plaque stability. Experiments demonstrated that statins inhibit MMPs activity and secretion, such MMP1, MMP-3 and MMP–9, by human and rabbit macrophages in vitro as well as in vivo w23,24x. Furthermore, statins have effects on the coagulation process. They have been reported to inhibit tissue factor expression and activity from activated macrophages in vivo and in vitro w25,26x, decrease endothelin-1 synthesis from endothelial cells w27x, improve fibrinolytic profile as measured by decrease in plasminogen activator-1 (PAI1) levels and increase in tissue plasminogen activator (tPA) levels, mostly via the inhibition of geranylgeranylated Rho proteins and disruption of the cytoskeleton w28–30x. Statins have never been shown to be involved in the immune response, although some clinical trials have suggested a better outcome of cardiac transplantation in patients under statin therapy. Reports from Kobashigawa et al., in 1995 w31x, confirmed by Wenke et al., in 1997 w32x revealed that statins therapy had a beneficial effect on the incidence of cardiac rejection causing hemodynamic compromise, coronary vasculopathy, as well as survival. Apart from these new clinical findings, to date, there was no in vitro scientific rational explaining this potential beneficial effect of statin on the immune system. Major Histocompatibility Complex class II (MHC class II) molecules, expressed on the surface of specialized cells, are directly involved in the control of
the immune response and thus determine rejection after organ transplantation. Whereas a limited number of specialized cell types express MHC class II constitutively, numerous other cells become MHC-II positive upon induction by the inflammatory mediator interferon-gamma (IFN-g). This complex regulation is under the control of the transactivator CIITA w33,34x. CIITA is the general controller of MHC class II expression and its own expression is tightly regulated w33,34x. Interestingly, expression of CIITA is controlled by several alternative promoters, operating under distinct physiological conditions w35x. CIITA promoter I controls constitutive expression in dendritic cells, promoter III controls constitutive expression in B lymphocytes, while CIITA promoter IV is specifically responsible for the IFN-g inducible expression of CIITA and thus of MHC class II w35x. Therefore, we hypothesize that statin may regulate IFN-g-induced MHC class II expression on antigen presenting cells and thus reduce T lymphocyte activation. We used human vascular endothelial cells (ECs), human monocytesymacrophages (Mf), the human Raji cell line (Epstein-Barr virus (EBV)-positive Burkitt lymphoma cell line) and human dendritic cells. Cells were stimulated with human recombinant IFN-g in presence or absence of the statins: Atorvastatin, Lovastatin, Pravastatin, or Simvastatin, which were all, obtained from commercial sources. To detect MHC class expression, we used flow cytometry (FACS), immunohistochemistry labeling. To analyze CIITA regulation, we performed RNAse protection assays and transfection experiments. For functional effects of statins on MHC class II expression, we performed mixed lymphocyte reactions and measured T lymphocytes proliferation by w3Hxthymidine incorporation and IL-2 production (ELISA). The effect of several statins were studied on the regulation of both constitutive MHC class II expression in highly specialized APC and inducible MHC class II expression by IFN-g in a variety of other cell types, including primary cultures of human ECs and Mf. These investigations have lead to the following conclusions (a) Statins effectively repress the induction of MHC class II expression by IFN-g and do so in a dosedependent manner (b) In the presence of l-mevalonate, the effect of statins on MHC class II expression is abolished, indicating that it is indeed the effect of statins as HMG-CoA reductase inhibitors that mediates repression of MHC class II. (c) Interestingly, repression of MHC class II expression by statins is highly specific for the inducible form of MHC-II expression and does not concern constitutive expression of MHC-II in highly specialized APCs, such as dendritic cells and B lymphocytes. (d) This effect of statins is specific for MHC class II and does not concern MHC class I expression. (e) In order to investigate functional consequences of
F. Mach / Transplant Immunology 9 (2002) 197–200
statin-induced inhibition of MHC class II expression, we performed mixed lymphocyte reactions (allogenic T lymphocytes incubated with IFN-g-pre-treated human ECs or Mf). T cell proliferation could be blocked by anti-MHC class II mAb. Pre-treatment of ECs or Mf with statins reduces subsequent T lymphocyte proliferation, as measured by w3Hxthymidine incorporation and IL-2 release. Repression of induction of MHC class II by IFN-g, in statin treated samples, is paralleled by a reduced induction of CIITA mRNA by IFN-g, which points to an inhibition of induction of the CIITA gene by statins. Interestingly, the different degrees of repression of CIITA mRNA induction observed with the different forms of statins are reflected in the different levels of repression of MHC class II expression observed with the same drugs. This confirms the quantitative nature of the control of CIITA over MHC class II gene activity w36x. Constitutive expression of MHC class II, known to be mediated by CIITA promoters I and III, is not affected by statins. The specificity of statins for repressing inducible and not constitutive MHC class II expression suggests an effect on CIITA promoter IV. Indeed, we show that induction of expression of the first exon specifically controlled by CIITA promoter IV is affected by statins. Finally, the statin effect is transcriptional, as demonstrated by actinomycin D experiments used to block de novo RNA synthesis and explore mRNA half-life. This effect is direct and does not require de novo protein synthesis, as seen by a lack of effect of cycloheximide experiments. All these effects of statins on MHC class II induction were observed with different forms of statins currently used in clinical medicine. Future studies, will tell us whether there is a link between mevalonate blockade due to statins and either the availability or the co-operative binding of the three well-defined transcription factors Stat1, USF-1, and IRF-1 required for the activity of CIITA promoter IV w37x. Regarding the HMG-CoA-mevalonate pathway, which yields a series of isoprenoids, experiments using down-stream synthetic inhibitor will demonstrate whether this effect is mediated via prenylation of different proteins, such as the farnesylated PPARs or Ras, or the geranlygeranlylated RhoyRacyCd42 w7x. Other mechanism unrelated to the HMG-CoA pathway could also be involved. Indeed, recent findings demonstrated that the statin Lovastatin selectively inhibits LFA-1-mediated adhesion, costimulation of lymphocytes, and thus suppress inflammatory response in vivo w38x. Two isolated clinical observations suggesting a beneficial effect of statin treatment on the outcome of heart transplantation were reported w31,32x. These observations were left unexplained, however, as statins have never before been connected to the immune system. We have discovered a novel effect of statins as an effective repressor of MHC class II expression and provided a detailed molecular explanation for this unexpected and
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novel effect of one of the most intensely used drug in medicine. The fact that statin-induced repression of MHC class II also represses MHC class II-dependent activation of T lymphocytes indicates that statin is likely to have an immunosuppressive effect. This discovery provides a firm scientific rationale to recommend the use of this drug as an immunosuppressor not only after heart transplantation, but also for organ transplantation in general. It also suggests numerous other practical clinical applications for using statins as immunomodulators, particularly in diseases where aberrant expression of MHC class II molecules are implicated. This ranges from autoimmune diseases such as type I diabetes, multiple sclerosis and rheumatoid arthritis to psoriasis and chronic inflammatory diseases like atherosclerosis. The high degree of patient tolerance of statins makes them potentially a welcome addition to the limited current arsenal of immunosuppressive agents. In vivo studies will be necessary to confirm such an effect. References w1x Maron DJ, Fazio S, Linton MF. Current perspectives on statins. Circulation 2000;101:207 –213. w2x Plana JC, Jones PH. The use of statins in acute coronary syndromes: the mechanisms behind the outcomes. Curr Atheroscler Rep 2001;3:355 –364. w3x Gotto AM. Statin therapy: where are we? Where do we go next? Am J Cardiol 2001;87:13B–18B. w4x Vaughan CJ, Gotto AM, Basson CT. The evolving role of statins in the management of atherosclerosis. J Am Coll Cardiol 2000;35:1 –10. w5x Gotto AM, Rundy SM. Lowering LDL cholesterol: questions from recent meta-analyses and subset analyses of clinical trial DataIssues from the Interdisciplinary Council on Reducing the Risk for Coronary Heart Disease, ninth Council meeting. Circulation 1999;99:E1 –E7. w6x Bellosta S, Ferri N, Bernini F, Paoletti R, Corsini A. Nonlipid-related effects of statins. Ann Med 2000;32:164 –176. w7x Corsini A, et al. New insights into the pharmacodynamic and pharmacokinetic properties of statins. Pharmacol Ther 1999;84:413 –428. w8x Farmer JA. Pleiotropic effects of statins. Curr Atheroscler Rep 2000;2:208 –217. w9x Kwak BR, Mach F. Statins inhibit leukocyte recruitment: new evidence for their anti-inflammatory properties. Arterioscler Thromb Vasc Biol 2001;21:1256 –1258. w10x Gotto AM, Farmer JA. Pleiotropic effects of statins: do they matter? Curr Opin Lipidol 2001;12:391 –394. w11x Maltese WA, Sheridan KM, Repko EM, Erdman RA. Posttranslational modification of low molecular mass GTP-binding proteins by isoprenoid. J Biol Chem 1990;265:2148 –2155. w12x Laufs U, La Fata V, Plutzky J, Liao JK. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 1998;97:1129 –1135. w13x Laufs U, Endres M, Liao JK. Regulation of endothelial NO production by Rho GTPase. Med Klin 1999;94:211 –218. w14x Laufs U, et al. Suppression of endothelial nitric oxide production after withdrawal of statin treatment is mediated by negative feedback regulation of rho GTPase gene transcription wIn Process Citationx. Circulation 2000;102:3104 –3110.
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w15x Ikeda U, Hojo Y, Katsuki T, Shimada K. Procoagulant and proinflammatory activity in acute coronary syndromes. Cardiovasc Res 1999;42(823):825. Discussion. w16x Ikeda U, Shimada K. Statins and monocytes. Lancet 1999;353:2070. w17x Bustos C, et al. HMG-CoA reductase inhibition by atorvastatin reduces neointimal inflammation in a rabbit model of atherosclerosis. J Am Coll Cardiol 1998;32:2057 –2064. w18x Weber C, Erl W, Weber KS, Weber PC. HMG-CoA reductase inhibitors decrease CD11b expression and CD11b-dependent adhesion of monocytes to endothelium and reduce increased adhesiveness of monocytes isolated from patients with hypercholesterolemia wsee commentsx. J Am Coll Cardiol 1997;30:1212 –1217. w19x Romano M, et al. Fluvastatin reduces soluble P-selectin and ICAM-1 levels in hypercholesterolemic patients: role of nitric oxide. J Invest Med 2000;48:183 –189. w20x Corsini A, Pazzucconi F, Pfister P, Paoletti R, Sirtori CR. Inhibitor of proliferation of arterial smooth-muscle cells by fluvastatin. Lancet 1996;348:1584. w21x Guijarro C, et al. 3-Hydroxy-3-methylglutaryl coenzyme a reductase and isoprenylation inhibitors induce apoptosis of vascular smooth muscle cells in culture. Circ Res 1998;83:490 – 500. w22x Baetta R, et al. Proapoptotic effect of atorvastatin on stimulated rabbit smooth muscle cells. Pharmacol Res 1997;36:115 –121. w23x Bellosta S, et al. Direct vascular effects of HMG-CoA reductase inhibitors. Atherosclerosis 1998;137:S101 –S109. w24x Bellosta S, et al. HMG-CoA reductase inhibitors reduce MMP9 secretion by macrophages. Arterioscler Thromb Vasc Biol 1998;18:1671 –1678. w25x Rosenson RS, Tangney CC. Antiatherothrombotic properties of statins: implications for cardiovascular event reduction. J Am Med Assoc 1998;279:1643 –1650. w26x Colli S, et al. Vastatins inhibit tissue factor in cultured human macrophages. A novel mechanism of protection against atherothrombosis. Arterioscler Thromb Vasc Biol 1997;17:265 –272. w27x Glorioso N, et al. Effect of the HMG-CoA reductase inhibitors on blood pressure in patients with essential hypertension and primary hypercholesterolemia. Hypertension 1999;34:1281 – 1286.
w28x Dangas G, et al. Pravastatin: an antithrombotic effect independent of the cholesterol-lowering effect. Thromb Haemost 2000;83:688 –692. w29x Essig M, et al. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase fibrinolytic activity in rat aortic endothelial cells. Role of geranylgeranylation and Rho proteins. Circ Res 1998;83:683 –690. w30x Essig M, Vrtovsnik F, Nguyen G, Sraer JD, Friedlander G. Lovastatin Modulates in vivo and in vitro the plasminogen activatoryplasmin system of rat proximal tubular cells: role of geranylgeranylation and Rho proteins. J Am Soc Nephrol 1998;9:1377 –1388. w31x Kobashigawa JA, et al. Effect of pravastatin on outcomes after cardiac transplantation. N Engl J Med 1995;333:621 –627. w32x Wenke K, et al. Simvastatin reduces graft vessel disease and mortality after heart transplantation: a four-year randomized trial. Circulation 1997;96:1398 –1402. w33x Steimle V, Otten LA, Zufferey M, Mach B. Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome). Cell 1993;75:135 –146. w34x Steimle V, Siegrist CA, Mottet A, Lisowska-Grospierre B, Mach B. Regulation of MHC class II expression by interferongamma mediated by the transactivator gene CIITA. Science 1994;265:106 –109. w35x Muhlethaler-Mottet A, Otten LA, Steimle V, Mach B. Expression of MHC class II molecules in different cellular and functional compartments is controlled by differential usage of multiple promoters of the transactivator CIITA. EMBO J 1997;16:2851 –2860. w36x Otten LA, Steimle V, Bontron S, Mach B. Quantitative control of MHC class II expression by the transactivator CIITA. Eur J Immunol 1998;28:473 –478. w37x Muhlethaler-Mottet A, Di Berardino W, Otten LA, Mach B. Activation of the MHC class II transactivator CIITA by interferon-gamma requires cooperative interaction between Stat1 and USF-1. Immunity 1998;8:157 –166. w38x Weitz-Schmidt G, et al. Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med 2001;7:687 –692.
Statins as immunomodulators Francois Mach* ¸ Cardiology Division, Department of Medicine, University Hospital Geneva, Foundation for Medical Research, 64 Avenue Roseraie, 1211 Geneva 4, Switzerland
Abstract HMG-CoA reductase inhibitors, or statins, are effective lipid lowering agents, extensively used in medical practice. Statins have never been shown to be involved in the immune response, although few clinical reports have suggested a better outcome of cardiac transplantation in patients under pravastatin therapy. Major histocompatibility complex class II (MHC-II) molecules are directly involved in the activation of T lymphocytes and in the control of the immune response. Whereas only a limited number of specialized cell types express MHC-II constitutively, numerous other cells become MHC-II positive upon induction by interferon gamma (IFN-g). We and others recently demonstrated that statins act as direct inhibitors of induction of MHC-II expression by IFN-g and thus as repressors of MHC-II-mediated T cell activation. This effect was observed in several cell types, including primary human endothelial cells and macrophages. Interestingly, this inhibition is specific for inducible MHC-II expression and does not concern either constitutive expression of MHC-II or expression of MHC-I. In repressing induction of MHC-II, and subsequent T lymphocyte activation, statins therefore behave as a novel type of immunomodulator. This unexpected effect provides a scientific rationale for suggesting the use of statins as novel immunosuppressors, not only in organ transplantation but in numerous other pathologies as well. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Statins (HMG-CoA reductase inhibitors); Immunology; MHC-II; Transplantation
A major challenge of contemporary medicine is to break the traditional compartmentalization that frequently separates different fields. The same holds between medical practice and basic biochemical mechanism. Unexpected linkages between different areas of medicine are indeed of particular interest. An unsuspected ‘bridge’ across cardiology practice and molecular immunology, as presented here, is a good example of such a linkage. In the last decades, substantial progress has been made in understanding the relationship between lipid disorders and prevention of cardiac ischemic disease. The identification of new therapeutic targets and new lipidmodifying agents expend treatment options. 3hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase inhibitors, the so-called statins, atorvastatin, cerivastatin, fluvastatin, pravavstatin, lovastatin and simvastatin, can induce relatively large reductions in plasma cholesterol levels and are established drugs for the treatment of hypercholesterolemia w1x. Clinical trials have demonstrated that statins can induce regression of *Corresponding author. Tel.: q41-22-382-7234; fax: q41-22-3475979. E-mail address: [email protected] (F. Mach).
vascular atherosclerosis as well as reduction of cardiovascular-related morbidity and mortality in patients with and without coronary artery disease w1–4x. The clinical beneficial results of HMG-CoA reductase inhibitors are usually assumed to result from their ability to reduce cholesterol synthesis w5x. However, because mevalonate, the product of the enzyme reaction, is the precursor not only of cholesterol but also of many nonsteroidal isoprenoid compounds, inhibition of HMGCoA reductase may result in pleiotropic effects w6–10x. Indeed, the mevalonate pathway yields a series of isoprenoids that are vital for diverse cellular functions w7x. These isoprenoids include isopentenyl adenosine, present in some types of transfer of RNA; dolichols required for glycoprotein synthesis; and polyisoprenoid side chains of ubiquinone and heme A, involved in electron transport. Several proteins have also been identified that are port-translationally modified by the covalent attachment of mevalonate-derived isoprenoid groups, either farnesyl or geranylgeranyl pyrophosphosphate w11x. These proteins must be prenylated as a prerequisite for membrane associate, which is required for their function. Members of this family are involved
0966-3274/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 6 6 - 3 2 7 4 Ž 0 2 . 0 0 0 3 0 - 8
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F. Mach / Transplant Immunology 9 (2002) 197–200
in a number of cellular process including cell signaling, cell differentiation and proliferation, myelination, cytoskeleton dynamics and endocytoticyexocytotic transport w6x. Hence, statins trough the inhibition of HMG-CoA reductase, could affect several processes that may help to explain their non-lipid-related pharmacological properties. Indeed, several recent in vitro and in vivo experiments demonstrated that HMG-CoA reductase inhibitors have anti-atherosclerotic effects non-related to lipid. Statins can directly up-regulating endothelial NOS (nitric oxide synthase) expression in vitro under cholesterol-clamped conditions w12–14x. This upregulation of eNOS expression by statins occurs predominantly at post-transcriptional level and is prevented by its isoprenoid derivatives, mevalonate and geranylgeranyl,w12,14x. From other in vitro studies, it has been demonstrated that statins decrease the secretion of the pro-inflammatory cytokines IL-6 and IL-8 but not TNF-a from activated macrophages w15,16x, inhibit chemokines release such as MCP-1 and IP-10 by endothelial cells w17x, inhibit adhesion molecules expression such as CD11 on monocytes w18x or ICAM-1 on endothelial cells w19x. On SMCs, statins inhibit proliferation and migration, possibly via apoptosis w20x. Indeed, statins have been reported to induce apoptosis in cultured SMCs and myocytes w21x. A proapoptotic effect of HMG-CoA reductase inhibitors was also demonstrated in vivo w22x. Statins may have direct effect related to atherosclerotic plaque stability. Experiments demonstrated that statins inhibit MMPs activity and secretion, such MMP1, MMP-3 and MMP–9, by human and rabbit macrophages in vitro as well as in vivo w23,24x. Furthermore, statins have effects on the coagulation process. They have been reported to inhibit tissue factor expression and activity from activated macrophages in vivo and in vitro w25,26x, decrease endothelin-1 synthesis from endothelial cells w27x, improve fibrinolytic profile as measured by decrease in plasminogen activator-1 (PAI1) levels and increase in tissue plasminogen activator (tPA) levels, mostly via the inhibition of geranylgeranylated Rho proteins and disruption of the cytoskeleton w28–30x. Statins have never been shown to be involved in the immune response, although some clinical trials have suggested a better outcome of cardiac transplantation in patients under statin therapy. Reports from Kobashigawa et al., in 1995 w31x, confirmed by Wenke et al., in 1997 w32x revealed that statins therapy had a beneficial effect on the incidence of cardiac rejection causing hemodynamic compromise, coronary vasculopathy, as well as survival. Apart from these new clinical findings, to date, there was no in vitro scientific rational explaining this potential beneficial effect of statin on the immune system. Major Histocompatibility Complex class II (MHC class II) molecules, expressed on the surface of specialized cells, are directly involved in the control of
the immune response and thus determine rejection after organ transplantation. Whereas a limited number of specialized cell types express MHC class II constitutively, numerous other cells become MHC-II positive upon induction by the inflammatory mediator interferon-gamma (IFN-g). This complex regulation is under the control of the transactivator CIITA w33,34x. CIITA is the general controller of MHC class II expression and its own expression is tightly regulated w33,34x. Interestingly, expression of CIITA is controlled by several alternative promoters, operating under distinct physiological conditions w35x. CIITA promoter I controls constitutive expression in dendritic cells, promoter III controls constitutive expression in B lymphocytes, while CIITA promoter IV is specifically responsible for the IFN-g inducible expression of CIITA and thus of MHC class II w35x. Therefore, we hypothesize that statin may regulate IFN-g-induced MHC class II expression on antigen presenting cells and thus reduce T lymphocyte activation. We used human vascular endothelial cells (ECs), human monocytesymacrophages (Mf), the human Raji cell line (Epstein-Barr virus (EBV)-positive Burkitt lymphoma cell line) and human dendritic cells. Cells were stimulated with human recombinant IFN-g in presence or absence of the statins: Atorvastatin, Lovastatin, Pravastatin, or Simvastatin, which were all, obtained from commercial sources. To detect MHC class expression, we used flow cytometry (FACS), immunohistochemistry labeling. To analyze CIITA regulation, we performed RNAse protection assays and transfection experiments. For functional effects of statins on MHC class II expression, we performed mixed lymphocyte reactions and measured T lymphocytes proliferation by w3Hxthymidine incorporation and IL-2 production (ELISA). The effect of several statins were studied on the regulation of both constitutive MHC class II expression in highly specialized APC and inducible MHC class II expression by IFN-g in a variety of other cell types, including primary cultures of human ECs and Mf. These investigations have lead to the following conclusions (a) Statins effectively repress the induction of MHC class II expression by IFN-g and do so in a dosedependent manner (b) In the presence of l-mevalonate, the effect of statins on MHC class II expression is abolished, indicating that it is indeed the effect of statins as HMG-CoA reductase inhibitors that mediates repression of MHC class II. (c) Interestingly, repression of MHC class II expression by statins is highly specific for the inducible form of MHC-II expression and does not concern constitutive expression of MHC-II in highly specialized APCs, such as dendritic cells and B lymphocytes. (d) This effect of statins is specific for MHC class II and does not concern MHC class I expression. (e) In order to investigate functional consequences of
F. Mach / Transplant Immunology 9 (2002) 197–200
statin-induced inhibition of MHC class II expression, we performed mixed lymphocyte reactions (allogenic T lymphocytes incubated with IFN-g-pre-treated human ECs or Mf). T cell proliferation could be blocked by anti-MHC class II mAb. Pre-treatment of ECs or Mf with statins reduces subsequent T lymphocyte proliferation, as measured by w3Hxthymidine incorporation and IL-2 release. Repression of induction of MHC class II by IFN-g, in statin treated samples, is paralleled by a reduced induction of CIITA mRNA by IFN-g, which points to an inhibition of induction of the CIITA gene by statins. Interestingly, the different degrees of repression of CIITA mRNA induction observed with the different forms of statins are reflected in the different levels of repression of MHC class II expression observed with the same drugs. This confirms the quantitative nature of the control of CIITA over MHC class II gene activity w36x. Constitutive expression of MHC class II, known to be mediated by CIITA promoters I and III, is not affected by statins. The specificity of statins for repressing inducible and not constitutive MHC class II expression suggests an effect on CIITA promoter IV. Indeed, we show that induction of expression of the first exon specifically controlled by CIITA promoter IV is affected by statins. Finally, the statin effect is transcriptional, as demonstrated by actinomycin D experiments used to block de novo RNA synthesis and explore mRNA half-life. This effect is direct and does not require de novo protein synthesis, as seen by a lack of effect of cycloheximide experiments. All these effects of statins on MHC class II induction were observed with different forms of statins currently used in clinical medicine. Future studies, will tell us whether there is a link between mevalonate blockade due to statins and either the availability or the co-operative binding of the three well-defined transcription factors Stat1, USF-1, and IRF-1 required for the activity of CIITA promoter IV w37x. Regarding the HMG-CoA-mevalonate pathway, which yields a series of isoprenoids, experiments using down-stream synthetic inhibitor will demonstrate whether this effect is mediated via prenylation of different proteins, such as the farnesylated PPARs or Ras, or the geranlygeranlylated RhoyRacyCd42 w7x. Other mechanism unrelated to the HMG-CoA pathway could also be involved. Indeed, recent findings demonstrated that the statin Lovastatin selectively inhibits LFA-1-mediated adhesion, costimulation of lymphocytes, and thus suppress inflammatory response in vivo w38x. Two isolated clinical observations suggesting a beneficial effect of statin treatment on the outcome of heart transplantation were reported w31,32x. These observations were left unexplained, however, as statins have never before been connected to the immune system. We have discovered a novel effect of statins as an effective repressor of MHC class II expression and provided a detailed molecular explanation for this unexpected and
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novel effect of one of the most intensely used drug in medicine. The fact that statin-induced repression of MHC class II also represses MHC class II-dependent activation of T lymphocytes indicates that statin is likely to have an immunosuppressive effect. This discovery provides a firm scientific rationale to recommend the use of this drug as an immunosuppressor not only after heart transplantation, but also for organ transplantation in general. It also suggests numerous other practical clinical applications for using statins as immunomodulators, particularly in diseases where aberrant expression of MHC class II molecules are implicated. This ranges from autoimmune diseases such as type I diabetes, multiple sclerosis and rheumatoid arthritis to psoriasis and chronic inflammatory diseases like atherosclerosis. The high degree of patient tolerance of statins makes them potentially a welcome addition to the limited current arsenal of immunosuppressive agents. In vivo studies will be necessary to confirm such an effect. References w1x Maron DJ, Fazio S, Linton MF. Current perspectives on statins. Circulation 2000;101:207 –213. w2x Plana JC, Jones PH. The use of statins in acute coronary syndromes: the mechanisms behind the outcomes. Curr Atheroscler Rep 2001;3:355 –364. w3x Gotto AM. Statin therapy: where are we? Where do we go next? Am J Cardiol 2001;87:13B–18B. w4x Vaughan CJ, Gotto AM, Basson CT. The evolving role of statins in the management of atherosclerosis. J Am Coll Cardiol 2000;35:1 –10. w5x Gotto AM, Rundy SM. Lowering LDL cholesterol: questions from recent meta-analyses and subset analyses of clinical trial DataIssues from the Interdisciplinary Council on Reducing the Risk for Coronary Heart Disease, ninth Council meeting. Circulation 1999;99:E1 –E7. w6x Bellosta S, Ferri N, Bernini F, Paoletti R, Corsini A. Nonlipid-related effects of statins. Ann Med 2000;32:164 –176. w7x Corsini A, et al. New insights into the pharmacodynamic and pharmacokinetic properties of statins. Pharmacol Ther 1999;84:413 –428. w8x Farmer JA. Pleiotropic effects of statins. Curr Atheroscler Rep 2000;2:208 –217. w9x Kwak BR, Mach F. Statins inhibit leukocyte recruitment: new evidence for their anti-inflammatory properties. Arterioscler Thromb Vasc Biol 2001;21:1256 –1258. w10x Gotto AM, Farmer JA. Pleiotropic effects of statins: do they matter? Curr Opin Lipidol 2001;12:391 –394. w11x Maltese WA, Sheridan KM, Repko EM, Erdman RA. Posttranslational modification of low molecular mass GTP-binding proteins by isoprenoid. J Biol Chem 1990;265:2148 –2155. w12x Laufs U, La Fata V, Plutzky J, Liao JK. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 1998;97:1129 –1135. w13x Laufs U, Endres M, Liao JK. Regulation of endothelial NO production by Rho GTPase. Med Klin 1999;94:211 –218. w14x Laufs U, et al. Suppression of endothelial nitric oxide production after withdrawal of statin treatment is mediated by negative feedback regulation of rho GTPase gene transcription wIn Process Citationx. Circulation 2000;102:3104 –3110.
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