Cytokine & Growth Factor Reviews 22 (2011) 359–365

Survey

Probing cytokines, chemokines and matrix metalloproteinases towards betterimmunotherapies of multiple sclerosis

Ghislain Opdenakker *, Jo Van Damme

Laboratories of Immunobiology and Molecular Immunology, Rega Institute for Medical Research, K.U. Leuven, B-3000 Leuven, Belgium

Contents

1. Multiple sclerosis as an autoimmune disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

2. The REGA model of autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

3. Additional examples of remnant epitopes in MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

3.1. aB-crystallin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

3.2. Beta-interferon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

4. A role for targeting matrix metalloproteinases in MS? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

5. A role for targeting chemokines? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

A R T I C L E I N F O

Keywords:

Autoimmunity

Immunodominant epitopes

Posttranslational modification

Extracellular proteolysis

Interferon

A B S T R A C T

Multiple sclerosis (MS) is an autoimmune disease with a spectrum of clinical evolutions. We here

summarize recent insights into the neuroinflammatory processes of demyelination, vascular cuffing,

destruction of the blood brain barrier (BBB), neuronal toxicity and the ensuing (re)activation of

autoreactive lymphocytes. Translation of these processes in molecular terms indicates that cytokines,

including interferons, ligands of the tumor necrosis factor receptor family and interleukins, and also

chemokines and matrix metalloproteinases play pivotal roles in MS. This not only helps to understand

disease mechanisms in the central nervous system of affected patients, but also forms a solid scientific

basis to improve present therapies. Treatment of MS with parenterally administered anti-inflammatory

agents may be improved, based on present knowledge and new insights obtained with animal models.

Such innovations include better use of knowledge about the formulation, administration, turnover and

glycosylation of interferon-b (IFN-b), combinations of IFN-b with inhibitors of IFN-b-degrading

proteinases in MS, and new ways to diminish vascular cuffs and the transmigration of leukocytes across

the two basement membranes of the BBB. Novel molecules interfering with matrix metalloproteinases

and chemokines, such as EMMPRIN, COAM and monoclonal antibodies are currently being investigated,

demonstrating continued efforts to find new drugs for MS treatment.

� 2011 Elsevier Ltd. All rights reserved.

Contents lists available at SciVerse ScienceDirect

Cytokine & Growth Factor Reviews

jo ur n al ho mep ag e: www .e lsev ier . c om / loc ate /c yto g f r

Abbreviations: BBB, blood brain barrier; CD, cluster of differentiation; COAM,

chlorite-oxydized oxyamylose; EAE, experimental autoimmune encephalomyelitis;

EMMPRIN, extracellular matrix metalloproteinase inducer; IFN-, interferon-; IL-,

interleukin-; MMP-, matrix metalloproteinase-; MOG, myelin oligodendrocyte

glycoprotein; MS, multiple sclerosis; REGA, remnant epitopes generate autoimmu-

nity; Th, T helper cell subset; TIMP-, tissue inhibitor of metalloproteinase-; TNF-,

tumor necrosis factor-.

* Corresponding author at: Rega Institute for Medical Research, Minderbroe-

dersstraat 10, B-3000 Leuven, Belgium. Tel.: +32 16 337385; fax: +32 16 337340.

E-mail addresses: Ghislain.opdenakker@rega.kuleuven.be (G. Opdenakker),

Jo.vandamme@rega.kuleuven.be (J. Van Damme).

1359-6101/$ – see front matter � 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.cytogfr.2011.11.005

1. Multiple sclerosis as an autoimmune disease

Multiple sclerosis (MS) is a chronic neuroinflammatory diseasewith unknown etiology and variable clinical evolution. Althoughneuroinflammation is a descriptive denominator in MS based onhistopathological observations, namely the penetration of leuko-cytes into the central nervous system, the clinical symptoms ofrelapses, remissions and progressive paralysis are the result oflosses of myelin and neurons [1,2]. In the absence of etiologicalfactors as targets for prevention and therapy, the definition of

G. Opdenakker, J. Van Damme / Cytokine & Growth Factor Reviews 22 (2011) 359–365360

molecular mechanisms that form the basis of inflammation,demyelination and toxicity for neurons have led to a number oftreatments that slow down disease progression in specific patientcohorts, but that do not cure the disease. Ideally, an MS drug shouldreverse the processes of neuroinflammation, demyelination andneuronal loss, but such substances do not exist. Like bacterialinfections, MS was once in history a diagnostic problem and notreatment existed. Now, diagnosis is improved, although the needfor better diagnostic and prognostic markers persists. Therapieshave been refined beyond anti-inflammatory glucocorticosteroids.The introduction of IFN-b, copolymer and, more recently,natalizumab (against a1b4 integrin) has considerably improvedthe life of many MS patients, but severe side-effects as well asintrinsic therapeutic limitations of these drugs must stimulatebasic and clinical researchers to define new, better and morepatient-compliant ways of treatment.

The neuroinflammatory MS process usually starts in patients intheir early twenties. This young age hints to a slow infectionaround adolescence age as a possible (co-)factor of the disease or toviruses being associated with MS. Many viruses have beensuspected and studied in this regard, but so far no specific virushas been found to be the cause of MS [3,4]. Associations ofdemyelination with virus infections have been detected in animalmodels (e.g. with Theiler’s virus) and by presently used treatments.Polyoma virus reactivation resulting in progressive multifocalleukoencephalopathy (PML) has been observed recently duringimmunosuppressive treatment of MS with the humanizedmonoclonal antibody natalizumab (Tysabri) [5,6]. Finally, one ofthe present treatments of MS is with recombinant IFN-b andnatural IFN-b was originally discovered as a broad spectrumantiviral agent [7,8]. MS strikes twice as many women than men ofCaucasian origin, implicating hormonal and genetic influences.Worldwide, about 2–3 million MS patients are mainly found inEurope (with a decreasing trend form Northern to SouthernEurope) and in countries with Caucasian immigration, includingthe USA and Canada, Australia and New Zealand and Northern Asia[9,10]. The possible influence of the geographic environment hasbeen studied with the help of monozygotic twins. Geneticallyidentical twins obtain the MS prevalence from their prepuberalgeographic destination [11].

In analogy with other multifactorial diseases, MS is influencedby genetic and environmental factors. With new tools of molecularbiology, such as whole genome sequencing, genome-wideassociation studies [12,13], gene expression profiling coupledwith protein and glycosylation analysis [14,15], we might in thefuture be able to define common identifiers for specific cohorts ofMS patients, to perform better and earlier diagnosis and hopefullydiscover etiological factors. However, this is presently not yetpossible and therapies are directed to block the immune processes,both innate and adaptive, that are associated with MS.

2. The REGA model of autoimmunity

Although we have modern technologies presently at ourdisposal to decipher a disease process at molecular and cellularlevels with unsurpassed detail, most insights are still based onhistopathological findings from long ago. MS is characterized bydemyelination, neuronal loss and neuroinflammation with vascu-lar cuffing [1,2]. Many of these processes can be brought back tomodel molecules and specific cell types. Demyelination may beviewed as a biochemical process with enzymes breaking down theconstituents of myelin, neuronal loss may be based on neurotoxicfactors and cells, neuroinflammation may be related to prototypicaspects of acute and chronic inflammation and vascular cuffs maybe related to the special nature of the blood brain barrier with twodifferent basement membranes.

About 15 years ago, we assembled the available informationfrom histopathological data and inflammation-associated mole-cules to generate a comprehensive model to understand MS and toplace MS therapies into a new context. We called this the REGAmodel for ‘‘Remnant Epitopes Generate Autoimmunity’’ on thebasis of our findings that proteinases, notably matrix metallopro-teinases, cleave substrate proteins into autoimmune peptides that(re)activate specific T cells [16]. We used MMP-2 and MMP-9 asexamples of proteinases in this process, because the earlypublications linked MMP-9 in the cerebrospinal fluid with multiplesclerosis, but also with optic neuritis and other neuroinflammatorydiseases [17,18] and because myelin basic protein was cleaved intoimmunodominant and encephalitogenic peptides by MMP-9[19,20]. Although most recognized for these two aspects, theREGA model contained much more information related toautoimmune diseases (e.g. about regulatory cytokines andchemokines) that was instrumental and will remain in the futureimportant for drug development [16]: (a) This REGA-modeladdressed the critical point that (unspecific) inflammatoryreactions with myeloid cells may be primordial and a possibletarget in MS. In 1994, this was rather controversial and led to someopposition in an era when most MS research was centered on T-cells. (b) It translated the central stage of inflammation in theautoimmune process into molecules and addressed the impor-tance of balances between pro-inflammatory versus anti-inflam-matory primary cytokines in the disease process. IL-1b, TNF-a andIFN-g were included as pro-inflammatory cytokines and IFN-a/band TGF-b as anti-inflammatory cytokines. (c) It demonstrated theimportance of chemokines in MS. This aspect has recently gainedimportance in MS research (vide infra). (d) It illustrated that thebalances between extracellular proteinases and proteinase inhi-bitors contribute to, even may determine, autoantigen generation.Most critically, it placed extracellular proteolysis at the same levelas lysosomal or proteosomal activities in antigen processing. (e) Itaddressed the role of myeloid antigen presenting cells in theactivation of T lymphocytes.

Meanwhile, insights into the producer cell types of thecytokines involved in the autoimmune process have increasedenormously, and Th1, Th2, Th17 and Treg cells have been definedin terms of regulation of transcription factors and resultingcytokine productions [21–24]. Therefore the original cartoon of theREGA model [16] is here adapted with the inclusion of theselymphocyte subsets and the corresponding key cytokines (Fig. 1).

3. Additional examples of remnant epitopes in MS

Remnant epitopes after extracellular cleavage in MS were firstillustrated with the hydrophilic myelin constituent myelin basicprotein (MBP) [16,19,20]. However, other immunodominant epi-topes have meanwhile been studied in detail and followedas examples. We here illustrate the concept with aB-crystallinand IFN-b.

3.1. aB-crystallin

aB-crystallin was described as a candidate pathogenicautoantigen in MS [25] and therefore it was intriguing to definethe ‘‘remnant epitopes’’ of this crystallin after cleavage by theMS-associated protease MMP-9 [26]. This study revealed variousfragments and several of these were defined as autoantigens inthe mouse, the rat and even in the human species (Fig. 2). Thisled to the conclusion that crystallin itself was not a pathogenicautoantigen. Instead, the remnant epitopes after extracellularcleavage into autoantigens were the culprits. Our conclusionwas that ab-crystallin should stay intact in order to preventautoimmune reactivation [26]. aB-crystallin, being a heat shock

Etiologicalagents ?

Trigger

Local primar y cytokines

Diseasepromotion

Diseaselimitation

IL- 1TNF-α

IFN-αIFN-βTGF β

IL-10IL-4

IL-17IL-12

Cytokine network

IL- 2IFN-γ

Gene tics, environme nt, immuni ty

Local secon dary cytokines

granulocytes monocytes

lymphoc ytes

variouscell type s

Proteinases Proteinase

TNF α TGF-β IL 4IL 12

Chemokines(e.g. CCLs, CXCLs)

IL- 6

Myel oid cel l recruitmentandstimulation

net work IFN γ

Proteinas es(e.g. gelati nase B,

granzymes)

Proteinas einhibitors

(e.g. TIMPs, PAIs )

Th17

Remnant pepti des

TregAntigen specificT Cell subsets

Enzyme cascades

Th1 Th2subsets

Fig. 1. The REGA model anno 2011. Multiple sclerosis is a multifactorial

autoimmune disease of unknown etiology. Influencing factors include host

genetics, environmental determinants, and the workings of the immune system.

The presented scheme illustrates all possible drug targets (disease-promoting

cytokines, chemokines, proteinases and cells) as well as compounds that limit

diseases (anti-inflammatory cytokines, proteinase inhibitors). On the basis of

definition of T cell subsets [21–24] and functional dichotomies between Th1 and

Th2, as well as Th17 and Treg cells, better therapies targeting or enhancing such

subsets will be possible. The flow chart is adapted from Ref. [16] and

complemented, according to recent insights on cytokines, chemokines and T cell

subsets, involved in the pathobiology of MS. PAI, plasminogen activator inhibitor.

G. Opdenakker, J. Van Damme / Cytokine & Growth Factor Reviews 22 (2011) 359–365 361

protein, is in fact protective in MS and this was later borne outby studies with aB-crystallin knockout mice [27]. The conclu-sions from all these studies were that aB-crystallin itself is notpathogenic, but instead protective. In fact, the REGA modelexplains the original findings about the involvement of aB-crystallin in MS. Indeed, in an inflammatory context, proteinasessuch as MMP-9 cleave the crystallin into remnant epitopes,

16*1

722

*23

54*5

560

*61

48*4

9

N

1 16 88372625Biozz i ABH

Human21 6040 41

Fig. 2. Remnant epitopes from aB-crystallin after cleavage by MMP-9. The horizontal ba

pointing to the major cleavage sites by gelatinase B/MMP-9. The numbers of the P1–P10

cleavage sites. The middle bars show the immunodominant epitopes as defined by epitop

the bottom bars indicate immunodominant fragments, as obtained by epitope scanning w

epitopes coincide with the immunodominant peptides. The scheme is adapted from St

which in turn stimulate autorecative T cells to enhance orperpetuate the autoimmune process [26]. This proteolysis alsoresults in less protective heat shock protein [27].

3.2. Beta-interferon

Another example is related to the commonly used IFN-b for thetreatment of MS [8,28,29]. Various commercial preparations ofIFN-b exist, but mainly two forms are defined biochemically:glycosylated and aglycosyl IFN-b. A simple analysis by gelfiltration chromatography indicates that presently used prepara-tions contain little bio-active protein. Indeed, the clinically usedpreparations contain mainly albumin as a stabilizer and to preventthe interferon to stick to recipients and syringes. In addition, aconsiderable amount of the recombinant IFN-b is aggregated, i.e.not necessarily bio-available but ideal to generate immunereactions [30]. A relevant question is related to what happenswhen such preparations are injected into patients with MS, whoare known to have increased levels of MMP-9 [17,18]. In addition,treatment with biologically active IFN-b may reduce the levels ofMMP-9 [31]. The aglycosyl IFN-b is readily degraded by MMP-9into fragments, that are, in fact, remnant epitopes. The IFN-bioactivity is killed and the remnant epitopes are ideal to generateT cell reactivity after antigen presentation [30]. No wonder thenthat the glycosylated interferon, which is better protected againstproteolysis by its attached N-linked oligosaccharide [32,33], is lessdegraded and yields less neutralizing antibodies in vivo [34]. Inaddition, the presence of a majority of aggregates in commercialpreparations [30] is an issue and may stimulate improvements offormulations, as such aggregates may influence immunogenicity[35]. Clinical studies demonstrate that the chance to developneutralizing antibodies is considerably higher with aggregatedversus soluble or with aglycosyl versus N-glycosylated interferon. Itis quite frustrating to observe that with this knowledge not moreefforts are made to generate better formulations of recombinantIFN-b and that still so many new patients are placed on aglycosylinterferon for their primary treatment. The societal costs and lossesby the health insurances are enormous and this when anopportunity exists to give the patients a better treatment at asimilar cost.

4. A role for targeting matrix metalloproteinases in MS?

On the basis of the above-mentioned literature, it seems logicthat direct inhibition of proteolysis or induction of a beneficialbalance between endogenous proteinases and their naturalinhibitors, for instance by transcriptional control mechanisms,are possible treatment strategies. However, further proof ofconcept was needed to demonstrate a pathogenic role ofextracellular proteolysis. One way to study this was with the

88*8

9

132*

133

150*

151

C

861351

131 15 0

r represents the protein sequence of human aB-crystallin with the vertical arrows

residue pairs of proteolysis are indicated on top and the asterisks (*) represent the

e scanning and activation of autoreactive T cells from Biozzi ABH mice [72], whereas

ith human lymphocytes from MS patients [73]. It is clear that several of the remnant

arckx et al. [26].

G. Opdenakker, J. Van Damme / Cytokine & Growth Factor Reviews 22 (2011) 359–365362

use of MMP knockout mice. So, it was found that young MMP-9knockout mice are partially resistant against the development ofexperimental autoimmune encephalomyelitis (EAE) [36,37]. How-ever, when both gelatinases, MMP-2/gelatinase A and MMP-9/gelatinase B were genetically knocked out, a complete resistanceagainst MOG-peptide-induced EAE was observed [38]. The latterstudy is important for several reasons. First, it underlines theoriginal findings that many proteinases act in cascades or innetworks [39,40]. Indeed MMP-2 had been shown to activateMMP-9 in vitro [41]. Both enzymes thus reinforce each other andboth enzymes share a number of substrates, including denaturedcollagen or gelatin [42] and b-dystroglycan [38]. These twoproteins are prototype substrates in the anchorage of basementmembranes. Second, the study by Agrawal et al. [38] points to thefact that high selectivity of inhibitors towards single MMPs, oncehailed as essential to make novel drugs for cancer therapy, isperhaps not necessary. This is hopeful because a vast knowledgeexists on MMP inhibitors in inflammatory or vascular diseases andmany phenotypes of MMP knockouts, including EAE resistance, arewell established [43]. Within the classes of small moleculeinhibitors, we have studied tetracyclines and their structure-function characteristics towards MMP-9 inhibition. Minocyclineand doxycycline were the best tetracycline MMP-9 inhibitors [44].Meanwhile, a number of studies have shown protection againstEAE by minocycline [45–48] and a limited number of (pre)clinicalstudies have been initiated that combine an MMP inhibitor (MMP-9 is known to destroy IFN-b) with IFN-b [47–50]. It may be thattetracyclines exert other effects than MMP-2 and MMP-9inhibition. Indeed, the context of an inflammatory reaction ismore complex and with many other proteolytic events than thosemediated by MMPs. In principle all classes of proteinases areinvolved, e.g. serine proteinases of the fibrinogenic and fibrinolyticcascades, proteasomal threonine proteinases, lysosomal asparticacid proteinases and caspases as thiol proteases in cell apoptosis.

Various ways exist to exploit the knowledge about proteinase/proteinase inhibitor balances in MS. In principle any good inhibitorof MMP-9/MMP-2 [43] may be combined with existing MS drugtherapies, e.g. corticosteroids, IFN-b, monoclonal antibodies

endothelial ce llendothelialbasementmembrane

intravascula rl k tleukocytes in

IFN

leukocyte

adhesion

astrocyte endfeet

parenchymalbasement

b

leukocytes invascular cuf f

Con taine d cuff

membrane

No parenchymalcell penet rati on

Fig. 3. The blood brain barrier as location for drug targeting. The schemes are adapted fro

leukocytes are contained within the parenchymal basement membrane produced by th

basement membrane, as happens in any inflammatory reaction. Type I interferons (IFN) a

including inhibition of proteolysis. As long as the leukocytes are contained within the cuff

with production of pro-inflammatory cytokines Il-1b, TNF-a and IL-17, chemokines and

and infiltration of the parenchyma of the central nervous system.

against adhesion molecules. A few examples are already substan-tiated with literature data. These include combination of anti-inflammatory and statin therapy with tetracyclines [48], combi-nation of IFN-b with tetracyclines [49]. Also neutralizingmonoclonal antibodies against MMPs [43,51] may follow as futuretherapies. With the present knowledge [38], a dual-specificantibody against MMP-2 and MMP-9 conceivably may make apotential novel therapy.

5. A role for targeting chemokines?

A second category of possible target molecules for thedevelopment of novel MS therapies is the chemokines [16,37].Chemokines are small chemotactic cytokines, usually less than10 kDa, and direct the migration of inflammatory cells throughoutthe host and assist in antigen presentation, mainly executed byshort-living myeloid cells (Fig. 1). Chemokines also define thespatial and temporal distribution and hence the education ofadaptive immune cells, the long living lymphocytes. As referred toin the introduction section, locoregional histopathology analysis ofthe cental nervous system reveals which cells are present in MSlesions. We can now translate the available neurohistopathologicalknowledge of MS lesions by the definition of chemokine expressionand the presence of chemokine receptors on the infiltratedleukocytes. Presently, this is intensively investigated in brainhomogenates [52,53] and in vascular cuffs [54–56]. To understandwell the structural and biological aspects of vascular cuffs, thereader is referred to a seminal review [57].

Fig. 3 illustrates schematically what supposedly takes place invascular cuffs and reconciles data from many laboratories. On theone hand, as long as the cuffing leukocytes are contained withinthe parenchymal basement membranes, no disease occurs (Fig. 3).Interferons, both IFN-a/b as well as IFN-g, help to retain cells inthe cuffs by stimulating adhesion molecules [58,59] and byswinging the balance to proteinase inhibition [43,60]. In mouseEAE models, IFN-g has indeed been demonstrated to be aprotective factor [61] and it is our opinion that IFN-g also needsto be reconsidered for therapy of MS patients (vide infra). On the

TNF-αIL- 1βIL-17

ChemokinesMMPs

Cell pen etration through parenc hymal basement membran e

Disease

m recent literature [38,57,74] and show at the left side a vascular cuff in which the

e astrocyte endfeet. These leukocytes have transmigrated through the endothelial

re anti-inflammatory cytokines and have protective effects by various mechanisms,

s, no disease occurs in animal models. The right panel demonstrates the disease state

matrix metalloproteinases (MMPs) leading to destruction of basement membranes

G. Opdenakker, J. Van Damme / Cytokine & Growth Factor Reviews 22 (2011) 359–365 363

other hand, in various mouse models IL-17 has turned out to bedetrimental and thus may become a good target for therapeuticmonoclonal antibodies, because this cytokine – alongside TNF-a –is upstream of the chemokine system (Fig. 1) [22].

Many pro-inflammatory cytokines, including IL-1b [62], TNFfamily members [63] and IL-17 [22] induce chemokine sets, thatrecruit leukocytes into the vascular cuffs of EAE and MS lesions.Therefore, neutralization of these chemokines with monoclonalantibodies may constitute a new therapeutic approach, providedthat specific dominant molecules are linked to the pathogenesis ofMS (rather than the whole chemokine families). Recently, eleganttricks have been developed to generate swiftly neutralizing(monoclonal) antibodies against cytokines [64].

In view of the multitude of chemokines and the redundancies intheir biological functions [65], it might be difficult to overcome theiroverlapping functions with selective agents, including antibodiesand specific chemokine receptor blockers. In addition, a generalchemokine blockage might seriously impair host defense. In abiological context, chemokines also interact with glycosaminogly-cans to build chemotactic gradients. The insight into thisphenomenon was used experimentally and theoretically to try todefine new anti-inflammatory agents [66]. By the study of theantiviral polymer COAM, previously thought to induce interferons,we discovered that COAM acts by inducing and binding chemokines.As such, COAM acts as a glycosaminoglycan mimetic [67]. By ectopicinjection (e.g. in the peritoneal cavity) COAM can diminish thenumber of inflammatory cells in the CNS (N. Berghmans, PhD Thesis,2008, University of Leuven). This view is presently elaboratedbecause COAM would constitute a drug with antiviral activity, thatcan reduce the amount of vascular cuffing. In other words, the newprinciple of deviating leukocytes by tricking the endogenouschemokine system deserves further study. Because COAM hasimmunostimulating and antiviral activities – rather than immuno-suppression observed with glucocorticosteroids, natalizumab andfingolimod – it deserves further analysis in EAE and MS and maybecome the first vascular cuff-modifying agent acting on theendogenous chemokine system.

6. Conclusions

From basic research efforts and preclinical findings in animalmodels of EAE it is clear that possibilities exist for better MStreatments. The designs of simple and good clinical studies bycaring neurologists, a courageous attitude by the pharmaceuticalindustry and political support for these new developments will beneeded, if we want to improve the quality of life of MS patients andreduce the societal costs needed for this chronic disease. Althoughno prevention of or cure for MS exists, patients may count onlimited drug discovery successes from the past and on continuedefforts to find new, more efficient and better tolerated ways to fightthis disease. First, treatment of MS with IFN-b has become anexample of the success of biotechnology. However, efficiency indrug development can be enhanced on the basis of combinationtherapies, whereas side-effects may be decreased by betterformulations and lower doses in combination therapies. Second,the formation of neutralizing antibodies against IFN-b may bereduced by the use of aggregate-free and proteinase-resistant N-glycosylated IFN-b. Third, application of the REGA model, forinstance by the use of proteinase inhibitors, may lead to noveltherapies in the forms of small molecular drugs and monoclonalantibodies [43]. A new wave within this concept is the identifica-tion of control molecules that influence the production of MMPsand their inhibitors, the tissue inhibitors of metalloproteinases(TIMPs). For instance, extracellular matrix metalloproteinaseinducer (EMMPRIN/CD147) induces MMPs and this constitutes anew therapeutic target [68]. In agreement with the concept to

interfere with the proteinase/inhibitor balances in MS, statins,which have been shown to reduce the expression level of MMP-9,are studied [69,70]. A fourth level in innovation is by controllingchemokine activity. In this case, the development of a peripheralchemokine sink or neutralizing monoclonal antibodies againstchemokines or chemokine receptor blockers may bring consider-able change in the therapeutic landscape for MS. A fifth level tospecifically block target (glyco)proteins may be by RNA interfer-ence. A final point is about patient compliance. The recent approvalof fingolimod (a sphingosine-1 phosphate receptor inhibitor) [71]is an example, together with tetracyclines, that more efforts arebeing done towards oral therapy. In addition, the use of parenteraldrugs may be made more compliant with fewer injections, as in thecase with many humanized neutralizing monoclonal antibodiesthat inhibit the named disease-promoting molecules and cells.

Conflict of interest

GO and JVD are inventors of intellectual property, owned byLeuven Research and Development (LRD) for the University ofLeuven.

Acknowledgements

The present study was made possible by grants from the Fundfor Scientific Research of Flanders (FWO-Vlaanderen), the Con-certed Research Actions (GOA) and from the Belgian CharcotFoundation for research on MS. We dedicate this manuscript toProfessor Raymond A. Dwek at the occasion of his 70th birthday.

References

[1] Murray TJ. Multiple sclerosis, the history of a disease. New York: DemosMedical Publishing; 2005.

[2] Compston A, McDonald I, Noseworthy J, Lassmann H, Miller D, Smith K, WekerleH, et al. McAlpine’s multiple sclerosis, 4th ed., Churchill Livingstone; 2005.

[3] Noseworthy JH. Progress in determining the causes and treatment of multiplesclerosis. Nature 1999;399(Suppl.):A40–7.

[4] Giraudon P, Bernard A. Chronic viral infections of the central nervous system:aspects specific to multiple sclerosis. Rev Neurol (Paris) 2009;165:789–95.

[5] Tan CS, Koralnik IJ. Progressive multifocal leukoencephalopathy and otherdisorders caused by JC virus: clinical features and pathogenesis. Lancet Neurol2010;9:425–37.

[6] Holmen C, Piehl F, Hillert J, Fogdell-Hahn A, Lundkvist M, Karlberg E, et al. ASwedish national post-marketing surveillance study of natalizumab treatmentin multiple sclerosis. Mult Scler 2011;17:708–19.

[7] Billiau A, Edy VG, Heremans H, Van Damme J, Desmyter J, Georgiades JA, et al.Human interferon: mass production in a newly established cell line, MG-63.Antimicrob Agents Chemother 1977;12:11–5.

[8] Weinstock-Guttman B, Ramanathan M, Zivadinov R. Interferon-beta treatmentfor relapsing multiple sclerosis. Expert Opin Biol Ther 2008;8:1435–47.

[9] Ebers GC, Sadovnick AD. The geographic distribution of multiple sclerosis: areview. Neuroepidemiology 1993;12:1–5.

[10] Goris A, Epplen C, Fiten P, Andersson M, Murru R, Sciacca FL, et al. Analysis ofan IFN-gamma gene (IFNG) polymorphism in multiple sclerosis in Europe:effect of population structure on association with disease. J Interferon Cyto-kine Res 1999;19:1037–46.

[11] Ebers GC, Bulman DE, Sadovnick AD, Paty DW, Warren S, Hader W, et al. Apopulation-based study of multiple sclerosis in twins. N Engl J Med1986;315:1638–42.

[12] Zuvich RL, McCauley JL, Pericak-Vance MA, Haines JL. Genetics and pathogen-esis of multiple sclerosis. Semin Immunol 2009;21:328–33.

[13] Oksenberg JR, Baranzini SE. Multiple sclerosis genetics—is the glass half full, orhalf empty? Nat Rev Neurol 2010;6:429–37.

[14] Steinman L, Zamvil S. Transcriptional analysis of targets in multiple sclerosis.Nat Rev Immunol 2003;3:483–92.

[15] Marino K, Bones J, Kattla JJ, Rudd PM. A systematic approach to proteinglycosylation analysis: a path through the maze. Nat Chem Biol 2010;6:713–23.

[16] Opdenakker G, Van Damme J. Cytokine-regulated proteases in autoimmunediseases. Immunol Today 1994;15:103–7.

[17] Gijbels K, Masure S, Carton H, Opdenakker G. Gelatinase in the cerebrospinalfluid of patients with multiple sclerosis and other inflammatory neurologicaldisorders. J Neuroimmunol 1992;41:29–34.

[18] Paemen L, Olsson T, Soderstrom M, Van Damme J, Opdenakker G. Evaluation ofgelatinases and IL-6 in the cerebrospinal fluid of patients with optic neuritis,

G. Opdenakker, J. Van Damme / Cytokine & Growth Factor Reviews 22 (2011) 359–365364

multiple sclerosis and other inflammatory neurological diseases. Eur J Neurol1994;1:55–63.

[19] Proost P, Van Damme J, Opdenakker G. Leukocyte gelatinase B cleavagereleases encephalitogens from human myelin basic protein. Biochem BiophysRes Commun 1993;192:1175–81.

[20] Gijbels K, Proost P, Masure S, Carton H, Billiau A, Opdenakker G, et al. B ispresent in the cerebrospinal fluid during experimental autoimmuneencephalomyelitis and cleaves myelin basic protein. J Neurosci Res1993;36:432–40.

[21] Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two typesof murine helper T cell clone. I. Definition according to profiles of lympho-kine activities and secreted proteins. J Immunol 1986;136:2348–57.

[22] Kebir H, Kreymborg K, Ifergan I, Dodelet-Devillers A, Cayrol R, Bernard M, et al.Human TH17 lymphocytes promote blood–brain barrier disruption and cen-tral nervous system inflammation. Nat Med 2007;13:1173–5.

[23] Venken K, Hellings N, Liblau R, Stinissen P. Disturbed regulatory T cellhomeostasis in multiple sclerosis. Trends Mol Med 2010;16:58–68.

[24] Gandhi R, Laroni A, Weiner HL. Role of the innate immune system in thepathogenesis of multiple sclerosis. J Neuroimmunol 2010;221:7–14.

[25] van Noort JM, van Sechel AC, Bajramovic JJ, el Ouagmiri M, Polman CH,Lassmann H, et al. The small heat-shock protein alpha B-crystallin as candidateautoantigen in multiple sclerosis. Nature 1995;375:798–801.

[26] Starckx S, Van den Steen PE, Verbeek R, van Noort JM, Opdenakker G. A novelrationale for inhibition of gelatinase B in multiple sclerosis: MMP-9 destroysalpha B-crystallin and generates a promiscuous T cell epitope. J Neuroimmu-nol 2003;141:47–57.

[27] Ousman SS, Tomooka BH, van Noort JM, Wawrousek EF, O’Connor KC, HaflerDA, et al. Protective and therapeutic role for alphaB-crystallin in autoimmunedemyelination. Nature 2007;448:474–9.

[28] Jacobs L, O’Malley J, Freeman A, Ekes R. Intrathecal interferon reduces exacer-bations of multiple sclerosis. Science 1981;214:1026–8.

[29] Jacobs L, Salazar AM, Herndon R, Reese PA, Freeman A, Josefowicz R, et al.Multicentre double-blind study of effect of intrathecally administered naturalhuman fibroblast interferon on exacerbations of multiple sclerosis. Lancet1986;2:1411–3.

[30] Nelissen I, Martens E, Van den Steen PE, Proost P, Ronsse I, Opdenakker G.Gelatinase B/matrix metalloproteinase-9 cleaves interferon-beta and is atarget for immunotherapy. Brain 2003;126:1371–81.

[31] Bartholome EJ, Van Aelst I, Koyen E, Kiss R, Willems F, Goldman M, et al.Human monocyte-derived dendritic cells produce bioactive gelatinase B:inhibition by IFN-beta. J Interferon Cytokine Res 2001;21:495–501.

[32] Rudd PM, Joao HC, Coghill E, Fiten P, Saunders MR, Opdenakker G, et al.Glycoforms modify the dynamic stability and functional activity of an enzyme.Biochemistry 1994;33:17–22.

[33] Rudd PM, Dwek RA. Glycosylation: heterogeneity and the 3D structure ofproteins. Crit Rev Biochem Mol Biol 1997;32:1–100.

[34] Scagnolari C, Bellomi F, Turriziani O, Bagnato F, Tomassini V, Lavolpe V, et al.Neutralizing and binding antibodies to IFN-beta: relative frequency in relaps-ing-remitting multiple sclerosis patients treated with different IFN-beta pre-parations. J Interferon Cytokine Res 2002;22:207–13.

[35] van Beers MM, Jiskoot W, Schellekens H. On the role of aggregates in theimmunogenicity of recombinant human interferon beta in patients withmultiple sclerosis. J Interferon Cytokine Res 2010;30:767–75.

[36] Dubois B, Masure S, Hurtenbach U, Paemen L, Heremans H, van den Oord J,et al. Resistance of young gelatinase B-deficient mice to experimental auto-immune encephalomyelitis and necrotizing tail lesions. J Clin Invest1999;104:1507–15.

[37] Opdenakker G, Nelissen I, Van Damme J. Functional roles and therapeutictargeting of gelatinase B and chemokines in multiple sclerosis. Lancet Neurol2003;2:747–56.

[38] Agrawal S, Anderson P, Durbeej M, van Rooijen N, Ivars F, Opdenakker G, et al.Dystroglycan is selectively cleaved at the parenchymal basement membraneat sites of leukocyte extravasation in experimental autoimmune encephalo-myelitis. J Exp Med 2006;203:1007–19.

[39] Cuzner ML, Opdenakker G. Plasminogen activators and matrix metallopro-teases, mediators of extracellular proteolysis in inflammatory demyelinationof the central nervous system. J Neuroimmunol 1999;94:1–14.

[40] Van den Steen PE, Dubois B, Nelissen I, Rudd PM, Dwek RA, Opdenakker G.Biochemistry and molecular biology of gelatinase B or matrix metallopro-teinase-9 (MMP-9). Crit Rev Biochem Mol Biol 2002;37(December(6)):375–536.

[41] Fridman R, Toth M, Pena D, Mobashery S. Activation of progelatinase B (MMP-9) by gelatinase A (MMP-2). Cancer Res 1995;55:2548–55.

[42] Van den Steen PE, Proost P, Grillet B, Brand DD, Kang AH, Van Damme J, et al.Cleavage of denatured natural collagen type II by neutrophil gelatinase Breveals enzyme specificity, post-translational modifications in the substrate,and the formation of remnant epitopes in rheumatoid arthritis. FASEB J2002;16:379–89.

[43] Hu J, Van den Steen PE, Sang QX, Opdenakker G. Matrix metalloproteinaseinhibitors as therapy for inflammatory and vascular diseases. Nat Rev DrugDiscov 2007;6:480–98.

[44] Paemen L, Martens E, Norga K, Masure S, Roets E, Hoogmartens J, et al. Thegelatinase inhibitory activity of tetracyclines and chemically modifiedtetracycline analogues as measured by a novel microtiter assay for inhibi-tors. Biochem Pharmacol 1996;52:105–11.

[45] Brundula V, Rewcastle NB, Metz LM, Bernard CC, Yong VW. Targeting leuko-cyte MMPs and transmigration: minocycline as a potential therapy for multi-ple sclerosis. Brain 2002;125:1297–308.

[46] Giuliani F, Metz LM, Wilson T, Fan Y, Bar-Or A, Yong VW. Additive effect of thecombination of glatiramer acetate and minocycline in a model of MS. JNeuroimmunol 2005;158:213–21.

[47] Giuliani F, Fu SA, Metz LM, Yong VW. Effective combination of minocycline andinterferon-beta in a model of multiple sclerosis. J Neuroimmunol 2005;165:83–91.

[48] Luccarini I, Ballerini C, Biagioli T, Biamonte F, Bellucci A, Rosi MC, et al.Combined treatment with atorvastatin and minocycline suppresses severityof EAE. Exp Neurol 2008;211:214–26.

[49] Minagar A, Alexander JS, Schwendimann RN, Kelley RE, Gonzalez-Toledo E,Jimenez JJ, et al. Combination therapy with interferon beta-1a and doxy-cycline in multiple sclerosis: an open-label trial. Arch Neurol 2008;65:199–204.

[50] Yong VW, Wells J, Giuliani F, Casha S, Power C, Metz LM. The promise ofminocycline in neurology. Lancet Neurol 2004;3:744–51.

[51] Paemen L, Martens E, Masure S, Opdenakker G. Monoclonal antibodies specificfor natural human neutrophil gelatinase B used for affinity purification,quantitation by two-site ELISA and inhibition of enzymatic activity. Eur JBiochem 1995;234:759–65.

[52] Gerard C, Rollins BJ. Chemokines and disease. Nat Immunol 2001;2:108–15.[53] Ransohoff RM, Glabinski A, Tani M. Chemokines in immune-mediated in-

flammation of the central nervous system. Cytokine Growth Factor Rev1996;7:35–46.

[54] Ubogu EE, Cossoy MB, Ransohoff RM. The expression and function ofchemokines involved in CNS inflammation. Trends Pharmacol Sci2006;27:48–55.

[55] Glabinski AR, Ransohoff RM. Targeting the chemokine system for multiplesclerosis treatment. Curr Opin Investig Drugs 2001;2:1712–9.

[56] Holman DW, Klein RS, Ransohoff RM. The blood–brain barrier, chemokinesand multiple sclerosis. Biochim Biophys Acta 2011;1812:220–30.

[57] Sorokin L. The impact of the extracellular matrix on inflammation. Nat RevImmunol 2010;10:712–23.

[58] Milner R. Understanding the molecular basis of cell migration; implicationsfor clinical therapy in multiple sclerosis. Clin Sci (Lond) 1997;92:113–22.

[59] Avolio C, Giuliani F, Liuzzi GM, Ruggieri M, Paolicelli D, Riccio P, et al. Adhesionmolecules and matrix metalloproteinases in multiple sclerosis: effects in-duced by interferon-beta. Brain Res Bull 2003;61:357–64.

[60] Nelissen I, Gveric D, van Noort JM, Cuzner ML, Opdenakker G. PECAM-1 andgelatinase B coexist in vascular cuffs of multiple sclerosis lesions. NeuropatholAppl Neurobiol 2006;32:15–22.

[61] Billiau A, Heremans H, Vandekerckhove F, Dijkmans R, Sobis H, Meulepas E,et al. Enhancement of experimental allergic encephalomyelitis in mice byantibodies against IFN-gamma. J Immunol 1988;140:1506–10.

[62] Dinarello CA. Immunological and inflammatory functions of the interleukin-1family. Annu Rev Immunol 2009;27:519–50.

[63] Ware CF. Network communications: lymphotoxins, LIGHT, and TNF. Annu RevImmunol 2005;23:787–819.

[64] Uyttenhove C, Van Snick J. Development of an anti-IL-17A auto-vaccine thatprevents experimental auto-immune encephalomyelitis. Eur J Immunol2006;36:2868–74.

[65] Mantovani A. The chemokine system: redundancy for robust outputs. Immu-nol Today 1999;20:254–7.

[66] Johnson Z, Proudfoot AE, Handel TM. Interaction of chemokines and glycosa-minoglycans: a new twist in the regulation of chemokine function withopportunities for therapeutic intervention. Cytokine Growth Factor Rev2005;16:625–36.

[67] Li S, Starckx S, Martens E, Dillen C, Lamerant-Fayel N, Berghmans N, et al.Myeloid cells are tunable by a polyanionic polysaccharide derivative and co-determine host rescue from lethal virus infection. J Leukoc Biol 2010;88:1017–29.

[68] Agrawal SM, Silva C, Tourtellotte WW, Yong VW. EMMPRIN: a novel regulatorof leukocyte transmigration into the CNS in multiple sclerosis and experimen-tal autoimmune encephalomyelitis. J Neurosci 2011;31:669–77.

[69] Stuve O, Prod’homme T, Slavin A, Youssef S, Dunn S, Steinman L, et al. Statinsand their potential targets in multiple sclerosis therapy. Expert Opin TherTargets 2003;7:613–22.

[70] Antel JP, Miron VE. Central nervous system effects of current and emergingmultiple sclerosis-directed immuno-therapies. Clin Neurol Neurosurg2008;110:951–7.

[71] Brinkmann V, Billich A, Baumruker T, Heining P, Schmouder R, Francis G, et al.Fingolimod (FTY720): discovery and development of an oral drug to treatmultiple sclerosis. Nat Rev Drug Discov 2010;9:883–97.

[72] Thoua NM, van Noort JM, Baker D, Bose A, van Sechel AC, van Stipdonk MJ,et al. Encephalitogenic and immunogenic potential of the stress proteinalphaB-crystallin in Biozzi ABH (H-2A(g7)) mice. J Neuroimmunol2000;104:47–57.

[73] Chou YK, Burrows GG, LaTocha D, Wang C, Subramanian S, Bourdette DN, et al.CD4 T-cell epitopes of human alpha B-crystallin. J Neurosci Res 2004;75:516–23.

[74] Wu C, Ivars F, Anderson P, Hallmann R, Vestweber D, Nilsson P, et al. Endo-thelial basement membrane laminin alpha5 selectively inhibits T lymphocyteextravasation into the brain. Nat Med 2009;15:519–27.

G. Opdenakker, J. Van Damme / Cytokine & Growth Factor Reviews 22 (2011) 359–365 365

Ghislain Opdenakker, MD, PhD, is a professor of im-munology in the Faculty of Medicine and leads theLaboratory of Immunobiology of the University of Leu-ven. His research is focused on the interplay betweencytokines, chemokines and matrix metalloproteinasesin inflammation and cancer. Together with Professor JoVan Damme and in the spirit of Piet De Somer, founderof the Rega Institute, he built a School of Immunologywith the aim of translating basic research to benefits forpatients. He is a member-elect of the Royal Academy ofMedicine (Belgium) and of the Royal College of Physi-cians (UK).

Jo Van Damme, Bio-Ir., PhD, is a professor of immunol-ogy in the Faculty of Medicine and head of the Labora-tory of Molecular Immunology at the Rega Institute inLeuven. He started his career in cytokine research byproducing the first batches of natural IFN-b for clinicaltesting. He also first purified and thus identified variousnatural interleukins and chemokines (IL-1, IL-6, IL-8). Inthe postgenomic era, he has developed a major researchline on posttranslational modifications of chemokinesand cytokines.