Views & Reviews
`
`CME Mechanisms of action of glatiramer
`acetate in multiple sclerosis
`
`Oliver Neuhaus, MD; Cinthia Farina, PhD; Hartmut Wekerle, MD; and Reinhard Hohlfeld, MD
`
`Article abstract—Glatiramer acetate (GA, Copaxone [Teva Pharmaceuticals, Kansas City, MO], formerly known as
`copolymer-1) and interferon- (IFN)-b are both used for the immunomodulatory treatment of multiple sclerosis, but they
`act in different ways. Four major mechanisms of GA have been identified: 1) competition with myelin-basic protein (MBP)
`for binding to major histocompatibility complex (MHC) molecules; 2) competition of GA/MHC with MBP/MHC for binding
`to the T-cell receptor; 3) partial activation and tolerance induction of MBP-specific T cells (action as an altered peptide
`ligand); and 4) induction of GA-reactive T-helper 2- (TH2)-like regulatory cells. Of these four mechanisms, 1 and 2
`presumably occur only in vitro and are therefore irrelevant for the in vivo effects of GA. In contrast, mechanisms 3 and 4
`could occur in vivo and both could contribute to the clinical effects of GA.
`NEUROLOGY 2001;56:702–708
`
`Glatiramer acetate (GA, Copaxone [Teva Pharma-
`ceuticals, Kansas City, MO], formerly known as
`copolymer-1) and interferon- (IFN)-b are now widely
`used for the immunomodulatory treatment of MS.1-4
`The mechanisms of action of these agents, although
`not completely understood, seem to be fundamen-
`tally different. Whereas IFN-b exerts its multiple
`immunomodulatory effects in an antigen-nonspecific
`way, GA seems to preferentially affect immune cells
`specific for myelin basic protein (MBP) and perhaps
`other myelin antigens.5-7 This view rests mainly on
`evidence obtained in experimental autoimmune en-
`cephalomyelitis (EAE), an animal model of MS. Until
`recently the effects of GA on the human immune
`system were largely unknown. However, several new
`articles have shed light on the mechanisms of action
`of GA in MS. In this article, we briefly review these
`novel findings and put them into perspective with
`the previous observations in animal models.
`
`Clinical effects of GA in EAE and MS. GA is a
`standardized, randomized mixture of synthetic
`polypeptides consisting of L-glutamic acid, L-lysine,
`L-alanine, and L-tyrosine with a defined molar resi-
`due ratio of 0.14 : 0.34 : 0.43 : 0.09 and an average
`molecular mass of 4.7 to 11.0 kDa, i.e., an average
`length of 45 to 100 amino acids. It has been known
`for a long time that GA has both suppressive and
`protective effects in EAE induced by various enceph-
`alitogenic antigens in different species.5,8-13 With
`some exceptions (murine graft-versus-host disease
`
`[in doses higher than required to suppress EAE],14
`experimental uveoretinitis,15 and inhibition of type II
`collagen-reactive T cells in vitro16), GA seems to be
`ineffective in other autoimmune models.6 In addition
`to the subcutaneous (s.c.) route of administration,
`the oral form of GA has also been shown to be effec-
`tive in EAE.17,18 Daily s.c. administration of GA has
`beneficial effects on the clinical and MRI-defined
`course of patients with MS.19-24
`
`Overview of the immunologic effects of GA in
`EAE. During the last three decades, the pioneering
`work of Michael Sela, Ruth Arnon, and their col-
`leagues has laid the foundations for the approval
`of GA for use in the treatment of MS.6,25 A large
`body of experimental evidence in numerous EAE
`models suggests that GA acts by several different
`mechanisms7:
`Results of in vitro studies suggest that GA com-
`petes in some way with MBP.26-28 Specifically, GA
`competes with MBP at the antigen-presenting cell
`(APC) level for binding to the major histocompatibil-
`ity complex (MHC), and GA/MHC competes with
`MBP/MHC for binding to the T-cell receptor (TCR).
`GA binds to many different alleles of MHC class II
`molecules (“promiscuous binding”).29 Interestingly,
`the stereoisomer of GA, D-GA, which is composed of
`D-amino acids, binds as effectively to MHC class II30
`but fails to suppress EAE.31 This suggests that com-
`petition for MHC binding alone is insufficient to ex-
`plain the beneficial effects of GA.5 At the TCR level,
`
`From the Department of Neuroimmunology (Drs. Neuhaus, Farina, Wekerle, and Hohlfeld), Max-Planck Institute of Neurobiology, Martinsried; and the
`Institute for Clinical Neuroimmunology and the Department of Neurology (Dr. Hohlfeld), Ludwig Maximilians University, Munich, Germany.
`Supported by a grant from TEVA. O.N. is a postdoctoral fellow supported by Deutsche Forschungsgemeinschaft and Max-Planck-Gesellschaft. The Institute
`for Clinical Neuroimmunology is supported by the Hermann and Lilly Schilling Foundation. R.H. is a consultant to TEVA.
`Received September 29, 2000. Accepted in final form December 10, 2000.
`Address correspondence and reprint requests to Dr. Reinhard Hohlfeld, Institute for Clinical Neuroimmunology, Klinikum Grosshadern, Ludwig Maximil-
`ians University, Marchioninistrasse 15, 81366 Munich, Germany; e-mail: hohlfeld@neuro.mpg.de
`
`702 Copyright © 2001 by AAN Enterprises, Inc.
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`GA has been reported to act as an antagonist of the
`antigenic peptide MBP 82-100, but not MBP 1-11
`and proteolipid protein (PLP) 139-151.32
`Results of in vivo studies indicate that GA induces
`regulatory T cells of the T-helper 2- (TH2)-type in
`the peripheral immune system outside the CNS.
`When spleen cells from GA-treated mice were adop-
`tively transferred into syngeneic animals, these cells
`protected from EAE induced by different CNS
`antigens.33-35 Earlier researchers demonstrated a
`similar inhibitory effect with a “soluble factor” ex-
`tracted from these cells,36 which were later identified
`as anti-inflammatory TH2 cytokines.34,35 Further
`support for the proposed protective role of GA-
`reactive regulatory T cells comes from the recent
`demonstration that GA-specific TH2 cells are
`present in the CNS of GA-treated mice.37 GA-specific
`T cells also have a neuroprotective effect after adoptive
`transfer into rats with experimental crush lesions of
`the optic nerve.38 This latter finding suggests the possi-
`bility that GA-specific TH2-like regulatory T cells not
`only provide protective cytokines such as interleukin-
`(IL)-4, IL-5, IL-13, and transforming growth factor-
`(TGF)-b, but also neurotrophic factors such as brain-
`derived neurotrophic factor (BDNF).38,39
`
`Immunologic effects of GA in human subjects.
`The different effects of GA are listed and compared
`with the effects of IFN-b in the table.
`High frequency of GA-reactive proliferating T cells
`In contrast to the lack of ef-
`in untreated subjects.
`fect of GA on immune cells isolated from untreated
`animals, GA induces vigorous proliferation of periph-
`eral blood lymphocytes (PBL)
`from untreated
`(unprimed) human subjects.40-42 Phenotypic analyses
`revealed that the GA-responsive T-cell population in
`untreated subjects is polyclonal43,44 and predomi-
`nantly originates from the memory T-cell pool.44 Re-
`cent findings indicate that the proliferative response
`to GA depends on both MHC class I- and MHC class
`II-restricted T cells.45
`Reduced proliferative response in GA-treated pa-
`tients. The proliferative response to GA decreases
`with time in GA-treated patients.6,42,46-48 Recent re-
`sults from our own group indicate that this decrease
`is specific to GA because it is not observed with re-
`call antigens like tetanus toxoid and tuberculin.49
`Using limiting dilution assays, Schmied et al. ob-
`served that the constitutively high frequency of GA-
`reactive T cells in untreated patients initially tends
`to increase during the first months of GA therapy
`and only later decreases below baseline.50 Theoreti-
`cally, the observed decrease in GA-reactive T cells
`could be caused by anergy induction or activation-
`induced cell death of GA-specific T cells.50
`Deviation from TH1 to TH2. TH cells can be di-
`vided into several types based on their characteristic
`cytokine secretion patterns and effector functions.51-55
`TH1 cells produce proinflammatory cytokines such as
`
`IL-2, IL-12, IFN-g, and tumor necrosis factor- (TNF)-a.
`In contrast, TH2 cells produce downregulatory cyto-
`kines such as IL-4, IL-5, IL-6, IL-10, and IL-13. TH1
`cells mediate proliferative and delayed hypersensitivity
`responses, whereas TH2 cells are involved in allergic
`pathways and support antibody production by B cells.52
`Different lines of evidence suggest that GA
`treatment induces a shift from TH1 to TH2. GA
`treatment was shown to increase serum IL-10 lev-
`els and TGF-b and IL-4 mRNA in PBL, whereas it
`suppressed TNF-a mRNA.55 Using intracellular
`double-immunofluorescence flow cytometry, we dem-
`onstrated that long-term GA-reactive T-cell lines
`(TCL) from patients with untreated MS and healthy
`controls predominantly produce IFN-g and are to be
`classified as TH1 cells, whereas GA-reactive TCL
`from patients with GA-treated MS predominantly
`produce IL-4, i.e., behave like TH2-cells.43 Recent ob-
`servations on short-term44,48 and long-term56 GA-
`reactive TCL by other groups are consistent with
`these findings. In contrast to MBP-reactive TCL,
`GA-reactive TCL secrete IL-6, a TH2-related cyto-
`kine.56 In addition, the IFN-g : IL-5 ratio was biased
`toward IFN-g in MBP-reactive TCL and toward IL-5
`in GA-reactive TCL, in both treated and untreated
`patients.42
`In contrast to the limiting dilution assay (which
`detects proliferation), results obtained with an auto-
`mated ELISPOT assay (which detects cytokine pro-
`duction of individual cells) indicate that during GA
`therapy, there is an increase of GA-reactive T cells
`producing IL-4 or IFN-g.49 Specifically, the study by
`Farina et al.49 demonstrated that patients with GA-
`treated MS show 1) a significant reduction of
`GA-induced proliferation of peripheral blood mono-
`nuclear cells; 2) a positive IL-4 ELISPOT response
`mediated predominantly by CD41 T cells after in
`vitro stimulation with a wide range of GA concentra-
`tions; and 3) an elevated IFN-g response partially
`mediated by CD81 T cells after stimulation with
`high GA concentrations. All three effects were GA-
`specific because they were not observed with control
`antigens.49 The GA-induced changes were stable over
`time and allowed the correct identification of GA-
`treated and untreated donors in most cases.49 It
`therefore appears that during therapy, GA-reactive
`T cells are not physically deleted, but rather they are
`modified in such a way that they respond to in vitro
`challenge with GA by secreting cytokines but not by
`proliferating.
`Effects on migratory potential of T cells. Using
`an in vitro model of lymphocyte migration, Prat et al.
`demonstrated that the migratory potential of lym-
`phocytes freshly isolated from GA-treated patients
`was reduced compared with untreated patients.57 In
`vitro treatment with IFN-b, but not GA, reduced
`lymphocyte migration rates, indicating that IFN-b
`acts directly on cell migration, whereas GA acts in-
`directly.57 GA did not change the expression of ad-
`hesion molecules on human brain microvascular
`endothelial cells.58 It is currently not known whether
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`Figure. Schematic view of the putative
`mechanism of action of glatiramer ace-
`tate (GA). In the periphery, outside the
`CNS, GA initially stimulates a popula-
`tion of TH1-like T cells. During treat-
`ment, the properties of the GA-stimulated
`T cells change, and they become more
`TH2-like (dotted arrow). The activated
`GA-specific T cells enter the CNS, where
`they encounter CNS antigens like MBP
`bound to MHC class II and presented on
`the surface of microglia cells. The GA-
`reactive T cells are stimulated to secrete
`downmodulatory cytokines like IL-4,
`which exert a bystander suppressive ef-
`fect on other T cells. TCR 5 T-cell recep-
`tor; MHC 5 major histocompatibility
`complex; Ag 5 antigen.
`
`the reduced migratory potential is related to the
`TH1-to-TH2
`shift
`observed with GA-specific
`TCL.42-44,48,56
`Cross-stimulation with MBP and other antigens.
`GA was originally developed to mimic MBP for EAE
`induction.6,8 Surprisingly, however, it turned out
`that GA inhibits MBP-induced EAE.6 Ever since
`then, some form of partial cross-reaction or competi-
`tion between MBP and GA has been considered cru-
`cial to the mechanism of GA. Most investigators of
`the human immune response to GA found that GA is
`not cross-reactive with MBP at the level of prolifera-
`tion.41,56 An exception was reported by Gran et al.,
`who observed that a small number of GA-reactive
`TCL isolated from a patient treated with GA for
`more than 6 years proliferated in response to whole
`MBP and peptide MBP 83-99.44
`Despite the lack of cross-stimulation at the prolif-
`eration level, there is clear evidence that GA and
`MBP may cross-stimulate T cells at the level of cyto-
`kine production. In our own study, about 10% of the
`tested GA-specific T cell
`lines could be cross-
`stimulated with MBP to produce low levels of cyto-
`In these experiments, TH1-type TCL
`kines.43
`preferentially produced IFN-g, whereas TH2-type
`TCL produced IL-4.43 Similar results were reported
`by others, using either MBP-specific TCL42 for cross-
`stimulation with GA, or GA-reactive TCL44 for cross-
`stimulation with MBP.
`Interestingly, two of our GA-specific T-cell lines
`could be stimulated to produce IFN-g with another
`myelin autoantigen, myelin-oligodendrocyte glyco-
`protein (MOG).43 This indicates that the cross-
`stimulatory effect on cytokine production is not
`entirely restricted to MBP. It may also occur with
`other autoantigens. Indeed, one group of investiga-
`tors found evidence that the immune response to GA
`in GA-treated patients becomes more “degenerate.”48
`The authors reported that with increasing duration
`of treatment, the surviving GA-reactive T cells re-
`sponded to an increasing number of components from
`a combinatorial peptide library.48 These observations
`704 NEUROLOGY 56 March (2 of 2) 2001
`
`would add an interesting facet to the mechanism of GA
`action, implying that GA-reactive, TH2-like (protective)
`T cells might be activated not only by MBP, but also by
`other cross-reactive antigens. This might help to ex-
`plain why GA had beneficial effects not only in EAE
`induced by MBP but also by MOG or PLP, and also in
`a few other experimental autoimmune diseases.6,14,15
`Inhibition of MBP-specific T cells by GA. GA was
`found to inhibit the MBP-induced proliferation and
`IL-2 secretion of human MBP-specific TCL.28 A triv-
`ial toxic effect of GA could be excluded because the
`inhibition was overcome by increasing the concen-
`tration of MBP.28 A control antigen, tuberculin pu-
`rified protein derivative (PPD), did not have any
`inhibitory effect.28 GA also inhibited influenza vi-
`rus hemagglutinin- and Borrelia burgdorferi-
`specific T-cell clones, although to a lesser extent
`than MBP-specific T cells.44 In addition, GA was
`shown to inhibit the cytolytic ability of human MBP-
`specific TCL restricted to MS-associated HLA-DR
`types.59 In a recent study, Gran et al. showed that
`GA inhibited IFN-g production in MBP-reactive T
`cells in a dose-dependent manner and had a less
`pronounced effect on the secretion of IL-4 and IL-5.44
`In a subset of the analyzed T-cell clones, GA had a
`differential effect, i.e., it inhibited proliferation and
`IFN-g production and induced IL-4 and IL-5 secre-
`tion. This indicates a differential influence of GA on
`the T-cell activation parameters.44 Furthermore, GA
`was shown to induce a state of nonresponsiveness
`(anergy) in MBP-specific T-cell clones.44 In principle,
`these inhibitory effects could occur at the level of
`binding of GA to the MHC or to the TCR.
`Interaction with MHC. GA binds to purified
`HLA-DR molecules without antigen processing29 and
`without any obvious preference for particular al-
`leles.60 Although distinct binding motifs of GA to MS-
`associated HLA-DR molecules could be defined,
`virtually all of the GA polypeptides seem to have a
`binding capacity to MHC class II, owing perhaps to
`their random composition.61 In addition to MHC
`class II molecules, GA also seems to be able to inter-
`
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`Table Comparison of major immunologic effects of glatiramer acetate (GA) and interferon (IFN)-b
`
`Effects
`
`T-cell proliferation in vitro
`
`Proliferation during
`treatment
`Regulation of MHC
`expression
`MHC binding
`
`T-cell migration
`
`TH2 shift in PBL
`
`TH2 shift in GA-specific T
`cells
`Cross-stimulation with
`MBP
`Cross-inhibition of MBP-
`specific TCL
`TCR antagonism
`
`APL effect on MBP-
`specific T cells
`Effects on antigen-
`presenting cells
`
`GA
`
`IFN-b71-73
`
`Suppression of proliferation of MBP-reactive T
`cells in vitro28
`Decreased proliferation of PBL to GA during
`treatment6,42,47-49
`No known effect
`
`Direct and promiscuous binding of GA to different
`HLA-DR alleles60,61
`Reduced migration of PBL from GA-treated
`patients (unknown mechanism)57; no effect on
`adhesion molecule expression on human brain
`microvascular endothelial cells58
`
`Increased levels of IL-10 in serum and of mRNA
`for TGF-b and IL-455; reduction of mRNA for
`TNF-a in PBL55
`Shift of GA-reactive T cells from TH1 towards TH2
`during GA treatment43,48
`Induction by GA of cytokine production in MBP-
`specific T cells and vice versa42-44
`Inhibition of proliferation of T cells specific for
`MBP and some other antigens28,44
`TCR antagonism with MBP 82–100 (controversial
`findings)32,44
`Induction of anergy in MBP-specific T-cell clones44
`
`Antigen-nonspecific suppression of T-cell
`proliferation74,75
`No known effect
`
`Inhibition of IFN-g-induced upregulation of MHC
`class II expression76,77
`No known effect
`
`Reduced T-cell migration caused by inhibition of
`matrix metalloproteinases (MMP),78-80 increase of
`soluble adhesion molecules (sICAM-1, sVCAM-
`1),80,81 and decrease of surface-expressed
`adhesion molecules (VLA-4)82
`Induction of TH2 cytokines and reduction of TH1
`cytokines in PBL83-85
`
`No known effect
`
`No known effect
`
`No known effect
`
`No known effect
`
`No known effect
`
`Inhibition of TNF-a and cathepsin-B production in
`a monocytic cell line62
`
`Several effects, e.g., inhibition of IFN-g induction
`of FcgRI expression in monocytes86
`
`Due to space limitations, only representative articles are cited for each mechanism.
`
`APL 5 altered peptide ligand; HLA 5 human histocompatibility leukocyte antigen; IL 5 interleukin; MBP 5 myelin-basic protein;
`MHC 5 major histocompatibility complex; mRNA 5 messenger ribonucleic acid; PBL 5 peripheral blood lymphocytes; sICAM 5 soluble
`intercellular adhesion molecule; sVCAM 5 soluble vascular cell adhesion molecule; TCL 5 T-cell lines; TCR 5 T-cell receptor; TGF 5
`transforming growth factor; TH 5 T-helper; TNF 5 tumor necrosis factor; VLA 5 very late antigen.
`
`act with MHC class I.45,49 It is therefore likely that
`part of the inhibitory effects described in the previ-
`ous section occur by competition between GA and
`other antigens for MHC binding. Clearly, this type of
`competition could occur with any antigen and is
`therefore antigen-nonspecific. However, for reasons
`explained below, this mechanism is probably irrele-
`vant for the in vivo effects of GA.
`In addition to competition
`Interaction with TCR.
`at the MHC class II level, and consistent with ani-
`mal data, GA was reported to act as a “TCR antago-
`nist” against the MBP 82-100 peptide.32 In contrast,
`in another study TCR-antagonistic effects were not
`observed.44 However, when MBP-specific TCL were
`stimulated with different concentrations of GA, their
`subsequent response to the nominal antigen MBP
`was turned off, i.e., the T cells had been “anergized”
`by GA.44 This suggests that a specific TCR engage-
`ment by GA does occur. This mechanism may be
`relevant to the in vivo effects of GA because it does
`
`not require the simultaneous presence of the nomi-
`nal antigen (e.g., MBP).
`Effect on monocytes. Although the vast majority
`of evidence suggests that GA acts primarily at the
`level of T cells, additional effects on other immune
`cells cannot be excluded. For example, GA was
`reported to inhibit a human monocytic cell line,
`THP-1.62 In THP-1 cells stimulated with lipopoly-
`saccharide or IFN-g, GA reduced the percentage of
`cells expressing HLA-DR and DQ antigen and inhib-
`ited the production of TNF-a and cathepsin-B. In
`contrast, the production of IL-1b was increased.62
`The mechanism of these effects and their relevance
`to the overall mechanism of action of GA currently
`are unknown.
`
`Conclusion: proposed mechanism of action of
`GA in multiple sclerosis. The results discussed
`in the previous sections suggest four major effects of
`GA on human T cells:
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`1. GA binds “promiscuously” to MHC class II and
`perhaps MHC class I molecules, thereby competing
`with the MHC binding of other antigens. This effect,
`which by its nature is antigen-nonspecific, is un-
`likely to play a role in vivo because after s.c. admin-
`istration GA is quickly degraded to free amino acids
`and small oligopeptides.63 Therefore it is not likely to
`reach the CNS where it could compete with the rele-
`vant auto-antigens for MHC binding.
`2. GA/MHC competes with MBP for binding to the
`antigen-specific surface receptor of MBP-specific T cells
`(“TCR antagonism”). The experimental evidence sup-
`porting this effect is controversial. If it occurs, it is
`unlikely to be relevant in vivo because GA is unlikely
`to reach sites where it could compete with MBP.
`3. GA/MHC binds to the TCR of T cells specific for
`MBP and, perhaps, other myelin antigens. In this
`view, GA acts like an “altered peptide ligand” (APL)
`relative to MBP. As a consequence, some of the
`myelin-specific, pathogenic T cells might become “an-
`ergic” or be otherwise changed in their properties,
`e.g., in their migratory potential. This effect would
`be relatively antigen-specific and presumably occur
`in the periphery at the injection sites or in their
`draining lymph nodes where the MBP-specific T cells
`might be confronted with GA. Although some in vitro
`findings support this mechanism, it is not yet known
`whether the functional properties of MBP-specific T
`cells are altered in GA-treated patients. It may be of
`relevance in this connection that we were unable to
`isolate MBP-specific TCL from GA-treated patients.43
`4. GA treatment induces a TH1-to-TH2 shift in GA-
`reactive T cells in vivo. The GA-reactive T cells act
`as regulatory cells and have beneficial effects on the
`pathogenic autoimmune reaction. Compared with
`the other putative mechanisms, this currently has
`the strongest experimental support. We would like to
`propose the following scenario (figure): GA-reactive
`TH2-like T cells are able to cross the blood-brain
`barrier because they are activated by daily immuni-
`zation.64 During treatment, the properties of the GA-
`reactive T cells are changed in such a way that they
`increasingly become TH2-like.43,48 Inside the CNS,
`the GA-reactive T cells are confronted with products
`of myelin turnover presented by local APC.65 Some of
`the GA-reactive cells cross-react with MBP or MOG
`and are therefore stimulated to release anti-
`inflammatory cytokines such as IL-4, IL-6, IL-10,
`and even neurotrophic factors.38,39,66 Subsequently,
`the production of pro-inflammatory cytokines such
`as IL-2 and IFN-g by other inflammatory cells is
`reduced via a suppressive bystander effect.34,35,67,68
`Hypothetically, a similar process might occur in the
`periphery: Any viral or bacterial antigens that stim-
`ulate peripheral MBP-specific T cells by molecular
`mimicry69,70 might also activate peripheral GA-
`specific, cross-reactive downregulatory T cells.
`
`Acknowledgment
`The authors thank Ms. Judy Benson for her helpful comments and
`careful reading of the manuscript.
`
`706 NEUROLOGY 56 March (2 of 2) 2001
`
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