Ž .Journal of Neuroimmunology 117 2001 1–8www.elsevier.comrlocaterjneuroin

Review article

Mechanism of action of glucocorticosteroid hormones: possibleimplications for therapy of neuroimmunological disorders

Ralf Gold a,), Frank Buttgereit b, Klaus V. Toyka a

a Clinical Research Group for Multiple Sclerosis and Neuroimmunology, Department of Neurology, Julius Maximilians UniÕersitat Wurzburg, Germany¨ ¨b ( )Department of Medicine Rheumatology , Charite UniÕersity Hospital, Humboldt UniÕersitat Berlin, Germany´ ¨

Received 9 March 2001; received in revised form 23 April 2001; accepted 23 April 2001

Abstract

Glucocorticosteroids are the most potent immunosuppressive and antiinflammatory drugs. Over the six decades that have passed sincetheir discovery, a variety of genomic effector mechanisms of steroid hormones has been described which are mediated by the cytosolicsteroid receptor. Recent evidence supports a direct effect of glucocorticosteroids on cellular membranes that occurs at higher hormoneconcentrations, termed nongenomic effects. These imply a qualitatively distinct mode of steroid action leading to cellular apoptosis. Inthis review, we discuss in vitro and in vivo data on nongenomic effects of glucocorticosteroids and their possible implications for thetherapy of human neuroimmunological diseases. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Multiple sclerosis; Polyneuritis; Myositis; Autoimmunity

1. Introduction

In 1938, natural steroid hormones were first extractedŽ .and purified from the suprarenal adrenal gland. Soon

after the discovery of natural glucocorticosteroids, synthe-sis of steroid hormones was achieved in 1947 and openedimmediate access to steroid therapy of human autoimmune

Ž .disorders Hench et al., 1949 . Even now, in the realm ofmolecular medicine, glucocorticosteroids belong to themost potent immunosuppressive drugs. In the 1950s, greatprogress has been made in the development of new andmore potent steroid derivatives. Despite that, treatmentwith ACTH of hypophyseal origin, which based on theAindirectB release of endogenous glucocorticosteroid andmineralocorticosteroid hormones, has still been widely useduntil 1970. ACTH has now been abandoned for virtuallyall neuroimmunological diseases. At present, the standard

AbbreÕiations: EAEsexperimental autoimmune encephalomyelitis;AT-EAEsadoptive transfer EAE; EAMsexperimental autoimmunemyositis; EANsexperimental autoimmune neuritis; GBSsGuillain–Barre syndrome; GSsglucocorticosteroids; MPsmethylprednisolone;´MRIsmagnetic resonance imaging; MSsmultiple sclerosis; PBLsperipheral blood leucocytes; TCRsT cell receptor

) Corresponding author. Neurologische Universitatsklinik, Josef-¨Schneider-Strasse 11, D-97080 Wurzburg, Germany. Tel.: q49-931-201-¨5755; fax: q49-931-201-3488.

Ž .E-mail address: r.gold@mail.uni-wuerzburg.de R. Gold .

approach to treat exacerbations of neuroinflammatory dis-Žorders is glucocorticosteroid pulse therapy see metaanaly-

ses in Brusaferri and Candelise, 2000; Kaufman et al.,.2000 . By using i.v. dosages in the range of 500–1000 mg

Ž .methylprednisolone MP per day, steroid peak levels are5–10 times higher than those achieved by endogenoussteroids released following ACTH.

Most of the mechanisms of glucocorticosteroid actionsat the genomic leÕel, i.e. those mediated by the cytosolicreceptor, have been described early after the discovery ofGS. For engagement with the cytosolic receptor, lowersteroid concentrations are sufficient, such as that achievedby endogenous steroid hormones released from the adrenalgland during ACTH therapy or by low-dose GS treatment.

Ž .We have now started to realize that other additionalsteroid effector pathways are active at very high dosesgiven during pulse therapy, probably mediated by a directeffect on cellular membranes and consequently ion trans-

Ž .port see review in Buttgereit et al., 1998 . The consequentreduction of the ATP availability is normally observed inapoptosis before DNA fragmentation ensues, and mayexplain the experimental findings of induction of apopto-sis. Thus, a qualitatively distinct mechanism of GS actionmay be achieved, which of course may act in concert withthe traditional genomic pathways. Here, we briefly reviewthe available evidence supporting the nongenomic actionof glucocorticosteroid hormones derived from in vitro data

0165-5728r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0165-5728 01 00330-7

( )R. Gold et al.rJournal of Neuroimmunology 117 2001 1–82

and from findings in disease models for human neuroin-flammatory disorders. In the animal models for Guillain–

Ž . Ž .Barre syndrome GBS and multiple sclerosis MS , exper-´Ž .imental autoimmune neuritis EAN and encephalomyelitis

Ž .EAE , high-dose glucocorticosteroid treatment clearly in-creases apoptosis of invading T cells and accelerates re-covery. Recently, these findings have been extended toexperimental myositis and may also apply to MP pulsetherapy in MS patients.

2. Nongenomic action of glucocorticosteroids on cellu-lar energy metabolism: therapeutical targeting of lym-phocytes by high-dose corticosteroids

The existence of steroid effects mediated by mecha-nisms distinct from genomic steroid action has been ap-parent from the very beginning of research into steroidphysiology and pharmacology, but was neglected for along time. These were collectively termed nongenomic

Ž .actions Buttgereit et al., 1998; Falkenstein et al., 2000a .The evidence came both from the basic sciences and frommany areas of clinical medicine showing rapid glucocorti-

costeroid effects that are incompatible with the more de-layed genomic modes of action leading to new concepts todescribe the therapeutic effects of glucocorticosteroidsŽ . Ž .Buttgereit et al., 1998; Falkenstein et al., 2000b Fig. 1 .

Nongenomic effects are suggested to result from inter-actions with biological membranes—either via membrane-

Ž .bound receptors specific nongenomic effects or via directŽphysicochemical interactions nonspecific or unspecific

.nongenomic effects . In 1991, a membrane corticosteroidreceptor was identified in synaptosomes prepared from anamphibian brain that was considered to participate in the

Ž .regulation of behavior Orchinik et al., 1991 . Further-more, the lysis of lymphoma cells has recently been at-tributed to membrane receptor-mediated mechanisms byassuming their participation in mediating the apoptotic

Žeffects of GS Gametchu, 1987; Gametchu et al., 1993;.Diba et al., 2001 . Although the induction of apoptosis by

Ž .GS is also mediated via genomic effects see below ,nongenomic effects are suggested by the relationship be-tween GS-induced apoptosis and mitochondrial function, atleast at high concentrations of GS. The latter suggestionrefers to the known relationship between glucocorticos-

Žteroid-induced apoptosis and mitochondrial function But-

Fig. 1. Putative molecular pathways of nongenomic and genomic steroid actions. APAFsapoptosis protease-activating factor.

( )R. Gold et al.rJournal of Neuroimmunology 117 2001 1–8 3

.tgereit et al., 2000 . It has been shown that glucocorticos-teroids and other inducers of apoptosis decrease and finallydisrupt the mitochondrial membrane potential. The conse-quent reduction of the ATP availability is normally ob-served in apoptosis before DNA fragmentation ensues, andis associated with alteration in mitochondrial structure andbioenergetic function.

With regard to the molecular mechanisms involved inthese effects on mitochondria and their function during thecourse of apoptosis, genomic glucocorticoid actions maybe implicated, since inhibitors of mRNA or protein synthe-sis can inhibit cell death. Recent observations point to

Žadditional nongenomic effects at high doses of GS Butt-.gereit et al., 2000 . It was shown that high-dose MP

considerably lowered mitochondrial membrane potential inthymocytes within 25 min, which is probably too fast to be

Ž .mediated by genomic effects Buttgereit et al., 1994 . Thisstudy was part of a series of experiments that aimed atinvestigating the unspecific nongenomic effects of gluco-corticoids on energy metabolism of different cell types. Inrat thymocytes and human lymphocytes, high, but thera-peutically relevant concentrations of MP and of other GSimmediately inhibit the respiration of Con A-stimulatedcells at concentrations that leave quiescent cells unaffectedŽ .Buttgereit et al., 1993, 1997; Schmid et al., 2000 . GSinstantly not only reverse but also prevent the mitogeniceffect on respiration. Furthermore, high concentrations of

Ž .GS were also found very rapidly i to reduce themitogen-induced increase of cytoplasmic calcium concen-

Ž .tration and ii to inhibit cation cycling across the plasmaŽ .membrane with iii only a small effect on intracellular

protein synthesis. These effects are thought to be causedby direct effects and not by a reduction in ATP production,even though substrate oxidation is slightly inhibited and

Žthe mitochondrial proton leak is slightly increased which,. Žtogether, decreased ATP availability . Buttgereit et al.,

.1994 . All of these effects may have therapeutic relevancein high-dose GS therapy, since the acute immune responsecould be diminished or prevented by interfering with theactivation of lymphocytes via processes like the above-mentioned rise in intracellular Ca2q concentration, whichare essential for the immediate and sustained activation oflymphocytes.

In clinical practice, the immediate effects produced byhigh-dose GS could be additive to the effects mediated bynuclear receptors. This is in line with the clinical observa-tion that often only high doses of glucocorticoids aresuccessful in acute exacerbations of autoimmune diseases

Ž .including MS Brusaferri and Candelise, 2000 . The initialeffects of high-dose therapy occur rapidly, i.e within thefirst week of starting the treatment. It is suggested, but notclear yet, that unspecific nongenomic glucocorticoid ef-fects are only present during high-dose treatment, and arelost when the GS dose is tapered. It is also not knownwhether a ceiling effect applies to the nongenomic actionsof high-dose GS, i.e. if one would constantly apply very

high doses of MP, would the therapeutic effects remain asgood as in the initial phase or fade away? This question,though of theoretical interest, may not be answered sincethe duration of treatment with high-dose GS is limited forthe well known side effects.

The observations summarized above led to the hypothe-sis that the relative potencies of nongenomic and genomiceffects within the spectrum of therapeutically used GS maydiffer substantially. Indeed, we have recently reported thatthe unspecific nongenomic potencies of five clinicallyimportant glucocorticoids are different from their classicalŽ . Ž .genomic potencies Buttgereit et al., 1999 . The relativedrug potencies for nongenomic effects were found to be in

Ž .the following order: prednylidene 3.0 )dexamethasoneŽ . Ž . Ž .1.2 )MP 1.0 ) prednisolone 0.4 ) betamethasoneŽ .0.2 , while the order for the relative potency of the

Ž .genomic effects is dexamethasone, betamethasone 25 )Ž . Ž . Ž .MP 5 )prednisolone 4 )prednylidene 3.5 . A similar

ratio of nongenomic potencies was recently also reportedŽ .in human cells Schmid et al., 2000 . Therefore, our

understanding of the therapeutic value has to be reformu-lated and the choice of the various GS derivatives inhigh-dose therapy may need reevaluation against this back-ground of new information.

3. Evidence for nongenomic action of glucocorticos-teroids in animal models for neuroimmunological disor-ders and its linkage to induction of T cell apoptosis insitu

3.1. Apoptosis in neuroinflammation

Apoptosis is a distinct mode of cell death. Although itŽ .was primarily defined by morphology Kerr et al., 1972 , it

has an array of pathophysiological and functional implica-tions. Typically, chromatin condensation and shrinkage ofthe cell occur in parallel and the integrity of the cellmembrane is preserved for a long time. These morphologi-cal events coincide with distinct biochemical events that

Ž .lead to caspase activation Nicholson, 1999 , changes inŽ .mitochondria and cellular membranes Vermes et al., 1995

and finally cause oligonucleosomal DNA fragmentationŽwhich can also be used for detection of apoptosis Gold et

.al., 1994 . Interestingly, the classical paper describingDNA laddering during apoptosis by gel electrophoresisŽ .Wyllie, 1980 took advantage of the susceptibility ofthymocytes to the genomic action of steroid hormones.

ŽThe importance of apoptosis for the immune system Cohen.et al., 1992 is thought to base on the preserved integrity of

apoptotic cells, which allows specific signalling to phago-Ž .cytic cells Savill et al., 1993; Chan et al., 2001 . Thus,

release of proinflammatory cytoplasmic constituents withinduction of secondary necrosis are avoided. It is conceiv-able that in vulnerable organs such as the nervous system,the parenchyma is extremely susceptible and has a lowregenerative capacity.

( )R. Gold et al.rJournal of Neuroimmunology 117 2001 1–84

EAE and EAN serve as animal models for the humanŽ .diseases MS and GBS see review in Gold et al., 2000 .

Both models can be induced by immunization with myelinŽ .antigen Aactive diseaseB or by intravenous transfer of

CD4 q T lymphocytes specific for myelin-antigensŽ Ž . .Aadoptive-transfer AT modelB . Accordingly, experi-

Ž .mental myositis EAM induced by repetitive immuniza-tion with myosin mimicks some features of polymyositisŽ .Kojima et al., 1997 . These disease models allow furtherstudies on pathogenesis and therapeutic mechanisms whichare active in situ. T cell apoptosis was identified as a majorphysiological mechanism of cell destruction in inflamma-

Žtory brain lesions of EAE in the Lewis rat Pender et al.,.1991 , and later on also in the peripheral nervous system inŽ .EAN Zettl et al., 1996 . T cell apoptosis in EAE also

occurs after adrenalectomy, although at a lower levelŽ .Smith et al., 1996 . Interestingly, spontaneous T cellapoptosis as determined by morphology and DNA frag-mentation is not observed in inflamed muscle or skinŽ .Schneider et al., 1996 .

3.2. Glucocorticosteroid-induced T cell apoptosis in cocul-ture models with glia cells

As yet the mediators of T cell apoptosis in the nervoussystem are still poorly understood. To some extent, TNF-a

Ž . Žand its p55 receptor TNF-R1 are involved Bachmann et.al., 1999 . Due to the tissue-specificity, resident neural

cells directly or through soluble factors may render T cellssusceptible for apoptosis or synergize with systemic hu-moral factors to deliver a proapoptotic stimulus. In thecentral nervous system, astrocytes and microglia cells mayexert a critical role. Astrocytes are only partially compe-tent antigen-presenting cells, unable to trigger the complete

Ž .T cell activation programme Weber et al., 1994 . Westudied antigen-driven effects of astrocytes on T cells and

Ž .evaluated the role of steroid hormones Gold et al., 1996a .Interestingly, astrocytes exerted a suppressive effect on Tcell activation, which was mediated by cell contact. Whenglucocorticosteroids were added after antigen presentation,they markedly augmented T cell apoptosis when given toT cellrastrocyte cultures during late stages of T cellactivation on day 3, but importantly, not when added atearlier time points or when thymus cells were used asantigen presenters. This effect was achieved with hydro-cortisone at a concentration of 5=10y5 M. Similar toastrocytes in the brain, Schwann cells prime pathogenic Tcells in the peripheral nervous system for cellular apopto-

Ž .sis Gold et al., 1996b .

3.3. Glucocorticosteroid-induced T cell apoptosis in ani-mal models

Steroid hormones have been used for a long time inEAE and EAN. In most cases, investigators focussed on a

low-dose regimen in the range of 1 mgrkg and primarilyaimed at the disease course or at inflammatory mecha-

Ž .nisms Hartung et al., 1988 . Our first experiments onsteroid pulse therapy were conducted in EAN. In theinflamed peripheral nerve, T cell apoptosis during disease

Žcourse is less frequent than in brain see review in Gold et.al., 1997 . Based on the therapeutic use of high-dose

glucocorticosteroids, we first investigated whether steroidŽApulse-therapyB could induce T cell apoptosis in situ Zettl

.et al., 1995 . Since a predictable and synchronized diseasecourse was highly desirable for these investigations, wefocussed on AT-EAN of the Lewis rat, which was pro-duced by transfer of activated, P2-specific T lymphocytes.To delineate whether the effect of steroid hormones is

Žstage-specific, two pulses of glucocorticosteroids 10 mg.prednisolone-21-hydrogen succinaterkg body weight were

administered, either after disease onset or at its presumedmaximum. Due to the rapid elimination of apoptotic cellsŽ .Bursch et al., 1990 , rats had to be sacrificed 6 h after thesecond steroid pulse, before a clinical effect became appar-ent. At both time points, there was a massive reduction ofT cell inflammation and a four- to five-fold increase of T

Ž .cell apoptosis in the inflamed sciatic nerve Fig. 2 . Inaddition, reduced cellular proliferation in lymphoid organsand loss of thymus weight were observed in steroid recipi-ents reflecting typical genomic actions of steroids.

Subsequently, steroid-induced apoptosis was surveyedin EAE, where dose–response relations were delineated

Ž .more clearly Schmidt et al., 2000 . In EAE doses of atleast 10 mgrkg body weight MP were needed to enhanceT cell apoptosis. Highest levels of apoptosis were achieved

ŽFig. 2. T cell apoptosis in sciatic nerve after i.v. steroid therapy 10 mg.prednisolone-21-hydrogen succinaterkg body weight at disease onset

Ž . Ž .day 5 or at the maximum of disease day 8 . Placebo-treated ratsreceived PBS by i.v. injection. Y-axis shows mean percentage of apop-

Ž .totic T cells"SD. modified from Zettl et al., 1995 .

( )R. Gold et al.rJournal of Neuroimmunology 117 2001 1–8 5

at 50 mgrkg MP, a dose which came close to high-doseŽsteroid therapy used in acute spinal cord injury Bracken et

.al., 1990 . Surprisingly, low-dose treatment with steroidsat 1 mgrkg body weight did not modulate T cell apopto-sis. Furthermore, we could show that serum levels of

y5 Ž .3.3=10 M MP were reached in individual rats Fig. 3 .These concentrations are clearly within the range wherethe above reported nongenomic glucocorticoid effects areto be observed in vitro. There was no unwanted side effectof high-dose glucocorticosteroids on oligodendrocyte sur-vival in EAE. In addition to these experiments, a dose-de-pendent effect of glucocorticosteroid-induced apoptosis inactive EAE could also be demonstrated in a recent studyŽ .Nguyen et al., 1997 . There is some evidence thatsteroid-induced apoptosis may interfere with apoptoticelimination of autoreactive T cells in the nervous systemŽ .McCombe et al., 1996 . This is supported by in vitro datashowing an antagonistic effect of steroids and activation

Ž .signals by the TCR Zacharchuk et al., 1990 . Similarly,when applied to a model of antigen-induced T cell apopto-sis simultaneous injection of MP may interfere with sig-nalling by the TCR and even caused T cell proliferation in

Ž .situ Weishaupt et al., 2001 .In human idiopathic inflammatory myopathies, apop-

totic T cells or muscle fibers are only very rarely de-Ž .tectable Behrens et al., 1997; Schneider et al., 1996 .

Furthermore, in polymyositis CD8q T cells exert a keyŽ .role in the pathogenesis Dalakas, 1991 . Both factors

mediating CD8q T cell apoptosis and steroid-susceptibil-ity of CD8q T cells in myositis may differ from themodels of EAN and EAE, where CD4q T cells play a

Ž .critical role see review in Gold et al., 2000 . Recently,Ž .Kojima et al. 1997 induced EAM by the combined

application of skeletal myosin from different species.Analysis of inflammatory infiltrates revealed features simi-lar to those observed in human autoimmune polymyositis.Although EAM only partly reflects features of humanpolymyositis, it appeared attractive to investigate whether

glucocorticosteroids, the mainstay therapy for humanŽ .polymyositis and dermatomyositis Dalakas, 1991 , may

act similarly by inducing apoptosis in inflamed muscletissue. The rate of spontaneous T cell apoptosis in ratEAM was low, even in muscle specimens with extensiveinflammation. Intravenous glucocorticosteroid pulse wasdelivered by injection of 10 mgrkg MP twice within 12 h.T cell apoptosis reached highest values with 33% of Tcells in rats of the steroid treated, but was only 5% in thecontrol group. Up to 50% of the apoptotic T cells in thesteroid treated rats were CD8q , leading to the assumptionthat not only CD8q T cells, but also CD4 positive T cells,were eliminated by apoptosis. Then, we evaluated markedlyinfiltrated muscle specimens of each group: 28 musclesfrom nine rats of the control groups, 13 muscles from sixanimals of the steroid-treated groups. The mean percentageof apoptotic T cells in the MP-treated group and in the

Ž .control group was 11.2"10.9% mean"SD and 0.8"Ž . Ž1.6% mean"SD , respectively p-0.0001, unpaired t-

.test . These results show that steroid-induced apoptosis isalso active in tissue where T cell apoptosis does not occurduring the natural disease course.

3.4. Glucocorticosteroid induced T cell apoptosis in hu-man therapy

Several clinical studies on glucocorticosteroid treatmentof MS yielded controversial results as to the most effective

Ždose and form of application Beck et al., 1992; Barnes et.al., 1997; Sellebjerg et al., 1999 . In view of the hetero-

Žgeneity of disease mechanisms in MS Lucchinetti et al.,.2000 and also of heterogeneity of available immunothera-Ž .pies Noseworthy et al., 1999 , it is conceivable that

controlled multicenter trials addressing clinical endpointssimilar to IFNb trials are hard to realize. Also, patents formost steroid preparations have expired. Thus, at present,the issue of low-dose, high- or ultrahigh steroid treatmentof MS relapse can only be further assessed on the basis of

Ž . Ž .Fig. 3. Methylprednisolone MP concentrations in serum, CSF, and spinal cord. MP levels ordinate, logarithmic scale , analysed by HPLC 2 h after singleŽ . Ži.v. injection of 50 mgrkg MP or 10 mgrkg MP in mild or severe EAE data are given as mean"SD : p-0.05 serum MP 50 mg vs. MP 10 mg in

. Ž . Žsevere EAE , p-0.05 serum and spinal cord in severe EAE with 10 mgrkg MP vs. mild EAE with 10 mgrkg MP , p-0.01 spinal cord MP 50 mg vs.. Ž .MP 10 mg in severe EAE , modified from Schmidt et al. 2000 .

( )R. Gold et al.rJournal of Neuroimmunology 117 2001 1–86

paraclinical parameters such as MRI. Oliveri and col-leagues have shown that pulse therapy with 2000 mg MPon 7 subsequent days was more efficent than 500 mg toreverse breakdown of the blood–brain barrier, as moni-tored by MRI and gadolinium enhancement. Recently, weconducted a clinical study focussing on apoptosis of pe-

Ž .ripheral blood leukocytes PBL , following MP pulse ther-apy in 66 patients with relapsing–remitting, secondary

Žprogressive or primary progressive MS Leussink et al.,.2001 . Also, cytokine secretion and cellular proliferation

was examined. Apoptosis of unstimulated PBL wasmarkedly and significantly augmented in all three MSsubgroups, typically increasing by 100%. FACS analysisshowed that apoptosis affected predominantly, but notexclusively CD4q T cells. Flow cytometric double label-ing also revealed a significant increase of apoptotic CD8qT cells, but no increase in apoptotic B cells or NK cells.The expression of bcl-2 in T cell subpopulations was notsignificantly modified by high-dose glucocorticosteroids.Culture supernatants of TCR-stimulated PBL after MPtherapy contained lower concentrations of IL-2, IFN-g,and TNF-a than those from PBL taken before pulsetherapy. Basically, similar changes in the rate of apoptosisand cytokine production were seen in non-MS patients,which received pulse therapy for other medical reasons.

In human T cell lines, glucocorticosteroids mediate Tcell apoptosis by a caspase-dependent manner and protect

ŽT cells against CD95-mediated apoptosis Zipp et al.,.2000 . Again, these data underscore that steroid hormones

may interact, or even antagonize with other stimuli mediat-ing T cell death or survival.

4. Conclusion

There is growing evidence on GS-action also occurringat the nongenomic level. These mechanisms can easily bedelineated in vitro. In vivo, nongenomic steroid effectsmay interfere with bioenergetic processes including sig-nalling pathways. It is often difficult to distinguish in vivoto which extent genomic action of GS, such as inhibitionof cytokine production, act in concert with nongenomiceffects. Yet, experimental data are available that supportthe use of high-dose steroid therapy to accelerate termina-tion of inflammation. We feel that these features stronglyencourage the use of glucocorticosteroid pulse therapy inpatients with active neuroimmunological diseases. In par-ticular, it applies to relapses of multiple sclerosis, severe

Ž .chronic polyneuritis CIDP and polyrdermatomyositis,but also to acute phases of rheumatic diseases, such assystemic lupus erythematosus and vasculitis.

The experimental findings with very high doses shouldgive rise to further controlled studies in selected popula-tions. At present, there is still uncertainty whether tradi-tional equivalence doses used for genomic steroid actionneed to be redefined for high-dose regimens, since under

high-dose treatment also nongenomic GS actions are in-volved.

Acknowledgements

We thank our colleagues, Dr. J. Schmidt, Dr. U.K.Zettl, Dr. V.I. Leussink, Dr. C. Schneider and Dr. M.D.Brand for their invaluable contributions to the experimen-tal work cited here. We are indebted to Dr. Marinos C.Dalakas for his critical reading and helpful suggestions.

Our research is supported by grants from the DeutscheForschungsgemeinschaft, Gemeinnutzige Hertie-Stiftung,¨Deutsche Multiple Sklerose Gesellschaft, DeutscherAkademischer Austauschdienst, Boehringer IngelheimFonds and by research funds from the State of Bavaria.

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