www.elsevier.com/locate/jneuroim

Journal of Neuroimmunology 152 (2004) 1–4

Conference report

The blood–brain barrier as target of multiple sclerosis

research and therapy

Report of an international scientific meeting,

Wurzburg, January 23/24, 2004

Ralf Golda,b,*, Richard M. Ransohoff c, Alfred Sandrockd,e, Peter Rieckmann a,*

aClinical Research Group for Multiple Sclerosis and Neuroimmunology, Department of Neurology, University of Wurzburg,

Josef-Schneider-Strasse 11, D-97080 Wurzburg, Germanyb Institute for Multiple Sclerosis Research, Medical Faculty and Gemeinnutzige Hertie-Stiftung, University of Gottingen, Germany

cDepartment of Neurosciences, The Lerner Research Institute and The Mellen Center for Multiple Sclerosis Treatment and Research,

The Cleveland Clinic Foundation, USAdBiogen Idec, Incorporation, Cambridge, MA, USA

eDepartment of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

Received 17 March 2004; accepted 17 March 2004

Multiple sclerosis (MS) is currently considered as a

heterogenous disease entity, where genetic and environmen-

tal factors initiate and modulate a chronic inflammatory

autoimmune reaction in the nervous system (reviewed in

(Hemmer et al., 2002)) Heterogeneity of pathogenic mech-

anisms is also suggested by studies on biopsy and autopsy

tissue from MS patients (Lassmann et al., 2001). Despite

considerable progress in unravelling the pathogenesis, there

is still no single therapy available with the potential to cure

this devastating disease. For relapsing–remitting MS, the

most frequent disease course, several promising therapies

have been developed and successfully studied throughout

the last decade. Most of these drugs target the blood–brain

barrier (BBB) as the prominent anatomical structure and

gatekeeper to the central nervous system (CNS). Thus, in his

welcome note Klaus Toyka, Wurzburg defined the goal of

this workshop that scientists from different backgrounds

should combine recent developments in basic research and

MS therapy focussing on the BBB.

Not too long ago, the CNS has been characterized as an

immunologically ‘‘privileged’’ site, but nowadays it is rather

recognized as an immunologically ‘‘specialized’’ site. Im-

doi:10.1016/j.jneuroim.2004.03.014

* Corresponding authors. Ralf Gold is to be contacted at Institute for

Multiple Sclerosis Research, Medical Faculty and Gemeinnutzige Hertie-

Stiftung, University of Gottingen, Waldweg 33, D-37073 Gottingen,

Germany. Tel.: +49-551-39-13331; fax: +49-551-39-13348. Peter Rieck-

mann, Tel.: +49-931-201-23766; fax: +49-931-201-23488.

E-mail addresses: r.gold@med.uni-goettingen.de (R. Gold),

rieckmann@mail.uni-wuerzburg.de (P. Rieckmann).

mune reactions do occur in the CNS, but take on a

distinctive character since leukocyte entry is restricted by

the BBB. Richard Ransohoff, Cleveland summarized recent

knowledge on the trafficking signals that guide migration of

leukocytes into the CNS (Ransohoff et al., 2003; Moser et

al., 2004). By using multicolor flow cytometry, his group

has characterized the surface phenotype of CSF cells and

defined the expression of selected adhesion molecules on

vasculature in the choroid plexus, the subarachnoid space

surrounding the cerebral cortex, and the cerebral parenchy-

ma. Cells in the cerebrospinal fluid consisted predominantly

of CD4+ CD45RA� CD27+ CD69+ activated central

memory T cells expressing high levels of CCR7 and L-

selectin. No naıve T cells were detected, and only 10% of T

cells exhibited an effector memory phenotype. P- and E-

selectin immunoreactivity was detected in large venules in

the choroid plexus and subarachnoid space, but not in

parenchymal microvessels. CD4+ T cells in the CSF

expressed high levels of P-selectin glycoprotein ligand 1,

and a subpopulation of circulating CD4+ T cells displayed

P-selectin binding activity. Based on these findings, he

proposed that activated memory T cells enter CSF directly

from the systemic circulation and monitor the subarachnoid

space, retaining their capacity to either initiate local immune

reactions or return to secondary lymphoid organs.

Peter Rieckmann, Wurzburg, focussed on human endo-

thelial cell culture as a model to address therapeutic

approaches. Using primary cultures from brain tissue with

several characteristics of the blood–brain barrier, his group

studied the modulation of adhesion molecule expression by

R. Gold et al. / Journal of Neuroimmunology 152 (2004) 1–42

proinflammatory cytokines and IFN-h (Kallmann et al.,

2000). In view of the increased expression of VCAM-1 in

active and chronic MS lesions (Cannella and Raine, 1995), a

time- and dose-dependent expression of VCAM-1 and re-

lease of soluble VCAM-1 was detected in response to TNF-

a. Interestingly, this release was further increased by co-

treatment with IFN-h and may represent another mechanism

by which IFN-h treatment exerts protective effects in MS.

He further addressed the modulatory effect of oncostatin M,

a member of the IL-6 cytokine family, on endothelial cells.

This cytokine blocks TNF-a-induced IL-8 expression by

cerebral endothelial cells. This could have an important

impact on the composition of the cellular infiltrate in

inflammatory diseases of the CNS. Using the cDNA micro-

array technique further, endothelial cell specific genes were

identified, which may serve as targets for therapeutic inter-

ventions at the BBB (Kallmann et al., 2002).

The permanent neurological impairment typical chronic

inflammatory demyelinating disorders of the CNS, such as

multiple sclerosis, is due to axonal loss resulting from

recurrent episodes of immune-mediated demyelination.

Gianvito Martino, Milan, used neurospheres as vehicles to

deliver regenerative therapy (Pluchino et al., 2003). Surpris-

ingly, even after i.v. injection, significant numbers of donor

cells entered into demyelinating areas of the central nervous

system and differentiated into mature brain cells. Within

these areas, oligodendrocyte progenitors markedly in-

creased, with many of them being of donor origin and

actively remyelinating axons. Furthermore, a significant

reduction of astrogliosis and a marked decrease in the extent

of demyelination and axonal loss were observed in trans-

planted animals. The functional impairment caused by EAE

was almost abolished in transplanted mice, both clinically

and neurophysiologically. Thus, adult neural precursor cells

promote multifocal remyelination and functional recovery

after intravenous or intrathecal injection in a chronic model

of multiple sclerosis. Interestingly, these neurospheres ex-

press high levels of VLA-4 and may enter the CNS via

interaction with VCAM on the surface of endothelial cells.

Mathias Maurer, Wurzburg, introduced the field of mo-

lecular imaging again targeting the BBB in EAE. Molecular

imaging requires the identification and spatial localization

of a specific marker and its quantitative analysis at high

resolution. In cooperation with Schering, Berlin, single air-

filled microparticles were coated with anti-ICAM-1 anti-

bodies and injected i.v. at different time points of rat EAE.

Based on the stimulated acoustic emission of these micro-

particles, an ultrasound-derived imaging method with spa-

tial resolution in the lower micrometer range was developed.

This platform technology allowed for high sensitivity,

specificity and resolution at the blood–brain barrier during

acute EAE in vivo in anesthetized rats. It also is sensitive to

treatment with corticosteroids (manuscript submitted for

publication).

Another approach to enable in vivo tracking of hema-

togenous macrophages was presented by Bendszus and Stoll

(2003) (Wurzburg). The spatiotemporal course of macro-

phage infiltration after acute peripheral nerve injury in

living rats was visualized by using superparamagnetic iron

oxide (SPIO) particles and magnetic resonance imaging. A

signal loss on MR images indicated iron accumulation. This

was observed in degenerating sciatic nerves between days 1

and 8 after a crush lesion, ceased thereafter, and corre-

sponded to the transient presence of iron-labeled ED1-

positive macrophages in tissue sections. Results were com-

pared with findings in stroke models and EAE. The authors

propose that SPIO-enhanced MRI provides a new tool to

selectively visualize active periods of macrophage transmi-

gration into the nervous system, thus enabling dynamic

views on a fundamental process in a multitude of nerve

disorders. Interestingly, macrophage imaging was not asso-

ciated with gadolinium enhancement, an indicator for

blood–brain barrier disturbance.

Naoto Kawakami, Martinsried, extended the molecular

imaging to ex vivo two-photon fluorescence microscopy. In

the extension of his previous elegant studies on the activa-

tion status of neuroantigen-specific T cells in the target

organ (Kawakami et al., 2004) he employed this technique

to study cellular movement in 300-Am spinal cord slices

from acute Lewis rat EAE. The migratory activity of

antigen-specific cells recognizing ovalbumin as model anti-

gen was much higher and random, as compared to enceph-

alitogenic MBP-specific T cells, a subset of which exhibited

a migration-arrest pattern, suggesting interaction with their

specific myelin antigen. T cells specific for S-100, a less

encephalitogenic autoantigen, exhibited an intermediate

migration behaviour. These findings may provide the first

direct visualization of antigen recognition and T-cell acti-

vation in the inflamed CNS.

Britta Engelhardt, Munster and Bern, reported a variety of

studies using intravital microscopy, EAE, and in vitro trans-

migration to analyze the potential of specific adhesion

molecules as therapeutic targets. Her group had previously

developed a novel spinal cord window preparation to directly

visualize CNS white matter microcirculation by intravital

fluorescence videomicroscopy (Vajkoczy et al., 2001). From

these experiments, she provided in vivo evidence that en-

cephalitogenic T-cell blasts interact with the spinal cord

white matter microvasculature without rolling and that a4-

integrin mediates the G protein-independent capture and

subsequently the G protein-dependent adhesion strengthen-

ing of T-cell blasts to microvascular VCAM-1. Next she

presented a novel approach of a ‘grey matter window’ to

observe cortical capillary fields during EAE. Interestingly,

she could not detect leukocyte capture events in the cortical

capillaries and also noted a much faster blood flow velocity

than in the spinal cord. This finding may contribute to a better

understanding of cortical lesions in chronic MS. Further

studies were presented which addressed the contribution of

adhesion molecules during EAE, modulation of integrin

conformation elicited by inside-out signalling following

engagement of chemokine receptors by chemokines, and

R. Gold et al. / Journal of Neuroimmunology 152 (2004) 1–4 3

integrin expression as examined by microarray studies. In

SJL mice, none of the selectin molecules was mandatory for

EAE induction. Two ‘lymphoid’ chemokines, CCL19 and

CCL21, were expressed in the inflamed murine BBB during

EAE. Furthermore, transendothelial migration in vitro was

dependent on ICAM-1/LFA-1 interaction (Laschinger et al.,

2002; Lyck et al., 2003).

Stephen Miller, Chicago, reported VLA4-directed treat-

ment strategies in the relapsing EAE model of SJL mice,

induced by immunization with PLP peptide spanning amino

acids 139–151 (Theien et al., 2001). Preclinical adminis-

tration of anti-VLA-4 either to naive recipients of primed

encephalitogenic T cells or to mice during the induction

phase of disease inhibited the onset and severity of clinical

disease. Surprisingly, Ab treatment either at the peak of

acute disease or during remission exacerbated disease

relapses and increased the accumulation of CD4+ T cells

in the CNS. Most significantly, anti-VLA-4 treatment either

before or during ongoing EAE enhanced Th1 responses to

both the priming peptide and endogenous myelin epitopes

released secondary to acute tissue damage. To further

investigate the potential costimulatory activity of intact

anti-VLA-4, the ability of BIO 5192, a small-molecule

VLA-4 antagonist (see below), was examined in relapsing

EAE (Theien et al., 2003). BIO 5192 administered 1 week

after peptide priming again delayed the clinical disease

onset but led to severe disease exacerbation upon treatment

removal. BIO 5192 treatment initiated during disease re-

mission moderately enhanced clinical disease while mice

were on treatment and also resulted in post-treatment

exacerbation. Enhanced disease was caused by the release

of encephalitogenic cells from the periphery and the rapid

accumulation of T cells in the CNS. Collectively, these

results further demonstrate the complexity of VLA-4/

VCAM interactions, particularly in a relapsing–remitting

autoimmune disease.

Ralf Gold, Wurzburg and Gottingen, focussed on thera-

peutic studies with monoclonal antibodies towards VLA-4

and VCAM-1 in experimental neuritis (EAN) (Enders et al.,

1998). EAN serves as model disease for human chronic

polyneuritis (CIDP) and acute polyradiculoneuropathy, the

Guillain-Barre syndrome. Disease was induced by intrave-

nous adoptive transfer of P2 specific T cells (AT-EAN), or

by immunization with bovine myelin (active EAN). VCAM-

1 in sciatic nerve was upregulated at early stages of EAN

(days 3–5 after T-cell transfer), while no expression was

noted in healthy controls. In both EAN models, blockade of

VLA-4 markedly attenuated disease severity, diminished T-

cell infiltration in sciatic nerve and demyelination. Blockade

of VCAM-1 was less effective. Thus, VLA-4 blockade may

also gain therapeutic importance in inflammatory neuropa-

thies. In a next step, the early effects of blockade of

signalling via VLA-4 and VCAM-1 on T-cell infiltration

and apoptosis were characterized (Leussink et al., 2002).

Anti-VCAM-1 led to a rapid, significant increase of apo-

ptotic T cells in the sciatic nerve with a maximum after 6 h,

preceding the significant decrease of T-cell infiltration seen

after 18 h. This was accompanied by a significant reduction

in mRNA levels for IFN-g and inducible nitric oxide

synthase. The early increase of T-cell apoptosis following

disruption of VLA-4/VCAM-1 interaction may reflect a

novel signaling component of proapoptotic pathways.

Bernd Kieseier, Dusseldorf, gave an overview about

clinical studies with adhesion molecule inhibition in MS.

Antegrenk (Natalizumab) is a humanized function block-

ing monoclonal antibody against a4 integrins. A first study

dealt with the effect of a4 integrin blockade on MRI lesion

activity in MS (Tubridy et al., 1999). After two IV infusions

of anti-alpha4 integrin antibody or placebo 4 weeks apart,

patients were followed up for 24 weeks with serial MRI and

clinical assessment. Short-term treatment with monoclonal

antibody against alpha4 integrin results in a significant

reduction in the number of new active lesions on MRI

during the first 12 weeks. In a subsequent randomized,

double-blind trial, a total of 213 patients with relapsing–

remitting or secondary progressive MS with relapse activity

were randomly assigned to receive 3 mg of intravenous

natalizumab per kg of body weight, 6 mg per kg, or placebo

every 28 days for 6 months (Miller et al., 2003). In this

study, treatment with natalizumab led to a 90% reduction of

inflammatory brain lesions and 50% fewer relapses occurred

over a 6-month period in the treatment arm. Similar to the

previous study, natalizumab was again well tolerated. Thus,

the results of this trial are encouraging, but longer-term data

are still needed. The results of two ongoing phase III studies

with Antegren, the AFFIRM and SENTINEL study are

awaited with high expectations.

Alfred Sandrock, Cambridge, presented the biochemical,

pharmacological, and pharmacodynamic properties and ef-

ficacy of the small molecule inhibitor of VLA-4 interaction,

BIO5192 [2(S)-{[1-(3,5-dichloro-benzenesulfonyl)-pyrroli-

dine-2(S)-carbonyl]-amino}-4-[4-methyl-2(S)-(methyl-{2-

[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-amino)-pentanoyla-

mino]-butyric acid] in EAE (Leone et al., 2003). BIO5192

was compared with the monoclonal anti-a-integrin antibody

TA-2 which has been extensively studied in rats. Both

inhibitors induced leukocytosis, an effect that was used as

a pharmacodynamic marker of activity, and both were

efficacious in the EAE model. Treatment with TA-2 caused

a decrease in a4-integrin expression on the cell surface,

which resulted from internalization of a4 integrin/TA-2

complexes. In contrast, BIO5192 did not modulate cell

surface a4h1. The results with BIO5192 indicated that

a4h7 did not play a role in this model and that blockade

of a4h1/ligand interactions without down-modulation is

sufficient for efficacy in rat EAE. BIO5192 is highly

selective and binds with high affinity to a4h1 from four

of four species tested. Sandrock concluded that BIO5192, a

novel, potent, and selective inhibitor of a4h1 integrin, will

be a valuable reagent for assessing integrin molecule biol-

ogy and may provide a new therapeutic tool for treatment of

human inflammatory diseases.

R. Gold et al. / Journal of Neuroimmunology 152 (2004) 1–44

In the final discussion, the attendees from several Euro-

pean countries agreed that the specific immunological

features of the BBB indeed make this barrier a very

promising target for effective immunotherapies in types of

MS, where the inflammatory component is prominent. The

meeting in the nice atmosphere of an old franconian wine

city allowed for a stimulating scientific interaction between

immunologists and clinicians and resulted in new ideas for

future collaborative projects.

Acknowledgements

Supported by educational grants from Biogen Idec

Europe and Biogen Idec Germany.

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