EAE in beta-2 microglobulin-deficient mice: axonal damage is not dependent on MHC-I restricted immune responses

www.elsevier.com/locate/ynbdi Neurobiology of Disease 19 (2005) 218 – 228

EAE in beta-2 microglobulin-deficient mice: Axonal damage is not dependent on MHC-I restricted immune responses Ralf A. Linker,a,1 Evelyn Rott,a H.H. Hofstetter,a T. Hanke,b Klaus V. Toyka,a and Ralf Gold,a,T,1 a

Department of Neurology, Clinical Research Group for Multiple Sclerosis, Julius-Maximilians-Universita¨t Wu¨rzburg, Josef-Schneider-Str. 11, D-97080 Wu¨rzburg, Germany b Department of Immunology, Julius-Maximilians-Universita¨t Wu¨rzburg, Josef-Schneider-Str. 7, D-97080 Wu¨rzburg, Germany Received 13 October 2004; revised 6 December 2004; accepted 8 December 2004 Available online 19 February 2005 There is accumulating evidence that CD8-positive (CD8+) T-cells and MHC-I expression may also play a role in neurodegeneration associated with multiple sclerosis (MS). We investigated the role of MHC-I and CD8+ T-cells by studying experimental autoimmune encephalomyelitis (EAE) in beta-2 microglobulin knockout mice induced by myelin oligodendrocyte glycoprotein (MOG) peptide 3555 or whole rat myelin basic protein (rMBP). For both encephalitogens and even after reconstitution of the immune system with MHC-I-positive bone marrow and transfer of mature CD8+ T-cells (iMHC-I+ CD8+ B2m / mice), the disease course in B2m / mice was significantly more severe with a 10-fold increased mortality in the B2m / mice as compared to wild-type C57BL/6 mice. EAE in B2m / mice caused more severe demyelination after immunization with MOG than with rMBP and axonal damage was more marked with rMBP as well as MOG even in iMHC-I+ CD8+ B2m / mice. Immunocytochemical analysis of spinal cord tissue revealed a significant increase in macrophage and microglia infiltration in B2m / and iMHC-I+ CD8+ B2m / mice. The different pattern of T-cell infiltration was underscored by a 2.5-fold increase in CD4positive (CD4+) T-cells in B2m / mice after induction of MOG 35-55 EAE. We conclude that lack of functional MHC-I molecules and CD8+ Tcells aggravates autoimmune tissue destruction in the CNS. Enhanced

Abbreviations: APP, amyloid precursor protein; ConA, Concanavalin A; CNS, central nervous system; CD, cluster of differentiation; Ci, Curie; EAE, experimental autoimmune encephalomyelitis; IL, interleukin; LFB, luxol fast blue; MBP, myelin basic protein; MHC-I, major histocompatibility complex-1; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; p.i., post-immunization; PHA, Phytohemagglutinin; PPD, purified protein derivative; vs., versus. T Corresponding author. Institute for Multiple Sclerosis Research, University of Goettingen and Gemeinnuetzige, Hertie-Stiftung, Waldweg 33, D-37073 Goettingen, Germany. Fax: +49 551 39 13348. E-mail address: [email protected] (R. Gold). 1

Present address: Institute for Multiple Sclerosis Research, University of Goettingen and Gemeinnuetzige Hertie-Stiftung, Waldweg 33, D-37073 Goettingen, Germany. Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2004.12.017

axonal damage speaks for pathways of tissue damage independent of CD8+ T-cells and neuronal MHC-I expression. D 2005 Elsevier Inc. All rights reserved. Keywords: Axonal damage; Knockout mouse; Macrophage; Multiple sclerosis; T-cell; MOG-EAE

Introduction Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) were traditionally thought to be mainly mediated by CD4-positive (CD4+) T-cells. Recently, there is accumulating evidence that also CD8-positive (CD8+) Tcells may play a role in disease pathogenesis. Clonal expansions of CD8+ T-cells dominate the T-cell infiltrate in active MS lesions (Babbe et al., 2000) and memory CD8+ T-cells are enriched in the CSF of multiple sclerosis patients (Jacobsen et al., 2002). Moreover, it was shown earlier that peptides from human myelin proteins can induce autoreactive CD8+ cytotoxic T-lymphocytes in vitro (Tsuchida et al., 1994). CD8+ T-cells are able to target several cell types in the CNS: MBP-specific human CD8+ T-cells are able to lyse MHC-Iexpressing oligodendrocytes (Jurewicz et al., 1998) but also attack neurons which can express MHC-I under specific conditions (Neumann et al., 1997) and thus can be prone to T-lymphocyte cytotoxicity (Medana et al., 2001). Thus CD8-mediated cytotoxicity is one possible pathogenic mechanism for axonal damage (Neumann et al., 2002). The pathogenic role of myelin-specific CD8+ T-cells was reproduced in rodent EAE models. CD8+ encephalitogenic T-cells produce severe autoimmunity in mice (Huseby et al., 2001; Sun et al., 2001). On the other side CD8+ T-cells may also exert disease modifying functions. Adoptive transfer of CD8+ T-cells from mice orally tolerized for MBP suppresses EAE in recipient mice (Chen et al., 1995). A possible mechanism was suggested by Jiang and coworkers implying that CD4+ Th1 cells can lead to activation of CD8+-regulatory T-cells which in turn promote a

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Th1–Th2 shift and thus protect from EAE (Jiang et al., 2001). The role of CD8+ T-cells in EAE has also been assessed using antibody-mediated depletion and knockout models. After antibody-mediated depletion of CD8+ T-cells, mice were no longer protected from relapses (Jiang et al., 1992). Additional work revealed that EAE in CD8 / mice revealed a lower mortality and more relapses (Koh et al., 1992) suggesting again a disease promoting as well as modifying function. To further assess the role of CD8+ T-cells and neuronal MHC-I expression, we investigated experimental autoimmune encephalomyelitis (EAE) in beta-2 microglobulin knockout mice (h2m / mice). These mice are deficient in functional major histocompatibility complex I (MHC-I) molecules and also lack MHC-Irestricted CD8+ T-cells (Zijlstra et al., 1990). h2m / mice do not exhibit a spontaneous phenotype. A Theiler virus model of demyelination in h2m / mice results in the absence of neurologic deficits following extensive demyelination (Rivera-Quinones et al., 1998). Other studies on Theiler’s encephalomyelitis in h2m / mice point to a more severe disease course with increased pathology (Begolka et al., 2001; Fiette et al., 1993). In previous studies focusing on the role of natural killer (NK)-cells, EAE in h2m / mice was reported to start earlier and to follow a more severe disease course (Fritz and Zhao, 2001; Zhang et al., 1997). We have induced the model disease experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, recombinant human MOG, or rat myelin basic protein (rMBP). Here we show that EAE in h2m / mice on a C57BL/6 background take a more severe disease course associated with increased macrophage infiltration and enhanced axonal damage.

Materials and methods Animals h2m / mice were backcrossed on a C57BL/6 background (Zijlstra et al., 1990) and bred at the in-house animal care facilities. In pilot experiments, the clinical course of MOG 35-55 and MBPEAE was compared between C57BL/6 wild-type mice and h2m+/ littermates showing no significant differences. Therefore, control C57BL/6 animals were purchased from Harlan Laboratories (Harlan Winkelmann, Borchen, Germany) for all following experiments. Animals were 8–12 weeks old and body weight was in a range of 20–30 g. Animals were housed in a room with controlled light cycle and were given commercial food pellets and water ad libitum. All experiments including bone marrow transfer were approved by the Bavarian state authorities for animal experimentation. Induction and clinical evaluation of EAE For induction of EAE, mice received a subcutaneous injection at flanks and tail base of 200 Ag MOG 35-55 peptide (Prof. Palm, Wurzburg, Germany), 200 Ag recombinant human MOG protein (rhMOG, prepared after Adelmann et al., 1995) or 200 Ag of rMBP (prepared after Oshiro and Eylar, 1970) in PBS emulsified in an equal volume of CFA containing Mycobacterium tuberculosis H37RA (Difco, Detroit MI, USA) at a final concentration of 1 mg/ml. Two injections of pertussis toxin (Sigma, Deisenhofen, Germany; 400 ng per mouse ip) were given 24 and 72 h later. Immunization with rMBP required a boost immunization on day 15

219

pi for disease induction, again followed by pertussis injections on day 16 and 18 pi. Animals were weighed and scored for clinical signs of disease on a daily basis. Disease severity was assessed using a scale ranging from 0 to 10; scores were as follows (Linker et al., 2002): 0 = normal; 1 = reduced tone of tail; 2 = limp tail, impaired righting; 3 = absent righting; 4 = gait ataxia; 5 = mild paraparesis of hindlimbs; 6 = moderate paraparesis; 7 = severe paraparesis or paraplegia; 8 = tetraparesis; 9 = moribund; 10 = death. Bone marrow transfer protocol Four weeks old h2m / and C57BL/6 control mice (n = 12 per group, pooled from 2 independent experiments) were sublethally irradiated with 5 Gy. They were then intravenously injected with 1 million of freshly prepared bone marrow cells and subsequently 3.5 million CD8+ T-cells each obtained from age- and gender-matched C57BL/6 mice. Preparation of bone marrow cells followed a protocol by Grauer et al. (2002), CD8+ T-cells were extracted from mouse spleen by anti-CD8+ columns (Qiagen, Hilden, Germany). Six weeks after bone marrow transfer, expression of MHC-I and CD8 on immune cells was investigated by FACS analysis of tail vein blood before and after induction of MOG 35-55 EAE (FITC anti-mouse CD8a, #553031, BD, via Pharmingen, Heidelberg, Germany; anti-mouse H2Db MHC, #MM 3801, Caltag, Hamburg, Germany). Histology At the maximum of disease (d23 pi for cryo-embedding, d27 pi for paraffin embedding of MOG 35-55, 30 pi for paraffin embedding of rMBP-diseased spinal cord, d23 pi for paraffin embedding of bone marrow transfer experiment), animals were anesthetized with pentobarbital and transcardially perfused with saline followed by 4% of paraformaldehyde. The complete spinal cord was carefully removed and 8–10 axial sections were further processed for routine paraffin embedding or snap frozen at 708C for cryosections. Paraffin sections were subjected to hematoxylin/eosin (H&E) and luxol fast blue (LFB) staining to assess parameters of inflammation and demyelination, respectively. Immunohistochemistry Immunohistochemistry was performed with 5 Am paraffin sections as described (Linker et al., 2002). If necessary, antigen unmasking was achieved by heat pretreatment of sections for 30 min in 10 mM citric acid buffer (Mac-3, APP) or 1 mM EDTA (CD3) in a microwave oven (850W). After inhibition of unspecific binding with 10% BSA, sections were incubated overnight at 48C with the appropriate primary antibody in 1% BSA. Secondary antibodies were used as indicated below. After blocking of endogenous peroxidase with H2O2, the peroxidase-based ABC detection system (DAKO, Hamburg, Germany) was employed with DAB as the chromogenic substrate. Specificity of staining was confirmed by omitting the primary antibody as a negative control. T-cells were labeled by rat anti-CD3 (Serotec; Wiesbaden, Germany; 1: 300) and macrophages by rat anti-mouse Mac-3 (Pharmingen; 1:200), each with a rabbit anti-rat secondary antibody (1:100, Vector via Linavis, Wertheim, Germany). CD4 and CD8 stainings were done on cryosections with hybridoma supernatants (1:50 and 1:700, kind gift from Prof. Zinkernagel,

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Zurich) with a rabbit anti mouse or rabbit anti-rat antibody (1:100, DAKO or 1:100, Vector) as secondary antibodies. Axonal damage was assessed by APP staining (1:1000, MAB348, Chemicon, Hofheim, Germany) with a rabbit anti mouse antibody (1:100, DAKO) as secondary antibody. Staining of neuronal cell bodies served as an internal positive control. Proliferation assay For lymph node and spleen-cell proliferation assays, single cell suspensions of spleen and inguinal lymph nodes from MOG 35-55 sensitized h2m / and wild-type C57BL/6 mice were prepared 12 days after immunization of animals (Linker et al., 2002). 2  105 cells were seeded in 96-well microtiter plates (Nunc, Wiesbaden, Germany) in 100 Al medium with addition of antigen. Antigen concentrations were 10 Ag/ml, except for ConA (2.5 Ag/ ml). Triplicate cultures were maintained at 378C in a humidified atmosphere with 5% CO2 for 56 h and harvested following a 16-h pulse with 0.2 ACi/well 3H-dT (tritiated thymidine, AmershamBuchler, Braunschweig, Germany). The cells were collected on fiberglass filter paper with a 96-well harvester (Pharmacia, Freiburg, Germany), and radioactivity was measured with a 96well Betaplate liquid scintillation counter (Pharmacia). ELISPOT assay ELISPOT assays were essentially performed as described elsewhere (Targoni et al., 2001). After sacrificing the mice on 12 days pi, the spinal cord was removed from the entire vertebral column, placed into DMEM medium, and disrupted with the back of a syringe. The resulting cell suspension was filtered through a Falcon Cell Strainer 2350 (BD, Heidelberg, Germany). The cells were washed twice with DMEM and subsequently counted. The cells were resuspended in HL-1 medium and plated at concentrations of 5  104–5  105 cells/well. ImmunoSpot M200 plates (Cellular Technology, Cleveland, USA) were coated overnight with the capture antibodies in sterile PBS. R46A2 at concentration of 4 mg/ml was used for capturing IFN-g, TRFK5 at 5 mg/ml for IL-5, JES6-1A12 for IL-2, and MP520F3 for IL-6 (Pharmingen, via BD, Heidelberg, Germany). The plates were then blocked for 1 h with 1% BSA in PBS and washed with PBS. Isolated cells from CNS or spleen were plated in HL-1/1% glutamine supplemented medium with or without MOG 35-55 at 10 Ag/ml in triplicate. After culture for 24 (IFN-g, IL-2) or 48 h (IL-5), the cells were discarded and washing with PBS and PBS/Tween 0.025% was performed. XMG1.2-biotin for IFN-g, biotinylated TRFK4 for IL-5, JES6-5H4 for IL-6, and MP5-32CH (all Pharmingen) were used as detection antibodies in an overnight incubation. After washing with PBST, steptavidine–alkaline phosphatase (DAKO, Hamburg, Germany) was added at a 1/1000 dilution in PBST for 2 h followed by thorough washing. The plates were developed using nitroblue tetrazolium 5-bromo-4-chloro-3indolyl phosphate substrate for 30 min. Quantification was done with the help of an ImmunoSpot Analyser (Cellular Technologies, Cleveland, USA). Digitized images were analyzed by gating and counting spots of defined size and shape (Targoni et al., 2001).

Coded sections were counted by blinded observers by means of overlaying a stereological grid onto the sections and counting inflammatory infiltrates per mm2 white matter (Eugster et al., 1999). The extent of demyelination was assessed by relating the number of grid squares with demyelination to the total number of grid squares containing white matter over an average of 8–10 independent levels of spinal cord per mouse. CD3-, CD4-, CD8-positive cells, Mac-3positive cells, and APP-positive axons were quantified on 3 representative sections, each one of cervical, thoracic, and lumbar spinal cord by counting 2 defined areas with the most intense pathology under a 400-fold magnification. For statistical evaluation of clinical course, data were pooled from different experiments. Analysis was performed using the Mann–Whitney U test or chisquare test for histology and clinical course and t test for ELISPOT data (SPSS program, SPSS, Chicago, USA). All data are given as mean values F SEM. P values were considered significant at *P b 0.05 and highly significant at **P b 0.01 or ***P b 0.001.

Results Disease course of MOG 35-55 and rMBP EAE in b2m /

mice

Mutant mice with a defect in MHC class I expression (h2m / mice) suffered from a more severe MOG 35-55 EAE than wild-type controls (n = 21 mice per group, data pooled from four independent experiments). At the maximum of disease (day 27 pi), h2m / mice exhibited severe quadriparesis whereas wild-type mice showed only a mild paraparesis (8.4 F 0.6 vs. 5.6 F 0.8, P = 0.004; Fig. 1A). Moreover, mortality in the h2m / group was higher and reached up to 52% in comparison to 5% in the control group ( P = 0.001, inset Fig. 1A). Similar results were obtained after immunization with the full recombinant human MOG protein (rhMOG, data not shown). There was no difference in onset of disease between h2m / mice and C57BL/6 control mice in any experiment. Experiments lasting until the late chronic disease phase were not feasible in view of the severe disease course with marked disability. rMBP-EAE was also studied. In rats, this encephalitogen induces inflammation and axonal damage without marked demyelination. Immunization with rMBP required the introduction of a modified immunization protocol with a boost immunization of 200 Ag rMBP on day 15 pi. Using this procedure, a monophasic disease course could be evoked with no relapses until the latest time point of observation on day 60 pi. At the maximum of disease h2m / mice exhibited severe paraplegia in comparison to control mice which showed only gait ataxia (7.3 F 0.7 vs. 3.4 F 0.6, P = 0.05; n = 9 vs.10 animals per group, animals grouped from 2 independent experiments; Fig. 1B). As expected with the milder disease course, in comparison to the MOG 35-55 EAE, no difference in mortality between both groups could be detected (inset Fig. 1B). No significant differences in disease incidence could be seen between knockout and wild-type mice for both immunization protocols (94% in h2m / mice vs. 81% in wildtype mice after immunization with MOG 35-55, P = 0.23; 75% for both groups after immunization with rMBP, P = 1.0).

Statistical analysis

Disease course of MOG 35-55 EAE in iMHC-I+ CD8+ b2m / mice

Quantitative evaluation of histopathological changes was essentially performed as described earlier (Linker et al., 2002).

To investigate the clinical course of h2m / mice with intact MHC-I and CD8 expression in the immune system

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Fig. 1. MOG 35-55 and rMBP-EAE in h2m / and C57BL/6 control mice. Dead mice were rated as b10Q from the day of death. Experiments lasting until the late chronic disease phase were not feasible in view of the local animal protection regulations. (A) Clinical course of MOG 35-55-induced EAE in h2m / and C57BL/6 control mice. Data are pooled from 3 independent experiments (n = 21 animals per group). h2m / mice exhibited severe quadriparesis at the maximum of disease, whereas wild-type mice showed moderate paraparesis only. After immunization with MOG 35-55, mortality in the h2m / group was higher and reached up to 50% in comparison to 5% in the control C57BL/6 group (see inset). (B) Clinical course of rMBP-induced EAE in h2m / and C57BL/6 control mice. Data are pooled from 2 independent experiments (n = 9/10 animals per group). h2m / mice suffered from a more severe disease course with paraparesis in comparison to control mice, which showed severe gait ataxia with only mild weakness of one hindlimb. No difference in mortality between h2m / and C57BL/6 control mice could be detected after immunization with rMBP (see inset).

(iMHC-I+ CD8+ h2m / mice), bone marrow chimeras were generated by injecting h2m / mice and C57BL/6 control mice (n = 12 per group, animals pooled from 2 independent experiments) with 1 million bone marrow cells and 3.5 million CD8+ T-cells from wild-type mice after sublethal irradiation. Expression of MHC-1 on immune cells was proven by FACS analysis before and after immunization with MOG peptide 35-55 (Figs. 2A and B). In the same way, the presence of CD8+ cells was shown by FACS analysis of peripheral blood cells (data not shown). Moreover, homing of CD8+ T-cells into the spleen and infiltration of the CNS could be demonstrated by immunohistochemistry on day 23 after induction of MOG 35-55 EAE (Figs. 2C and D). Staining of spleen and spinal cord of nontransplanted h2m / mice did not show CD8-positive cells neither in immune organs nor in the spinal cord at any time (data not shown).

The clinical course of EAE in iMHC-I+ CD8+ h2m / mice and C57BL/6 mice in this experiment did not differ significantly from the clinical course in the experiments without bone marrow transfer up to day 23 pi. iMHC-I+ CD8+ h2m / mice still exhibited a more severe disease course with severe paraplegia in contrast to mild paraparesis in the wild-type mice (8.1 F 0.4 vs. 5.4 F 0.5, P = 0.005; Fig. 2E). On day 23 pi, the experiment was terminated for histologic examination. Histologic evaluation of rMBP-EAE in b2m /

mice

Spinal cord was prepared at the maximum of disease (d30 pi). At first, the number of perivascular or intraparenchymal inflammatory infiltrates per mm2 cross section was assessed on hematoxylin/eosin stained sections over several levels of spinal cord thereby generating the inflammatory index (Eugster et al.,

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Fig. 2. Bone marrow and CD8 cell transfer in h2m / mice and C57BL/6 control mice. (A and B) FACS analysis of tail vein blood from a representative h2m / mouse before (A) and after bone marrow and CD8 cell transfer (biMHC-I+ CD8+ h2m / mouseQ; B) showing no MHC-I expression before and a clear MHC-I expression after transfer. The MHC expression in C57BL/6 control mice remained unchanged before and after transplantation. (C and D) Immunohistochemistry for CD8 in spleen and spinal cord cross sections from a representative, transplanted iMHC-I+ CD8+ h2m / mouse. CD8+ T-cells could be identified in the spleen (C) and as a minor part of the infiltrate in the spinal cord (D) after induction of MOG 35-55 EAE. Staining of spleen and spinal cord of non-transplanted h2m / mice does not show CD8+ cells neither in immune organs nor in the spinal cord after induction of EAE. (E) Clinical course of MOG 35-55-induced EAE in iMHC-I+ CD8+ h2m / mice and C57BL/6 control mice after bone marrow and CD8 cell transfer. Data are pooled from 2 different experiments (n = 12 mice per group). On day 23 pi, the clinical course does not significantly differ from non-transplanted h2m / mice in Fig. 1A. iMHC-I+ CD8+ h2m / mice suffer from tetraparesis whereas wild-type C57BL/6 mice only show mild paraparesis.

1999). Numbers of inflammatory infiltrates per mm2 were higher in knockout mice as compared to wild-type mice (see Table 1). To further characterize the inflammatory infiltrate, staining for T-cells was performed. After CD3 staining, we found no difference in numbers of infiltrating T-cells between h2m / and wild-type mice (Table 1). Macrophages constitute the prevailing immune cells of the inflammatory infiltrate in most EAE lesions. There was a significant increase in numbers of infiltrating Mac-3-positive macrophages in h2m / mice in comparison to wild-type mice (Figs. 3A and B). To assess the extent of demyelination, we performed LFB staining of spinal cord cross sections However, LFB staining revealed only very mild demyelination with no difference between h2m / and wild-type mice. We next investigated the extent of axonal damage in h2m / and wild-type mice using APP as a marker (Kornek et al., 2000).

After immunization with rMBP, a significant increase of damaged, APP-positive axons could be observed in the lesions of h2m / mice (Table 1). In summary, rMBP-EAE is characterized by a significant amount of inflammation and axonal damage in the absence of massive demyelination. In particular, h2m / mice exhibit increased macrophage infiltration and a higher amount of APPpositive axons (Figs. 3C and D). Histologic evaluation of MOG 35-55 EAE in b2m /

mice

After immunization with MOG 35-55, spinal cord was prepared at the maximum of disease (d27 pi) and analyzed for parameters of inflammation and tissue damage. There was no significant difference in inflammatory infiltrates in the spinal cord of h2m / mice in comparison to wild-type mice. We performed Mac-3 staining to

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Table 1 Histologic analysis of parameters of inflammation and tissue damage on paraffin sections after induction of MOG 35-55 or rMBP-EAE in chimeric iMHC-I+ CD8+ h2m / mice, untransplanted h2m / mice, and C57BL/6 wild-type mice Animal group

MOG 35-55 EAE C57BL/6 mice (n = 5) h2m / mice (n = 4) MOG 35-55 EAE after bone marrow and CD8 cell transfer C57BL/6 mice (n = 8) h2m / mice (n = 6) rMBP-EAE C57BL/6 mice (n = 4) h2m / mice (n = 4)

Inflammatory index (infiltrates/mm2 F SEM)

Macrophages (cells/mm2 F SEM)

Demyelination (% F SEM)

Axonal injury (APP-positive axons/mm2 F SEM)

11.5 F 1.4 14.0 F 1.4

1336 F 106 2135 F 82TTT

40.5 F 5.6 93.6 F 3.2TTT

293 F 33 340 F 41

n.d. n.d.

1372 F 89 1833 F 105T

17.5 F 1.8 21.2 F 2.0

281 F 34 478 F 56TTT

No demyelination No demyelination

123 F 18 223 F 21TT

10.0 F 0.4 12.0 F 0.7

595 F 35 782 F 70T

Analyses were done at the maximum of disease, respectively. T P b 0.05. TT P b 0.01. TTT P b 0.001.

assess the number of macrophages and found a significant increase in numbers of infiltrating Mac-3-positive cells in h2m / mice in comparison to wild-type mice (Table 1). To further characterize the inflammatory infiltrate, staining for T-cells was performed. Since CD4 and CD8 staining in mice is only feasible with cryopreserved tissue, another experiment was carried out to assess the number of infiltrating CD3+, CD4+, and CD8+ T-cells in the spinal cord of h2m / and wild-type mice on cryosections after immunization with MOG 35-55 (Table 2). At the

maximum of disease (day 23 pi), numbers of CD3+ T-cells showed no difference. The numbers of infiltrating CD4+ T-cells at the maximum of disease were significantly higher in h2m / mice than in wild-type mice (Figs. 4A and B), whereas CD8+ T-cells were only found in wild-type mice. These data corroborate a qualitatively different composition of the inflammatory infiltrate in h2m / mice. Overall, numbers of CD3+ T-cells and macrophages were significantly higher after immunization with MOG 35-55 than with rMBP.

Fig. 3. Histologic analysis of rMBP EAE in h2m / and C57BL/6 mice. Scale bar 50 Am for all sections. (A and B) Macrophage staining (Mac-3) of spinal cord cross sections after induction of rMBP-EAE reveals a larger macrophage infiltration in h2m / mice (A) than in BL/6 control mice (B). Representative cross sections depicting the anterior columns are shown. (C and D) Representative spinal cord cross sections stained for amyloid precursor protein (APP) as an indicator of axonal damage. APP-positive axons are more abundant in lesions of h2m / mice (C) than wild-type mice (D).

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Table 2 Histologic analysis of the T-cell infiltration pattern on cryosections after induction of MOG 35-55 EAE in h2m / mice and C57BL/6 wild-type mice

MOG 35-55 EAE C57BL/6 (n = 4) h2m / (n = 5)

CD3+ T-cells/ mm2 F SEM

CD4+ T-cells/ mm2 F SEM

CD8+ T-cells/ mm2 F SEM

axons were as abundant in h2m / mice as in wild-type mice (Table 1). These results indicate that despite the lack of CD8+ T-cells and MHC-I in h2m / mice, there is still a significant amount of axonal damage pointing at other destructive mechanisms than interaction of CD8+ T-cells with neuronal MHC-I.

786 F 74 848 F 80

468 F 49 970 F 149T

277 F 29 none

Histologic evaluation of MOG 35-55 EAE in iMHC-I+ CD8+ b2m / mice

Analysis is done at the maximum of disease (day 23 pi). T P b 0.05.

In summary, the more severe clinical course in h2m / mice after immunization with MOG 35-55 is paralleled by an increase in infiltrating Mac-3-positive macrophages and microglia and a qualitatively different T-cell infiltration. We next were interested in the type of pathology associated with the observed increase in Mac-3-positive and CD4+ cells in h2m / mice. The extent of demyelination in LFB staining correlated well with the area of Mac-3 positivity underscoring the importance of macrophage effector function for myelin disintegration. Extensive demyelination was observed after immunization with MOG 35-55 and demyelinated areas were significantly larger in h2m / mice in comparison to wild-type mice (Table 1). After immunization with MOG 35-55, the amount of APPpositive axons in inflamed areas was higher than after immunization with rMBP. In MOG 35-55 EAE, APP-positive

Histologic analysis was performed on day 23 pi. Similar to h2m / mice, MOG 35-55 EAE in iMHC-I+ CD8+ h2m / mice was characterized by a massive increase in Mac-3-positive microglia and macrophages in the spinal cord in comparison to transplanted control mice (Figs. 5A and B). However, in LFB staining, the demyelinated area in iMHC-I+ CD8+ h2m / mice was significantly reduced in comparison to non-transplanted h2m / mice. Although the amount of demyelination did not differ between iMHC-I+ CD8+ h2m / mice and transplanted wild-type mice (Table 1), APP-positive axons were significantly increased in the spinal cord of chimeric iMHC-I+ CD8+ h2m / mice in comparison to transplanted wild-type mice (Figs. 5C and D). Taken together, bone marrow and CD8 cell transfer led to a reduction of demyelination in iMHC-I+ CD8+ h2m / mice. The more severe disease course in chimeric iMHC-I+ CD8+ h2m / mice was paralleled by an increase in Mac-3-positive macrophages and APP-positive damaged axons.

Fig. 4. Histologic analysis of MOG 35-55 EAE in h2m / and C57BL/6 mice. (A and B) CD4 staining of representative spinal cord cross cryosections from h2m / (A) and C57BL/6 mice, day 23 pi (B). Note the larger CD4+ infiltrate in h2m / mice. Scale bar = 50 Am. (C and D) Luxol fast blue (LFB) staining of representative spinal cord paraffin sections from h2m / (C) and C57BL/6 mice (D). Lumbosacral sections are shown. Note the massive demyelination in h2m / mice (marked by arrows) in comparison to wild-type mice. Scale bar = 100 Am.

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Fig. 5. Histologic analysis of MOG 35-55 EAE after bone marrow and CD8 cell transfer in iMHC-I++ CD8+ h2m / mice and C57BL/6 mice. Representative spinal cord cross sections depicting the anterior columns are shown. Scale bar = 50 Am for all sections. (A and B) Mac-3 staining for macrophages in iMHC-I+ CD8+ h2m / mice (A) and control transplanted C57BL/6 mice (B). Note that Mac-3-positive cells are more abundant in iMHC-I+ CD8++ h2m / mice (A). This pattern of macrophage infiltration is similar to h2m / mice without transplantation. (C and D) APP staining for axonal damage in iMHC-I+ CD8+ h2m / mice (C) and C57BL/6 mice (D). Numerous damaged axons are seen in lesions of iMHC-I+ CD8+ h2m / mice (C, marked by arrows).

Production of IFN-c To detect differences in priming of immune cells in MOG 35-55 and rMBP-EAE, proliferation assays with lymph node cells were performed ex vivo. We found no difference in the proliferative response to rMBP and MOG 35-55 between h2m / and wild-type mice (data not shown). In further experiments, we monitored cytokine production from mononuclear cells that had infiltrated the CNS. Mononuclear cells were extracted from spinal cord tissue and production of several Th1 and Th2 cytokines was assessed in ELISPOT assays. At the maximum of disease, frequencies of MOG 35-55-specific, IFN-g-producing cells were clearly more abundant in cultures taken from h2m / mice than from wild-type mice (Fig. 6; mean F SD: 2541 F 753 spots per million cells in h2m / mice vs. 1420 F 1291 spots per million cells in wild-type control animals, P = 0.026). There was a significant reduction of MOG 35-55specific, IL-5-producing cells in CNS-infiltrating cells in h2m / mice although at generally low levels (mean F SD: 83 F 34 spots per million cells in h2m / mice vs. 40 F 27 spots per million cells in wild-type control animals, P = 0.004). In the same way, MOG 35-55 triggered IL-2 and IL-6 levels tended to be higher in h2m / mice although with a larger interindividual variation (mean F SD: 1034 F 584 spots per million cells in h2m / mice vs. 832 F 626 spots per million cells in wild-type control animals, P = 0.47 for IL-2; 1658 F 979 spots per million cells in h2m / mice vs. 1296 F 753 spots per million cells in wild-type control animals, P = 0.38 for IL-6). In summary, cytokine production from CNS-infiltrating MOG 35-55-specific cells ex vivo is mainly characterized by an increased number of MOG 35-55-specific, IFN-g-producing cells.

Fig. 6. Ex vivo ELISPOT analysis of MOG 35-55-specific IFN-g-producing cells from the spinal cord of h2m / mice and C57BL/6 wild-type mice 20 days after induction of MOG 35-55 EAE. The number of IFN-gproducing cells is significantly increased in the knockout mice (black columns, left side) in comparison to the wild-type mice (white columns, right side) after stimulation with MOG 35-55. Hatched columns represent control stimulation with cell culture medium only. Each column represents an individual animal, data were pooled from two experiments.

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Discussion Our study reveals that the disease course of MOG 35-55 EAE and rMBP-EAE is more severe in h2m / mice, but also in bonemarrow chimeric h2m / mice with intact CD8+ T-cells and MHC-I on immune cells (iMHC-I+ CD8+ h2m / mice). These data correlate well with previous studies on MOG-EAE in h2m / mice investigating the role of NK-cells in EAE (Fritz and Zhao, 2001; Zhang et al., 1997). Here we add new data to characterize axonal damage and the inflammatory reaction in situ in these mice. First, we show that APP-positive axons are abundant in h2m / mice lacking neuronal MHC-I and CD8+ T-cells after induction of EAE with two basically different encephalitogens: rMBP leading to a predominant inflammatory response while MOG-induced EAE also results in marked demyelination mainly mediated by macrophage cytotoxicity (Calida et al., 2001; Svensson et al., 2002). Therefore, axonal damage can occur in the absence of CD8+ T-cells, neuronal MHC-I, and, in the rMBP-model, even demyelination. Axonal damage occurs to the same degree in iMHC-I+ CD8+ h2m / mice in the presence of CD8+ T-cells and MHC-I in the immune system. In h2m / mice and iMHC-I+ CD8+ h2m / mice, the extent of axonal damage correlates mainly with an increase in the number of macrophages. These data are in accordance with the correlation of acute axonal injury with inflammation, especially macrophage infiltration, in multiple sclerosis lesions (Bitsch et al., 2000; Kuhlmann et al., 2002). In recent in vitro studies interaction between CD8+ T-cells and MHCI-expressing axons was shown to result in axonal injury (Medana et al., 2001). Our observations are in line with the idea that in the situation of massive macrophage infiltration and/or demyelination CD8/MHC-I independent mechanisms like Fas/FasL interaction (Neumann et al., 2002), TNF-a cytotoxicity, perforin-mediated damage, free radicals, matrix metalloproteinases, or NO production as well as lack of trophic support by the myelin sheath may mainly contribute to axonal injury (for a review, see Bjartmar and Trapp, 2001; Coleman and Perry, 2002; Redwine et al., 2001). Moreover, the larger number of APP-positive axons in h2m / mice correlates well with the more severe disease course for both autoantigens. The amount of axonal damage is higher in the MOG 35-55 than in the MBP-immunized animals, which does well connect with the more severe disease course after immunization with MOG. These data underscore the role of axonal damage as a determining factor of disability in inflammatory CNS diseases (Wujek et al., 2002). Second, we show that in MOG 35-55 and rMBP-induced EAE in C57BL/6 mice, disease onset and progression is not necessarily dependent on the presence of CD8+ T-cells. These results are consistent with earlier observations on Theiler’s murine encephalomyelitis in h2m / mice (Begolka et al., 2001; Fiette et al., 1993). In inflammatory CNS diseases, CD8+ T-cells can play a pathogenic (cytotoxic CD8+ T-cells) or regulatory role (CD8+ bsuppressorQ T-cells). Recent studies suggested mainly a pathogenic role for CD8+ T-cells in EAE and particularly MS (Babbe et al., 2000; Jacobsen et al., 2002). h2m / mice are characterized by a lack of MHC-I and therefore all MHC-I-restricted cytotoxic and regulatory CD8+ T-cells and, importantly, also display a defective NK-cell function (Liao et al., 1991). h2m / mice show a more severe disease course, an increase in CNS-infiltrating macrophages and microglia, and a relative increase of CD4+ cells in situ with a predominance of Th1 cytokines, in particular an increase in IFN-gproducing CNS-infiltrating cells. This might be attributed to the

lack of regulatory CD8+ T-cells or, more recently in the focus of interest, NK-cells controlling the pathogenic CD4+ T-cells (Jahng et al., 2001; Mars et al., 2002; Singh et al., 2001; Zhang et al., 1997). iMHC-I+ CD8+ h2m / mice display a similar disease course and pathology than h2m / mice. iMHC-I+ CD8+ h2m / mice are characterized by intact CD8+ cells and MHC-I on immune cells but still have a defective NK-cell function in the setting of a persisting MHC-I deficiency on cells outside the blood stream. The similar findings in h2m / mice and iMHC-I+ CD8+ h2m / mice point to the importance of NK-cells as regulatory mechanisms in inflammatory demyelinating diseases of the CNS. These results are consistent with earlier observations on NK-cell function in EAE (Matsumoto et al., 1998; Miyamoto et al., 2001) and some more recent studies in MS patients (Takahashi et al., 2004). Finally, we show that EAE can be evoked after immunization of C57BL/6 mice with a new boost immunization protocol using rMBP and not guinea pig MBP as antigen. An immunization regimen with a boost protocol can overcome tolerance in C57BL/6 mice on a H2b background as shown for various other regimens like injection of IFN-g, combination of active and passive immunization, or high doses of antigen (Faunce et al., 2004; Shaw et al., 1992, 1996). Immunization of C57BL/6 mice with rMBP results in a monophasic disease course similar to that in rats (Gold et al., 2000) with inflammation, a minor degree of demyelination and axonal damage in the histopathologic analysis.

Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (DFG), SFB 581, TPA1, and the Institute for Multiple Sclerosis Research, University of Goettingen Bereich Humanmedizin and gemeinnuetzige Hertie-Stiftung. We wish to thank Prof. H. Lassmann for helpful discussions and H. Brqnner and V.T. Wfrtmann for expert technical assistance.

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