Interferon-b Treatment of ExperimentalAutoimmune Encephalomyelitis Leads toRapid Nonapoptotic Termination of T CellInfiltration

Jens Schmidt,1 Steffen Sturzebecher,2 Klaus Viktor Toyka,1 and Ralf Gold1*1Department of Neurology, Clinical Research Group for Multiple Sclerosis and Neuroimmunology, Universityof Wurzburg, Wurzburg, Germany2Clinical Development Cardiovascular and Central Nervous System, SBU Therapeutics, Schering AG,Berlin, Germany

We investigated the possible mechanisms how interferon(IFN)-b may control T cell infiltration in the CNS in exper-imental autoimmune encephalomyelitis (EAE). Adoptivetransfer (AT) EAE was induced in groups of six femaleLewis rats. Animals were treated with 3 3 105 units ofrecombinant rat IFN-b s.c. once at 18 hr, or with 10mg/kg methylprednisolone (MP) i.v. twice at 18 and 6 hrprior to dissection, or with a combination of both. T cellapoptosis was detected by immunohistochemistry onparaffin sections of spinal cord, using morphological cri-teria and TUNEL staining. Double labeling of immunecells was done for tumor necrosis factor (TNF)-a andmetalloproteinase (MMP) 2. Disruption of the blood–brain barrier (BBB) was visualized by staining for albu-min. In severe EAE, an increase of T cell apoptosis wasseen after IFN-b alone (all data presented as mean 6 SD:24.5% 6 2.2%, P , 0.05, vs. 19.4% 6 3.1% in controls),and in combination with MP (29.4% 6 7.3%, P , 0.05 vs.controls). Only the combination therapy decreased T cellinfiltration (53.9 6 17.7 cells/mm2, P , 0.05, vs. 99.5 635.2 cells/mm2 in controls). In moderate EAE, the rate ofT cell apoptosis was slightly increased after IFN-b(21.2% 6 5.2% vs. 17.4% 6 5.0% in controls), whereasMP alone (25.5% 6 3.5%, P , 0.01 vs. controls) and thecombination therapy (22.4% 6 4.8%, P , 0.05 vs. con-trols) had a clear augmenting effect. IFN-b tended todecrease T cell infiltration (46.1 6 12.7 cells/mm2) com-pared to controls (59.2 6 18.5 cells/mm2). The rate ofTNF-a-expressing T cells was significantly decreased byIFN-b and in combination with MP. Also, TNF-a expres-sion in macrophages was significantly reduced by IFN-band by the combination therapy. The rate of MMP2-expressing macrophages was lower after IFN-b butclearly decreased only in combination with MP. BBBdisruption was ameliorated after IFN-b but significantlyonly in combination with MP. Our study indicates thatIFN-b affects the immunopathological process in EAE inseveral ways, but apoptosis appears as a minor compo-nent. In view of treatment of MS relapses, the synergisticeffects in this study corroborate the use of a combination

therapy with high-dose MP and IFN-b. J. Neurosci. Res.65:59–67, 2001. © 2001 Wiley-Liss, Inc.

Key words: interferons; T cell apoptosis; autoimmunedisorders; central nervous system; inflammation

INTRODUCTIONFollowing the first description in 1957 by Isaacs and

Lindenmann, the current classification of interferons (IFN)by gene analysis includes more than 20 cytokines. In 1981,Jacobs et al. reported the efficacy of intrathecal applicationof IFN-b in multiple sclerosis (MS) patients. Since then,several studies have revealed beneficial effects in MS. Inrelapsing-remitting disease, the relapse rate was reduced(IFNB Multiple Sclerosis Study Group, 1993) and therewere fewer active lesions on MRI scans (Paty and Li,1993). Also, in secondary-progressive MS, a therapeuticeffect could be demonstrated (European Study Group onInterferon b-1b in Secondary Progressive MS, 1998). Inexperimental models, IFN-b has been shown to inhibitprogression of relapsing-remitting experimental autoim-mune encephalomyelitis (EAE; Yu et al., 1996), and earlydiscontinuation of IFN-b led to exacerbation of activeEAE (van der Meide et al., 1998).

There are several putative mechanisms for IFN-baction: counteraction of IFN-g effects, decrease ofMHC-II molecules, modulation of the cytokine milieu,reduced expression of adhesion molecules, down-regulation of metalloproteinases (MMP; for review seeHohlfeld, 1997; Rieckmann, 1997; Yong et al., 1998a). InMS patients, IFN-b has been shown to down-modulate

Contract grant sponsor: State of Bavaria; Contract grant sponsor: WilhelmSander Stiftung.

*Correspondence to: Ralf Gold, MD, Neurologische Universitatsklinik,Josef-Schneider-Str. 11, 97080 Wurzburg, Germany.E-mail: r.gold@mail.uni-wuerzburg.de

Received 26 December 2000; Revised 1 February 2001; Accepted 27February 2001

Journal of Neuroscience Research 65:59–67 (2001)

© 2001 Wiley-Liss, Inc.

the spontaneous TNF-a and IFN-g gene expression(Gayo et al., 1999) and soluble CD 95 (APO-1/Fas) wasshown to be elevated in serum (Zipp et al., 1998). Re-cently IFN-b was found to increase TNF-a-induced re-lease of soluble vascular cell adhesion molecule 1 (Kall-mann et al., 2000).

It is still unclear whether the therapeutic benefit ofIFN-b is also based on the induction of apoptosis. In astudy with a fibroblast model for virus-induced apoptosisin vitro, IFN-b clearly enhanced apoptosis (Balachandranet al., 2000). The apoptosis rate of peripheral blood mono-nuclear cells (PBMCs) from MS patients after in vitrostimulation with anti-CD 3 antibodies was increased byIFN-b (Kaser et al., 1999). In another study in PBMCsfrom healthy donors, IFN-a increased cell-surface CD95L and led to enhanced apoptosis of sensitive (lymphoid)cells by natural killer (NK) cells (Kirou et al., 2000).However, these findings were not confirmed in a recentstudy with permanent T cell lines (Zipp et al., 2000).Other antiapoptotic effects of type 1 IFNs have also beendescribed (Akbar et al., 2000).

Here we characterized the immediate in situ effectsof IFN-b in adoptive transfer (AT)-EAE. Our interest wasfocussed on IFN-b-induced T cell apoptosis. At the sametime, we studied the modulation of the cytokine profile inthe lesion and the integrity of the blood–brain barrier(BBB). Furthermore, we investigated a potential synergyof IFN-b with methylprednisolone (MP), which is themainstay for therapy of MS relapses.

MATERIALS AND METHODS

Animals

For all experiments, female Lewis rats, aged 6–8 weeks ata body weight of 140–160 g, were obtained from Charles River(Sulzfeld, Germany). Animals were housed in plastic cages with-out grid floors and given commercial food pellets and water adlibitum. All experiments were carried out in accordance withBavarian state regulations for animal experimentation and wereapproved by the responsible authorities.

Cell Culture

All culture media and supplements were obtained fromGibco BRL (Eggenstein, Germany). Encephalitogenic T cellsfor in vivo experiments were generated and maintained aspreviously described in detail (Gold et al., 1995). Primed T cells(3 3 105/ml) were restimulated with guinea pig MBP(20 mg/ml) in RPMI 1640 supplemented with 1% normal ratserum, 100 U/ml penicillin, 100 mg/ml streptomycin, and2 mM glutamine, using freshly isolated and irradiated (3,000 rad)thymocytes (1.5 3 107/ml) as antigen-presenting cells. Seventy-two hr later, activated T cell blasts were separated from celldebris by centrifugation on Ficoll gradients (Nycomed AS, Oslo,Norway).

Induction of EAE and Clinical Score

AT-EAE was induced by tail vein injection of 8–12 3106 freshly activated, MBP-specific T cells. We used groups of

five rats for all experiments except for the pilot studies (three ratsper group) and for the Evan’s blue experiment (four rats pergroup). Animals were weighed and inspected daily, using asix-grade score for clinical staging: 0, healthy; 1, weight loss,limp tip of tail; 2, limp tail, mild paresis; 3, moderate paraparesis,ataxia; 4, tetraparesis; 5, moribund; 6, dead (Lassmann, 1983).Disease onset with first clinical signs was on day 2, reaching amaximal of grade 4–5 on day 5.

Treatment Protocol and Tissue Sampling for ApoptosisStudies

We used methylprednisone-21-hydrogensuccinate (MP;Urbason solubile; Aventis, Frankfurt, Germany), which wasapplied i.v. at 10 mg/kg body weight, essentially following aprotocol that had been established in earlier studies (Zettl et al.,1995; Schmidt et al., 2000). Recombinant rat IFN-b, producedin Chinese hamster ovary cells, from the Biomedical PrimateResearch Center (Rijswijk, The Netherlands; Ruuls et al.,1996), was injected s.c. at 3 3 105 units as described by van derMeide et al. (1998). At the peak of the disease on day 4 or 5, twopulses of MP were injected i.v. 18 and 6 hr prior to sacrifice orPBS for controls. IFN-b was given s.c. once 18 hr prior tosacrifice together with the first i.v. treatment. In pilot experi-ments, the time schedule for IFN-b was varied between 12 and48 hr to find the optimal interval for investigation of rapidIFN-b actions.

Animals were anesthesized with 200 mg/kg body weightpentobarbital (Narcoren; Rhone Merieux, Laupheim, Ger-many) and perfused through the left cardiac ventricle withHAES-steril 6% (Fresenius, Bad Homburg, Germany) for 5 min,followed by fixation with paraformaldehyde 4% in 0.1 M phos-phate buffer at pH 7.4. The spinal cord was removed, postfixedfor 1 hr in the same fixative, cut into 5 mm slices, dehydrated,and embedded in paraffin.

Immunohistochemistry: T Cells and Albumin Staining

Five micrometer cross-sections were deparaffinized withxylene and rehydrated. Hydroxylamine pretreatment (0.9% hy-droxylamine in 0.05 M PBS) for 30 min was required only forthe albumin staining. Nonspecific binding was reduced by in-cubation with 10% bovine serum albumin (BSA) for 30 min.Immunohistochemistry was performed using a monoclonalmouse antibody labeling a pan-T cell antigen (B115-1; HyCultBiotechnology b.v., via Sanbio, Beutelsbach, Germany), dilutedat 1:500 in 0.05 M Tris-buffered saline (0.15 M sodium; TBS)with 1% BSA for 1 hr at room temperature. To detect BBBdisruption, we used an antialbumin antibody (Nordic, Bochum,Germany) diluted at 1:200 in 0.05 M PBS (0.15 M sodium) asdescribed elsewhere (Morrissey et al., 1996). After blocking ofendogenous peroxidase activity with 3% H2O2 and 0.2 Msodium azide in methanol, primary antibodies were detectedusing the ABC system (Dako, Hamburg, Germany) with 3,39-diaminobenzidine tetrahydrochloride as substrate. All sectionswere counterstained with hematoxylin for 30 sec, dehydrated,and mounted in Vitro-clud (R. Langenbrinck, Emmendingen,Germany).

60 Schmidt et al.

Double Labeling Techniques for Detection ofApoptosis, MMP2-Positive Macrophages, and TNF-a-Positive T Cells and Macrophages

Apoptotic T cells were identified by in situ tailing (IST;synonym: TUNEL) as described previously (Gold et al.,1994) with nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP). Tissue sections were depar-affinized, rehydrated, and treated with chloroform for 1 sec.For labeling of apoptotic cells in the tissue, we used thedetection kit from Roche, following the instructions givenby the supplier. For T cell immunohistochemistry, we fol-lowed the protocol given above, except that alkalinephosphatase-based detection was used with New-fuchsin(Dako) or with Vector reu (Vector) as chromogenic substrate.Sections were mounted in aqueous mounting media Aquatex(Merck, Darmstadt, Germany).

Double staining for MMP2-positive macrophages and forTNF-a-positive T cells or macrophages was performed on 5mm paraffin sections, which were dewaxed and rehydratedfollowing the protocol described above. Protease pretreatmentwas performed only before MMP2 staining by incubation with0.4% Protease XXIV (Sigma, Steinheim, Germany) for 7 min at37°C, followed by washing with 100% ethanol three times.After blocking of nonspecific binding with 10% BSA, sectionswere incubated with a polyclonal rabbit anti-TNF-a antibody(Serotec, via Biozol, Munchen, Germany) or with a polyclonalrabbit anti-MMP2 antibody (Chemicon, Hofheim, Germany),both at a dilution of 1:100 in TBS with 1% BSA overnight at4°C. As secondary antibody, we used a biotinylated goat anti-rabbit IgG antibody (Vector, Wertheim, Germany) diluted 1:50in TBS with 1% BSA. Visualization was achieved by an alkalinephosphatase AB system (Dako) and Vector red (Vector) assubstrate. After blocking of the AB binding sites with an ABblocking kit (Vector), the next primary antibodies were appliedfor detection of T cells or macrophages. The T cell staining wasperformed as described above. Macrophages were detected bythe monoclonal antibody ED 1 (Serotec) for 1 hr at roomtemperature, diluted 1:500 in TBS with 1% BSA. Primary T cellor macrophage antibodies were detected by a biotinylated goatanti-mouse IgG antibody, which was preabsorbed with serafrom rat and rabbit for 15 min at 37°C and then diluted 1:200in TBS with 1% BSA. The peroxidase-based AB system (Dako)was used with DAB-nickel (Vector) as a chromogenic substrate.

Quantification of Cellular Infiltration and DoubleLabeling

Analysis of inflammatory infiltrates and of T cell apoptosiswas performed by an observer blinded to the treatment rating2 3 10 visual fields (2 3 1.6 mm2) of lumbar spinal cord at3250, two sections per animal. Apoptosis was assessed by mor-phological criteria (Wyllie, 1980) or labeling by IST. Analysis ofMMP2-positive macrophages or TNF-a-positive T cells andmacrophages was performed as described above, except that onesection with 10 visual fields (1.6 mm2) was counted per animal.

Quantification of the BBB Disruption byImmunohistochemistry

For quantification of the BBB disruption by immunohis-tochemistry, we performed computer-aided grey-scale measur-

ing (Scion Image software; Scioncorp) with an Axiovert 100microscope (Zeiss, Gottingen, Germany) and a CCD DXC950P camera (Sony, Koln, Germany). We measured the meanand the maximal signal intensity of the perivascular area of threeequally sized spinal cord vessels per animal. Data were thenexpressed as mean of the three vessels per animal. In addition,we used a previously established five-grade scale for semiquan-titative analysis by eye, with 0, no staining; 1/2, thin/thick ringof signal around the endothelial layer; 3, diffuse halo aroundvessels; 4, more or less homogeneous staining signal (Morrisseyet al., 1996).

Statistical Analysis

Statistical analysis of the data was performed with theStudent’s t-test (Excel, Microsoft, Germany), consideringP , 0.05 and P , 0.01 as significant.

RESULTS

IFN-b Causes Rapid Reduction of T CellInfiltration

Pilot studies with three rats per group were per-formed to establish the optimal timing for investigation ofearly effects of recombinant rat IFN-b. The density of Tcell infiltration in spinal cord was reduced within 12 hr(32.9 6 8.1 cells/mm2) compared to the control group(71.5 6 24.9 cells/mm2, Fig. 1A,B). Extending the periodto 24 hr p.i. (52.3 6 17.7 cells/mm2), or, in anotherexperiment, 48 hr p.i. (81.6 6 23.9 vs. 129.6 635.7 cells/mm2 in controls) did not result in a strongereffect on diminished T cell infiltration. In only one ex-periment was there a marked increase in the rate of T cellapoptosis (Fig. 1C) 24 hr after IFN-b s.c. compared to thecontrol group. For the following experiments, we there-fore selected an interval of 18 hr prior to perfusion.

In an experiment with severe EAE (all rats exhibitinga clinical score of 5 on day of perfusion) and a correspond-ingly strong cellular infiltration, we observed an increasedT cell apoptosis in all three treatment groups compared toPBS-treated controls. This was statistically significant forIFN-b given alone and for the combination therapy(P , 0.05). However, no significant result was achievedwith MP alone because of high variability and loss of oneanimal in this group. The T cell infiltration was decreasedin all treatment groups, but only with the combinationtherapy was this significant compared to PBS (P , 0.05,Fig. 2).

In two experiments with moderately severe EAE(clinical score of 3–4), four groups of rats received either10 mg/kg body weight MP, 3 3 105 units IFN-b, or acombination of both, and controls were injected withPBS. Both experiments revealed similar results, so datawere analyzed together. IFN-b alone led to a slightlyhigher rate of apoptosis compared to controls. MP mono-therapy showed a highly increased rate of T cell apoptosiscompared to PBS (P , 0.01). Also, in combination withIFN-b, the rate of apoptotic T cells significantly increased(P , 0.05 vs. control). In all three treatment groups, the Tcell infiltration was reduced compared to the control

IFN-b Effects In Situ in AT-EAE 61

Figure 1.

group, yet this did not reach statistical significance becauseof the high variability in the control group. In theseexperiments, we observed no synergy between IFN-b andMP in terms of induction of T cell apoptosis or reductionof cellular infiltration (Fig. 3).

IFN-b Treatment Decreases TNF-a-Positive TCells and Macrophages In Situ

For assessment of IFN-b effects on cytokine produc-tion in situ, we investigated the colocalization of T cellsand TNF-a expression or macrophages and TNF-a ex-pression by immunohistochemical double labeling (Fig.1E,F). Data represent two experiments mentioned above,which yielded similar results. The rate of TNF-a-positiveT cells was significantly reduced by IFN-b (P , 0.05) andby MP plus IFN-b (P , 0.05) compared to PBS. Noeffect could be demonstrated after monotherapy with MP(Fig. 4).

The rate of TNF-a-positive macrophages was re-duced by IFN-b (P , 0.05) and even moreso by thecombination therapy (P , 0.01) compared to controls.MP alone led to a slight decrease in the rate of TNF-a-positive macrophages, which was not statistically signifi-cant (Fig. 5). The combination of MP and IFN-b was notsuperior to either monotherapy regarding any of theseparameters.

IFN-b Reduces the Rate of MMP2-PositiveMacrophages In Situ

For investigation of IFN-b effects on MMP2 pro-duction by macrophages in situ, we performed immuno-histochemical double labeling for MMP2 and macro-phages (Fig. 1D). After monotherapy with MP or withIFN-b, the rate of MMP2-expressing macrophages wasreduced compared to the control group (P 0.09 for IFN-bvs. PBS). However, the combination therapy was superiorand led to a significant decrease of MMP2-expressingmacrophages compared to sham treatment (P , 0.01; Fig. 6).

Detection of BBB Disruption byImmunohistochemistry

To investigate the integrity of the BBB by immu-nohistochemistry, we performed staining for albumin onthe same experiments mentioned above. Grey-scale anal-ysis revealed a reduction of the mean and maximal perivas-cular signal intensity in all three treatment groups. How-

Š

Fig. 1. T cell infiltration, apoptosis, and immunohistochemical doublelabeling of immune cells and TNF-a or MMP2. Five micrometerparaffin sections of spinal cord from AT-EAE rats on day 5.A,B: Staining of T cells (DAB, brown) by the monoclonal antibodyB115-1 and hematoxylin counterstaining (blue). Treatment with PBS(A) or 3 3 105 units recombinant rat IFN-b (B). Solid arrows indicatenormal T cells, open arrow in A depicts nuclear fragmentation of anapoptotic T cell. Note the reduced cellular infiltration in B.C: TUNEL stain (NBT/BCIP, black) for detection of apoptosis, co-visualized with the antibody B115-1 for T cells (Vector red). Solidarrows denote apoptotic T cells, and the open arrow indicates a normal

T cell. D: Double labeling for MMP2 (Vector red) by a polyclonalantibody to MMP2 and macrophages (DAB-nickel, black) by themonoclonal antibody ED 1. MMP2-positive macrophages are indicatedby solid arrows and an MMP2-negative macrophage by an open arrow.E,F: Double labeling for TNF-a (Vector red) by a polyclonal antibodyto TNF-a, and T cells (E; DAB-nickel, black) by the monoclonalantibody B115-1 or macrophages (F; DAB-nickel, black) by the mono-clonal antibody ED 1. TNF-a-positive immune cells are depicted bysolid arrows and TNF-a-negative immune cells by open arrows. Scalebars 5 30 mm in B (applies to A,B); 20 mm for in F (applies to C–F).

Fig. 2. Treatment of severe EAE (clinical score of 5) with 3 3 105 unitsIFN-b s.c., 10 mg/kg methylprednisolone (MP) i.v., or with a com-bination of both (MP 1 IFN), or with PBS for controls. Immunohis-tochemical detection of T cells in spinal cord. A: Number of infiltratingT cells/mm2. B: Percentage of apoptotic T cells as rated by morpho-logical criteria and TUNEL staining. A: *P , 0.05 (MP 1 IFN vs.PBS). B: *P , 0.05 (IFN vs. PBS and MP 1 IFN vs. PBS). Eachsymbol represents one animal; n 5 5 per group; numbers and barsindicate mean and SD.

IFN-b Effects In Situ in AT-EAE 63

ever, only the combination therapy showed a significantreduction of the mean intensity compared to the controlgroup (P , 0.01). The maximal signal intensity was sig-nificantly reduced by MP (P , 0.05) and slightly reducedby IFN-b. The combination therapy of MP and IFN-bled to a less intense perivascular signal compared to themonotherapies with both methods of analysis (P , 0.01vs. PBS). Regarding the mean perivascular signal, thecombination therapy was superior compared to MP(P , 0.05; Fig. 7). The data indicate an additive effect ofMP and IFN-b on restitution of the integrity of the BBB.

DISCUSSIONThe aim of this study was to investigate the imme-

diate effects of IFN-b in situ in AT-EAE. An increased

rate of T cell apoptosis was typically seen after 10 mg/kgMP, which is in accordance with previous results (Schmidtet al., 2000). IFN-b given alone or in combination withMP did not lead to a consistently elevated rate of apopto-sis, although it caused a rapid reduction of inflammatory Tcells. Only in one experiment with a severe disease courseand strong cellular inflammation was the rate of apoptoticT cells increased in the groups that received IFN-b. Thus,IFN-b leads to rapid, nonapoptotic reduction of T cellinfiltration and does not synergize with MP in this respect.

Immunohistochemical analysis of proinflammatorycytokine expression and MMP2 with double labeling re-vealed a clear decrease in the rate of TNF-a-positive Tcells and macrophages after IFN-b, given alone or incombination with MP. The rate of MMP2-expressingmacrophages was diminished after MP or IFN-b givenalone, whereas the combination therapy was more effec-tive. To our knowledge this is the first demonstration ofthese effects of IFN-b in situ. Also, IFN-b and MPexhibited additive effects on the BBB disruption as assessedby albumin staining. Thus the combination of IFN-b andMP, which reflects daily clinical practice during relapses ofMS, has some potentiating and no antagonistic therapeuticeffects.

Especially over the last few years, IFN-b has shownbeneficial effects, not only for EAE (Yu et al., 1996),which serves as an animal model for MS (Gold et al.,2000), but also for different subgroups of MS patients(IFNB Multiple Sclerosis Study Group, 1993; EuropeanStudy Group on Interferon b-1b in Secondary Progressive

Fig. 3. Pooled data from two experiments with treatment of moder-ately severe EAE (clinical score of 3–4) with 3 3 105 units IFN-b s.c.,10 mg/kg methylprednisolone (MP) i.v., or with a combination of both(MP 1 IFN), or with PBS for controls. Immunohistochemical detec-tion of T cells in spinal cord section. A: Number of infiltrating Tcells/mm2. B: Percentage of apoptotic T cells as rated by morpholog-ical criteria and TUNEL staining. B: **P , 0.01 (MP vs. PBS),*P , 0.05 (MP 1 IFN vs. PBS). Each symbol represents one animal;n 5 5 per group in each of two experiments; numbers and bars indicatemean and SD.

Fig. 4. Pooled data from two experiments with treatment of moder-ately or severe EAE (clinical score of 3–4 or 5) with 3 3 105 unitsIFN-b s.c., 10 mg/kg methylprednisolone (MP) i.v., or with a com-bination of both (MP 1 IFN), or with PBS for controls. Immunohis-tochemical double labeling for TNF-a and T cell markers in spinalcord. Percentage of TNF-a-expressing T cells as visualized by colo-calization. *P , 0.05 (IFN vs. PBS and MP 1 IFN vs. PBS); n 5 5 pergroup in each of two experiments; numbers and bars represent meanand SD; squares indicate median.

64 Schmidt et al.

MS, 1998). At present, numerous mechanisms of action ofIFN-b are known (for review see Hohlfeld, 1997; Riec-kmann, 1997; Yong et al., 1998a). Some recent in vitroand ex vivo studies addressed the question of whether

IFN-b could also induce apoptosis. In a fibroblast modelfor virus-induced apoptosis in vitro, IFN-b clearly en-hanced apoptosis (Balachandran et al., 2000). In PBMCsfrom IFN-b-treated MS patients, the surface expressionsof the CD 95 (Fas) molecule (Rep et al., 1999) and of itsligand CD 95L (FasL; Kaser et al., 1999) were increased.Also, the rate of apoptotic PBMCs from MS patients afterin vitro stimulation with anti-CD 3 antibodies was foundto be elevated by IFN-b (Kaser et al., 1999). However,these findings were not confirmed in a recent study per-formed with permanent T cell lines (Zipp et al., 2000).Also, an increased soluble vascular cell adhesion molecule

Fig. 5. Pooled data from two experiments with treatment of moder-ately or severe EAE (clinical score of 3–4 or 5) with 3 3 105 unitsIFN-b s.c., 10 mg/kg methylprednisolone (MP) i.v., or with a com-bination of both (MP 1 IFN), or with PBS for controls. Immunohis-tochemical double labeling for TNF-a and macrophage markers inspinal cord. Percentage of TNF-a-expressing macrophages as visualizedby colocalization: *P , 0.05 (IFN vs. PBS), **P , 0.01 (MP 1 IFNvs. PBS); n 5 5 per group in each of two experiments; numbers andbars represent mean and SD; squares indicate median.

Fig. 6. Treatment of severe EAE (clinical score of 5) with 3 3 105 unitsIFN-b s.c., 10 mg/kg methylprednisolone (MP) i.v., or with a com-bination of both (MP 1 IFN), or with PBS for controls. Immunohis-tochemical double labeling for MMP2 and macrophage markers inspinal cord. Percentage of MMP2-expressing T cells as visualized bycolocalization: **P , 0.01 (MP 1 IFN vs. PBS); n 5 5 per group;numbers and bars represent mean and SD; squares indicate median.

Fig. 7. Treatment of moderately severe EAE (clinical score of 3–4)with 3 3 105 units IFN-b s.c., 10 mg/kg methylprednisolone (MP)i.v., or with a combination of both (MP 1 IFN), or with PBS forcontrols. Immunohistochemical detection of blood–brain barrier(BBB) disruption by staining for albumin followed by grey-scale anal-ysis (densitometry). A: Mean signal intensity. B: Maximal signal inten-sity. Diamond shapes represent six vessels of two animals per group;numbers and bars indicate mean and SD. Values for statistical signifi-cance are indicated and explained in the text.

IFN-b Effects In Situ in AT-EAE 65

1 level after IFN-b, as was shown in vitro after TNF-astimulation (Kallmann et al., 2000), could have a ratherproapoptotic potential. In view of these data, we wereinterested in whether the therapeutic benefit of IFN-b inautoimmune diseases of the CNS could also be exerted byinduction of apoptosis in situ. In most of our AT-EAEexperiments, we could not demonstrate significant induc-tion of T cell apoptosis 18 hr after IFN-b s.c., but thecellular infiltration was reduced rapidly, which led us tolook for other mechanisms of action.

Further beneficial effects of IFN-b are mediated bymodulation of the cytokine milieu, e.g., by a decrease oftypical Th1 cytokines such as IFN-g and TNF-a and byan increase of Th2 cytokines such as interleukin (IL)-10production (for review see Yong et al., 1998a). Immuno-histochemical double labeling revealed a reduced rate ofTNF-a-positive T cells and macrophages in situ afterIFN-b, but not after MP monotherapy. The reduction ofTNF-a-producing cells after IFN-b together with thereduced T cell infiltration results in a partial removal ofthis important mediator of apoptosis, which might explainwhy the apoptotic effect of IFN-b on T cells is seen onlyin severely affected animals.

Important effectors of disruption of the BBB areMMPs (Yong et al., 1998b), which appeared to be down-regulated by IFN-b, accompanied by a reduced T cellmigration in in vitro models of the BBB (Stuve et al.,1996; Lou et al., 1999). To our knowledge this is the firstreport of the in situ reduction of MMP2-positive macro-phages after IFN-b, and even moreso in combination withMP, as reflected by immunohistochemical double label-ing. This could be one explanation for the beneficialsynergy of IFN-b and MP on the disrupted BBB function,which has been demonstrated by MRI studies in MSpatients (Gasperini et al., 1998).

This study extends our knowledge of IFN-b effectsin the inflamed nervous system. In AT-EAE, T cell infil-tration was reduced without induction of apoptosis. Areduction of proinflammatory cytokines and macrophageactivation in situ may also prevent ongoing neurodegen-eration such as axonal loss (Rudick et al., 1999). Fortherapy of autoimmune disorders of the CNS such as MS,our results clearly support the use of s.c. IFN-b and,owing to synergistic effects on the disrupted BBB, favor acombination with i.v. MP for therapy of acute MS re-lapses.

ACKNOWLEDGMENTSWe thank Mrs. Helga Brunner, Daniela Seemann,

and Verena Wortmann for excellent technical assistance.

REFERENCESAkbar AN, Lord JM, Salmon M. 2000. IFN-alpha and IFN-beta: a link

between immune memory and chronic inflammation. Immunol Today21:337–342.

Balachandran S, Roberts PC, Kipperman T, Bhalla KN, Compans RW,Archer DR, Barber GN. 2000. Alpha/beta interferons potentiate virus-induced apoptosis through activation of the FADD/caspase-8 death sig-naling pathway. J Virol 74:1513–1523.

European Study Group on Interferon b-1b in Secondary Progressive MS.1998. Placebo-controlled multicentre randomised trial of interferonbeta-1b in treatment of secondary progressive multiple sclerosis. Lancet352:1491–1497.

Gasperini C, Pozzilli C, Bastianello S, Koudriavtseva T, Galgani S, Mille-fiorini E, Paolillo A, Horsfield MA, Bozzao L, Fieschi C. 1998. Effect ofsteroids on Gd-enhancing lesions before and during recombinant betainterferon 1a treatment in relapsing remitting multiple sclerosis. Neurol-ogy 50:403–406.

Gayo A, Mozo L, Suarez A, Tunon A, Lahoz C, Gutierrez C. 1999.Interferon beta-1b treatment modulates TNFalpha and IFNgamma spon-taneous gene expression in MS. Neurology 52:1764–1770.

Gold R, Schmied M, Giegerich G, Breitschopf H, Hartung HP, ToykaKV, Lassmann H. 1994. Differentiation between cellular apoptosis andnecrosis by the combined use of in situ tailing and nick translationtechniques. Lab Invest 71:219–225.

Gold R, Giegerich G, Hartung HP, Toyka KV. 1995. T-cell receptor(TCR) usage in Lewis rat experimental autoimmune encephalomyelitis:TCR beta-chain-variable-region V beta 8.2-positive T cells are notessential for induction and course of disease. Proc Natl Acad Sci USA92:5850–5854.

Gold R, Hartung HP, Toyka KV. 2000. Animal models for autoimmunedemyelinating disorders of the nervous system. Mol Med Today 6:88–91.

Hohlfeld R. 1997. Biotechnological agents for the immunotherapy ofmultiple sclerosis. Principles, problems and perspectives. Brain 120:865–916.

IFNB Multiple Sclerosis Study Group. 1993. Interferon beta-1b is effectivein relapsing-remitting multiple sclerosis. I. Clinical results of a multi-center, randomized, double-blind, placebo-controlled trial. The IFNBMultiple Sclerosis Study Group. Neurology 43:655–661.

Isaacs A, Lindenmann J. 1957. Virus interference: I. The interferon. Proc RSoc Lond [Biol] 147:258–267.

Jacobs L, O’Malley J, Freeman A, Ekes R. 1981. Intrathecal interferonreduces exacerbations of multiple sclerosis. Science 214:1026–1028.

Kallmann BA, Hummel V, Lindenlaub T, Ruprecht K, Toyka KV, Riec-kmann P. 2000. Cytokine-induced modulation of cellular adhesion tohuman cerebral endothelial cells is mediated by soluble vascular celladhesion molecule-1. Brain 123:687–697.

Kaser A, Deisenhammer F, Berger T, Tilg H. 1999. Interferon-beta 1baugments activation-induced T-cell death in multiple sclerosis patients[letter]. Lancet 353:1413–1414.

Kirou KA, Vakkalanka RK, Butler MJ, Crow MK. 2000. Induction of Fasligand-mediated apoptosis by interferon-alpha. Clin Immunol 95:218–226.

Lassmann H. 1983. Comparatative neuropathology of chronic experimentalallergic encephalomyelitis and multiple sclerosis. Berlin: Springer-Verlag.

Lou JN, Gasche Y, Zheng L, Giroud C, Morel P, Clements J, Ythier A,Grau GE. 1999. Interferon-beta inhibits activated leukocyte migrationthrough human brain microvascular endothelial cell monolayer. LabInvest 79:1015–1025.

Morrissey SP, Stodal H, Zettl U, Simonis C, Jung S, Kiefer R, Lassmann H,Hartung HP, Haase A, Toyka KV. 1996. In vivo MRI and its histologicalcorrelates in acute adoptive transfer experimental allergic encephalomy-elitis. Quantification of inflammation and oedema. Brain 119:239–248.

Paty DW, Li DK. 1993. Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. II. MRI analysis results of a multicenter,randomized, double-blind, placebo-controlled trial. UBC MS/MRIStudy Group and the IFNB Multiple Sclerosis Study Group. Neurology43:662–667.

Rep MHG, Schrijver HM, van Lopik T, Hintzen RQ, Roos ML, Ader HJ,Polman CH, van Lier RAW. 1999. Interferon (IFN)-beta treatmentenhances CD95 and interleukin 10 expression but reduces interferon-gamma producing T cells in MS patients. J Neuroimmunol 96:92–100.

66 Schmidt et al.

Rieckmann P. 1997. The Effects of interferon-b on cytokines and immuneresponses. In: Reder AT, editor. Interferon therapy of multiple sclerosis.New York: Marcel Dekker, Inc. p 161–191.

Rudick RA, Fisher E, Lee JC, Simon J, Jacobs L. 1999. Use of the brainparenchymal fraction to measure whole brain atrophy in relapsing-remitting MS. Multiple Sclerosis Collaborative Research Group. Neu-rology 53:1698–1704.

Ruuls SR, de Labie MCDC, Weber KS, Botman CA, Groenestein RJ,Dijkstra CD, Olsson T, van der Meide PH. 1996. The length of treatmentdetermines whether IFN-beta prevents or aggravates experimental auto-immune encephalomyelitis in Lewis rats. J Immunol 157:5721–5731.

Schmidt J, Gold R, Schonrock L, Zettl UK, Hartung HP, Toyka KV.2000. T-cell apoptosis in situ in experimental autoimmune encephalo-myelitis following methylprednisolone pulse therapy. Brain 123:1431–1441.

Stuve O, Dooley NP, Uhm JH, Antel JP, Francis GS, Williams G, YongVW. 1996. Interferon beta-1b decreases the migration of T lymphocytesin vitro: effects on matrix metalloproteinase-9. Ann Neurol 40:853–863.

van der Meide PH, de Labie MCDC, Ruuls SR, Groenestein RJ, BotmanCD, Olsson T, Dijkstra CD. 1998. Discontinuation of treatment withIFN-beta leads to exacerbation of experimental autoimmune encephalo-myelitis in Lewis rats. Rapid reversal of the antiproliferative activity of

IFN-beta and excessive expansion of autoreactive T cells as diseasepromoting mechanisms. J Neuroimmunol 84:14–23.

Wyllie AH. 1980. Glucocorticoid-induced thymocyte apoptosis is associ-ated with endogenous endonuclease activation. Nature 284:555–556.

Yong VW, Chabot S, Stuve O, Williams G. 1998a. Interferon beta in thetreatment of multiple sclerosis: mechanisms of action. Neurology 51:682–689.

Yong VW, Krekoski CA, Forsyth PA, Bell R, Edwards DR. 1998b. Matrixmetalloproteinases and diseases of the CNS. Trends Neurosci 21:75–80.

Yu M, Nishiyama A, Trapp BD, Tuohy VK. 1996. Interferon-beta inhibitsprogression of relapsing-remitting experimental autoimmune encephalo-myelitis. J Neuroimmunol 64:91–100.

Zettl UK, Gold R, Toyka KV, Hartung HP. 1995. Intravenous glucocor-ticosteroid treatment augments apoptosis of inflammatory T cells inexperimental autoimmune neuritis (EAN) of the Lewis rat. J NeuropatholExp Neurol 54:540–547.

Zipp F, Weller M, Calabresi PA, Frank JA, Bash CN, Dichgans J, McFar-land HF, Martin R. 1998. Increased serum levels of soluble CD95(APO-1/Fas) in relapsing-remitting multiple sclerosis. Ann Neurol 43:116–120.

Zipp F, Beyer M, Gelderblom H, Wernet D, Zschenderlein R, Weller M.2000. No induction of apoptosis by IFN-beta in human antigen-specificT cells. Neurology 54:485–487.

IFN-b Effects In Situ in AT-EAE 67