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The data set the stage for definition .... bout of paresis lasted for 3–6 days, followed by recovery for 3–5 ... cal EAE symptoms such as floppy tail and/or slight gait.
Eur. J. Immunol. 2003. 33: 1907–1916

Chromosome 4 regions regulating MOG-EAE

1907

Paradoxical effects of arthritis-regulating chromosome 4 regions on myelin oligodendrocyte glycoprotein-induced encephalomyelitis in congenic rats Kristina Becanovic1, Liselotte Bäckdahl2, Erik Wallström1, Fahmy Aboul-Enein3, Hans Lassmann3, Tomas Olsson1 and Johnny C. Lorentzen2 1 2 3

Neuroimmunology Unit, Department of Medicine, Karolinska Hospital, Stockholm, Sweden Rheumathology Unit, Department of Medicine, Karolinska Hospital, Stockholm, Sweden Brain Research Institute, University of Vienna, Vienna, Austria

Immunoregulatory gene loci in different organ-specific inflammatory diseases often colocalize. We here studied myelin oligodendrocyte glycoprotein (MOG)-induced EAE in rat strains congenic for arthritis-regulating genome regions on chromosome 4. We used congenic rats with a 70-centimorgan (cM) fragment from the EAE- and arthritis-resistant PVG.1AV1 rat strain on the arthritis- and EAE-permissive Dark Agouti (DA) rat background. In addition, we evaluated three recombinant strains, C4R1–C4R3, which overlap with arthritis-linked loci. PVG.1AV1 alleles in the C4R1 recombinant did not affect arthritis, but conferred protection against MOG-EAE. PVG.1AV1 alleles in the C4R2 recombinant downregulated arthritis but had no effect in MOG-EAE. Paradoxically, PVG.1AV1 alleles in the C4R3 recombinant down-regulated arthritis, but the same fragment increased serum levels of anti-MOG Ab and aggravated clinical MOG-EAE. Thus, we provide original evidence that the same genome regions can have opposite effects in different organ-specific inflammatory diseases. Interestingly, no apparent difference in the MOG-EAE phenotype was observed in full-length congenic rats and parental DA rats, suggesting that the disease amelioration in C4R1 and aggravation in C4R3 functionally counteract each other. The data set the stage for definition of the mechanisms and positioning of the genes regulating two organ-specific inflammatory diseases differently. Key words: Experimental autoimmune encephalomyelitis / Multiple sclerosis / Quantitative trait locus / Autoimmunity / Arthritis

1 Introduction Multiple sclerosis (MS) is characterized by chronic inflammation of the central nervous system (CNS), which causes demyelination, axonal damage and neurological deficits. The disease is generally referred to as an autoimmune disorder, but the etiopathogenesis is largely unknown. There is, however, evidence for genetic influence on MS from twin, adoption and family studies [1, 2]. One way to increase insight into disease pathways is

[DOI 10.1002/eji.200323692] Abbreviations: MOG: Myelin oligodendrocyte glycoprotein QTL: Quantitative trait locus p.i.: Post-immunization Cia: Collagen-induced arthritis Pia: Pristane-induced arthritis Aia: Adjuvant-induced arthritis Oia: Oil-induced arthritis DA: Dark Agouti MS: Multiple sclerosis © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received Revised Accepted

28/11/02 24/3/03 22/4/03

therefore to identify disease-regulating genes. Certain HLA haplotypes are known to increase susceptibility for MS [3–5], but these genes only explain part of the genetic susceptibility, leaving an important role for nonMHC genes and environmental factors. Genetic heterogeneity and uncontrollable environmental conditions complicate genetic dissection of MS in humans. Therefore, disease-regulating non-MHC genes can be more easily mapped in EAE in mice and rats, where heterogeneity is reduced and the environment is controlled [6, 7]. Knowledge of disease-linked rodent genes may point to disease-associated human homologs and to disease pathways. We therefore initiated genetic dissection of EAE induced in Dark Agouti (DA) rats by immunization with myelin oligodendrocyte protein (MOG). MOGEAE is particularly suitable for such analysis since the disease closely mimics MS with prominent demyelination, axonal damage and a chronic relapsing disease course [8, 9]. 0014-2980/03/0707-1907$17.50 + .50/0

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Linkage analyses suggest that MOG-EAE is regulated by a large number of quantitative trait loci (QTL) [10], some of which overlap with QTL for experimental rheumatoid arthritis on rat chromosome 4 [11–14]. Recently, congenic strains for arthritis-linked chromosome 4 intervals were produced and tested in experimental arthritis models. The present study had two major aims: firstly, to determine whether the chromosome intervals isolated in congenic rats affect MOG-induced EAE; secondly, to determine the manner of the regulation since a QTL may affect the two diseases in the same or in opposite ways. To determine the influence of rat chromosome 4 loci on EAE, we established a congenic strain, DA.C4(PVG.1AV1), by selective back-crossing of a 70cM PVG.1AV1 chromosome 4 interval onto DA background. In addition, three recombinant strains were developed, covering different intervals of the QTL-rich 70-cM region (Fig. 1). The first recombinant, C4R1, overlaps with the QTL Pia2 (pristane-induced arthritis 2) [15], Aia2 (adjuvant-induced arthritis 2) [16], Cia3 (collageninduced arthritis 3) [13], Pia5 [15] and Aia3 [16]. This recombinant also harbors a QTL regulating MOG-EAE survival [11]. C4R2 overlaps with Cia3, Aia3, Pia5, Pia7 [14] and Cia13 [17]. C4R3 overlaps with Oia2 (oilinduced arthritis 2) [12], Cia13 and Pia7. C4R3 also harbors a QTL regulating anti-MOG IgG isotypes in EAE [11]. We monitored MOG-induced EAE in DA rats and the different congenic strains and evaluated the following disease phenotypes: (1) clinical signs of encephalomyelitis, i.e. paresis, (2) histopathology, including inflammation and demyelination in the CNS, (3) the humoral immune response with regard to serum levels of total anti-MOG IgG and IgG isotypes, and (4) antigen-specific cellular responses recorded as proliferation and IFN- + secretion.

2 Results 2.1 Clinical EAE phenotypes in DA, PVG.1AV1 and C4 congenic strains A survey of the outcome of the clinical signs is presented in Table 1 and Fig. 2. In general, weight loss preceded the paresis by 1–2 days. DA rats displayed a high disease incidence (17/19) and severe disease course, while PVG.1AV1 rats displayed low incidence (5/17) and mild neurological deficits in affected individuals. Twelve out of 17 affected DA rats displayed a clinical course with relapses of hind leg paresis. The first neurological signs appeared at day 17±5 (mean ± SD; Table 1). The first bout of paresis lasted for 3–6 days, followed by recovery for 3–5 days and then a second bout of paralysis. The

Fig. 1. Schematic illustration of the distal part of chromosome 4, the congenic C4 and the different recombinant strains C4R1, C4R2 and C4R3. In the center, the vertical bar entitled RN04 indicates the microsatellite markers used to map the recombinants. Vertical bars to the left show the homozygous C4 and recombinant intervals tested in functional studies. A dashed line indicates the interval within which recombination has occurred. To the right, the light bars indicate the chromosomal location of the arthritislinked QTL Aia2, Pia2, Aia3, Cia3, Pia5, Oia2, Pia7 and Cia13 [11–17]. Microsatellite marker positions in cM (from an SHRSP×BN F2 intercross) were retrieved from http://rgd.mcw.edu/.

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Chromosome 4 regions regulating MOG-EAE

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cal EAE symptoms such as floppy tail and/or slight gait disturbance (10/19).

Fig. 2. Monitoring of clinical EAE signs during 40 days p.i. DA rats (open diamonds) displayed a chronic relapsing EAE in contrast to the relatively resistant PVG.1AV1 rats (open squares). C4R3 rats (filled squares) developed EAE with an earlier onset compared to DA rats (p=0.012, Mann-Whitney U-test) as well as more severe disease (day 13, p=0.039; and day 17, p=0.0064). C4R1 rats (filled triangles) displayed a benign disease course, with less severe disease at days 24–30 p.i. (p p 0.05); cumulative EAE score (p=0.015), maximum score (p=0.025) and duration (p=0.010) were lower compared to DA rats. Clinical EAE score was graded from 0 to 5.

second bout lasted longer and tended to be more severe with intervals of complete para-paralysis, consistent with the previously described disease course in DA rats [8, 9]. Furthermore, 9/17 affected DA rats displayed paralysis until the day of sacrifice, day 40 post-immunization (p.i.). The incidence in C4R1 rats was 14/19, but only 4/19 C4R1 developed hind leg paralysis with a relapsingremitting disease course. The rest of the animals remained either healthy (5/19) or displayed weaker clini-

C4R3 rats displayed EAE with an incidence of 16/19 and with an earlier onset (day 13±1.9) compared to DA rats (p=0.012, Mann-Whitney U-test). In addition, the clinical disease score was higher in C4R3 compared to DA rats at day 13 (p=0.039) and day 17 (p=0.0064). Thirteen out of 16 affected C4R3 rats displayed a clinical course with relapses of hind leg paresis. Furthermore, 11/16 C4R3 rats displayed paralysis until the day of sacrifice, day 40 p.i. Before the day of sacrifice, 1/21 C4, 1/19 C4R1, 2/17 C4R2, 5/19 C4R3 and 4/20 DA rats died. The C4R2 recombinant and the full-length congenic C4 strain displayed intermediary clinical EAE, with an incidence of 14/ 17 and 15/20, respectively. None of the clinical EAE phenotypes tested differed for either C4 or C4R2 when compared to DA. Nine out of 14 C4R2 and 8/15 affected C4 rats developed hind leg paralysis with a relapsingremitting clinical course, and 4/14 C4R2 and 5/15 C4 affected rats displayed hind leg paralysis until day 40 p.i.

2.2 Histopathological evaluation Extensive spinal cord and/or brain demyelination and inflammation was recorded in DA, C4R3 and C4R2 rats. C4 rats were intermediate, while C4R1 and PVG.1AV1 rats displayed a lower degree of demyelination (C4R1 vs. DA: p=0.075; PVG.1AV1 vs. DA: p=0.022; Mann-Whitney U-test) and inflammation (C4R1 vs. DA: p=0.0073; PVG.1AV1 vs. DA: p=0.016) (Fig. 3). Patterns of demye-

Table 1. Summary of clinical EAE phenotypes in chromosome 4 congenic rat strains and PVG.1AV1 rats compared to DA

a)

Mean day of onset in affected rats on the first day of clinical EAE signs. Mean maximum score for affected and unaffected rats. Rats dead before day 40 p.i. were given a maximum EAE score of 5. c) Mean severity for affected rats. d) Calculated by adding all the daily EAE scores for affected and unaffected rats within each strain. e) Mean disease duration for affected rats. f) Number of animals dead before day 40. p values were calculated with the Mann-Whitney U test; corresponding to *p p 0.05, **p p 0.005, ***p p 0.0005. b)

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Fig. 3. Inflammation and demyelination assessed by histopathological evaluation of brain and spinal cord sections at day 40 p.i. Six to nine rats from each strain were analyzed semi-quantitatively. Demyelination is shown on the y axis and inflammation on the x axis. Data are presented as mean ± SEM. DA, C4R2 and C4R3 rats displayed intense inflammation and demyelination in contrast to C4, C4R1 and PVG.1AV1 rats.

lination were in agreement with previously published histopathological observations in DA and PVG.1AV1 rats [8, 9].

2.3 Anti-MOG antibody serum levels C4R1 rats (n=18) displayed lower IgG1 (p=0.044, MannWhitney U-test) and IgG2c (p=0.00013) isotype serum levels compared to DA rats (n=18) (Fig. 4). C4R3 rats (n=18) produced higher total IgG (p=0.0065), IgG1 (p=0.040) and IgG2a (p=0.0082) isotype serum levels. Furthermore, both C4 (n=18) and PVG.1AV1 (n=11) rats displayed higher total IgG (C4 vs. DA: p=0.0011; PVG.1AV1 vs. DA: p=0.0012) and IgG1 (C4 vs. DA: p=0.000034; PVG.1AV1 vs. DA: p=0.000029) isotype serum levels compared to DA rats. C4 rats also displayed higher IgG2c isotype levels compared to DA rats (p=0.015). C4R2 rats (n=17) did not show any differences in IgG isotype levels compared to DA. Interestingly, C4R1 rats, which were more similar to PVG.1AV1 than DA rats in clinical disease, produced lower levels of total IgG (p=0.00027), IgG1 (p=0.000010), IgG2a (p=0.043), IgG2b (p=0.041) and IgG2c (p=0.0034) isotypes compared to PVG.1AV1 rats.

2.4 Anti-MOG T cell responses Numbers of IFN- + -producing cells and proliferative responses were measured in DA, C4R1, C4R2, C4R3, C4 and PVG.1AV1 strains to assess the MOG-specific T cell response [18–20]. The variability in specific responses

Fig. 4. Anti-MOG Ab serum levels at day 12 p.i. measured by ELISA. C4R1 rats (n=18) displayed lower IgG1 (p=0.044, Mann-Whitney U-test) and IgG2c (p=0.00013) isotype serum levels compared to DA rats (n=18). C4R3 rats (n=18) produced higher total IgG (p=0.0065), IgG1 (p=0.040) and IgG2a (p=0.0082) isotype serum levels. Furthermore, both C4 (n=18) and PVG.1AV1 (n=11) rats displayed higher total IgG (C4 vs. DA: p=0.0011; PVG.1AV1 vs. DA: p=0.0012) and IgG1 (C4 vs. DA: p=0.000034; PVG.1AV1 vs. DA: p=0.000029) isotype serum levels compared to DA rats. C4 rats also displayed higher IgG2c isotype levels compared to DA (p=0.015). C4R2 rats (n=17) did not show any differences in IgG isotype levels compared to DA. Data are presented in arbitrary units. The ends of the box plots show the 25th and 75th quartiles. The line across the middle of the box identifies the median sample value. The whiskers extend from the ends of the box to the outermost data point. p values were calculated with the Mann-Whitney U-test; *p p 0.050, **p p 0.005, ***p p 0.0005.

between individuals within a strain was considerable. Strain comparisons did not reveal differences except that C4R2 rats displayed a lower proliferative response to the MOG (aa91-108) peptide compared to DA rats (data not shown). However, neither the proliferative response to rMOG (aa1-125) nor numbers of rMOG- or MOG peptide-specific IFN- + -secreting cells differed between DA and C4R2 rats.

Eur. J. Immunol. 2003. 33: 1907–1916

3 Discussion Compared to arthritis experiments in the same congenic recombinant strains used here, we observed paradoxical effects in the regulation of MOG-EAE (Table 2). Thus, rats carrying PVG.1AV1 alleles in the C4R3 recombinant region are protected from oil-induced arthritis, and are less susceptible to arthritis induced with collagen type II, squalene and pristane [21]. Here, PVG.1AV1 alleles in the C4R3 region aggravated MOG-EAE with a significantly earlier onset of disease and a more conspicuous humoral autoimmune response. C4R3 rats also displayed a tendency of increased disease susceptibility, severity, cumulative score, duration and mortality when compared to DA rats. This same tendency was also detected for inflammation and demyelination. Although not statistically significant, this trend suggests that PVG alleles in C4R3 result in a more severe disease compared to DA rats. In contrast, PVG.1AV1 alleles in C4R1 rats exerted a powerful down-regulation of MOG-EAE, although collagen type II- and pristane-induced arthritis was not affected [21]. In contrast to C4R1 and C4R3 rats, PVG.1AV1 alleles in C4R2 did not affect MOG-EAE in males or females, although they down-regulated collagen-induced arthritis in males. The absence of influence from C4R2 strongly suggests that EAE regulation from C4R1 and C4R3 is not due to contaminating genes outside the congenic regions, since all recombinant strains were derived from the same DA.C4(PVG.1AV1) congenic strain. Interestingly, the 70-cM C4 congenic fragment did not display any evident up- or downregulation of EAE compared to DA rats, although it harbors both C4R1 and C4R3 fragments. This could be due to genetic interactions. C4R1 and C4R3 fragments within the full-length congenic fragment may counteract and/or neutralize each other’s regulatory effects. This phenomenon, i.e. the absence of phenotypic differences in a large congenic fragment, although apparent in smaller fragments, may be of general interest for scientists in the field of genetic dissection of disease regulation using congenic strains.

Table 2. Overview of MOG-induced EAE and experimental arthritis in C4-congenic rat strains and PVG.1AV1 compared to DA

Chromosome 4 regions regulating MOG-EAE

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Previous linkage studies of experimental diseases in F2 populations [11, 22] or meta-analysis of both experimental and human organ-specific inflammatory diseases [6, 23, 24] have suggested clustering of disease-regulating genes. In addition, it was previously demonstrated in a mouse congenic strain that a locus on chromosome 3 regulates both type 1 diabetes and EAE [25]. In this case, both diseases were down-regulated. We believe it is of principle interest that the regulatory influence on MOGEAE, which was detected in the C4R3 recombinant, worked in an opposite manner compared to arthritis. While the findings presented here concern non-MHC gene regulation, it is well known that HLA-DR2(15),DQ6 may predispose for MS [5], whereas the same haplotype protects against type 1 diabetes [26]. This is a phenomenon that may have to be considered in gene-mapping studies of inflammatory diseases affecting different organs. Although apparently paradoxical, it is possible to imagine genetically regulated mechanisms that may be protective in one inflammatory disease but detrimental in another. An example of such a phenomenon may be the outcome of TNF-blocking agents, which ameliorate rheumatoid arthritis [27, 28] but aggravate MS [29]. It is also important to note that the present study uses strains in which each congenic chromosome interval contains many genes. It therefore remains to be investigated whether the two organ-specific inflammatory diseases are controlled by the same gene or by closely located, but different genes, in each congenic strain. If the regulating gene is the same, the difference in disease outcome should be due to opposite or different effects of strain-specific allelic variants of the gene. In our study, these are PVG.1AV1 alleles within the described C4R1, C4R2 and C4R3 recombinant regions, on a DA background. There could possibly also be epistasis between DA and PVG.1AV1 alleles, which enhance or reduce the regulatory effect of these genome fragments. Anti-MOG Ab responses have been proven to be of importance in rodent EAE [30–32]. We measured the humoral anti-MOG IgG isotype response in the recombinant strains as a possible marker for immunological events important for disease outcome. The quantitative levels of total IgG were determined, as well as IgG isotype Ab levels, which reflect qualitative differences in T cell responses. Th1-biased T cell responses are characterized by production of pro-inflammatory cytokines such as IFN- + and TNF- § , while Th2-biased responses are characterized by production of B cell helpassociated cytokines such as IL-4, IL-5, IL-6 and IL-10 [33, 34]. In EAE, Th1 responses are associated with aggravated autoimmune neuroinflammation, while Th2biased responses under certain circumstances may be protective [35, 36]. In the rat, IgG2b and IgG2c are asso-

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ciated with Th1, and IgG1 is associated with Th2 responses [37]. Here we demonstrate higher levels of total IgG and IgG1 isotype in PVG.1AV1 compared to DA rats, suggesting a Th2-biased response, which coincides with decreased disease susceptibility. In contrast, C4R3 rats produced higher serum levels of total IgG, IgG1 (Th2-driven) and IgG2a (not linked to Th1 or Th2) compared to DA rats, but they developed more severe EAE than DA rats. One explanation could be that the very severe disease in C4R3 rats depends on a generally more intense humoral anti-MOG immune response. Accordingly, C4R1 rats developed less severe disease than DA rats and had lower serum levels of both IgG1 (Th2-driven) and IgG2c (Th1-driven) compared to DA rats. The recorded differences between the strains in anti-MOG Ab production may be directly related to the different clinical outcomes or may not be causally related to clinical disease, but still caused by gene polymorphisms in the congenic fragments. Interestingly, we noted that DA and PVG.1AV1 rats displayed equivalent levels of anti-MOG IgG2c, but C4R1 rats displayed lower levels than both these strains. This may indicate that PVG.1AV1 alleles in the C4R1 fragment interact with DA alleles but not with PVG.1AV1 alleles to produce lower levels of IgG2c. Furthermore, C4R1 rats also displayed lower levels of total IgG, IgG1, IgG2a and IgG2b when compared to PVG.1AV1 rats. These observations suggest that the PVG.1AV1 alleles within the C4R1 fragment exhibit a strong down-regulatory effect on anti-MOG Ab production, but only when integrated on a DA background. In conclusion, we have established experimental situations that enable the use of congenic strains for genetic dissection of QTL linked to MOG-EAE, a model for multifactorial MS. The EAE down-regulating QTL encompassed in C4R1 congenics shows homology to human 7q34–36, a region that shows evidence of linkage to MS (Fig. 5) [38]. The QTL present in C4R3 congenics show homology to human 12p12-p13, for which there is evidence of linkage to both MS [39] and rheumatoid arthritis [40, 41]. For the QTL in the C4R3 region (Oia2, Cia13, Pia7), we provide original evidence of paradoxical disease regulation, since the QTL down-regulates arthritis but up-regulates neuroinflammation. An important task for the future is to identify and functionally characterize the disease-regulating genes, since this may enable development of rational strategies for disease treatment and prevention. The results described for C4R1 and C4R3 congenic strains represent an important step in this direction, and the unique strains represent a valuable tool for future studies.

Fig. 5. Genetic map of rat chromosome 4 (RNO4), aligned with congenic intervals, arthritis-linked QTL, and homologous human chromosome regions. The map contains mapped genes and microsatellite markers used for the construction of the congenic fragments and for the definition of their boundaries. Vertical black lines to the left indicate the chromosomal intervals for the congenic (C4) and recombinant (R1–R3) strains. To the right, the human syntenic regions and homologous genes known within these intervals are shown. Microsatellite marker positions in cM (from SHRSP×BN F2 intercross) and gene information were retrieved from http://rgd.mcw.edu/, http://www.ncbi.nlm.nih.gov/genemap99/, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM, and http://www.ncbi.nlm.nih.gov/genome/guide/human/

4 Materials and methods 4.1 Animals Inbred DA rats were originally derived from the Zentralinstitut für Versuchstierzucht, Hannover, Germany. MHCidentical PVG.1AV1 rats were originally obtained from Harlan

Eur. J. Immunol. 2003. 33: 1907–1916 UK Limited, (Oxon, GB). The genetics and characteristics of these rat strains have been described [42]. The congenic strains were produced by backcrossing for ten generations including the F1 generation, which is in agreement with the International Committee on standardized genetic nomenclature for mice and rat genome and nomenclature committee. For each generation, one rat, heterozygous for the PVG.1AV1 genotype in the interval between D4Rat155 and D4Mgh21, was selected for further backcrossing onto DA. This selective backcross breeding procedure was repeated for nine consecutive generations. Thereafter heterozygous (F1N10) littermates were intercrossed, and one male and one female offspring (F1N10F1) homozygous for PVG.1AV1 genes in the 70-cM C4 region were selected and used as founder animals for the DA.C4(PVG.1AV1) congenic strain. To produce sub-congenic strains, the founder was bred with DA rats to render heterozygous DA.C4(PVG.1AV1) animals. Sub-congenic animals stem from a cross between the F1N10 heterozygous DA.C4(PVG.1AV1) male and female DA rats to allow recombination, followed by intercrossing between heterozygous recombinant males and females for each C4 subregion. Three homozygous recombinant strains were used: DA.C4(PVG.1AV1) harbors PVG.1AV1 genes between the centromeric marker D4Rat155 and the telomeric marker D4Rat84, but it harbors DA genes at D4Mgh14, centromeric of D4Rat155. DA.C4R1(PVG.1AV1) harbors PVG.1AV1 genes in the interval D4Rat155 to SPR, but not outside of these boundary markers, at D4Mgh14 and D4Rat106, respectively. DA.C4R2(PVG.1AV1) is defined by SPR to D4Rat56, not Fabp1 and D4Rat141. DA.C4R3(PVG.1AV1) is defined by D4Rat63 to D4Rat203, not D4Rat137 and D4Mgh11. All animals were bred at the Biomedical Center in Uppsala or the Center of Molecular Medicine (CMM) in Stockholm, and were kept at CMM under specific pathogen-free conditions according to a health monitoring program for rats at the National Veterinary Institute in Uppsala. They were maintained in polystyrene cages containing aspen wood shavings, and had access to food and water ad libitum.

4.2 Genetic analysis Individual DNA was prepared from tailtips, and purified according to a standard protocol [43]. Genotypes were determined by PCR amplification of polymorphic simplesequences length polymorphisms as previously described, except [ + -33P]ATP was used to label one of the primers in each pair. The following genomic markers were used: D4Mgh21, D4Mit22, D4Mit27, D4Mgh11, D4Rat203, D4Rat66, D4Wox14, D4Rat63, D4Rat56, D4Rat57, D4Rat53, D4Rat106, D4Mgh17, D4Mit12, D4Rat35, D4Mit24, D4Mit6, D4Wox23, D4Wox24, D4Rat155, D4Rat141, D4Rat137 and D4Mgh14. Primers were purchased from Research Genetics (Huntsville, AL).

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4.3 Induction and clinical evaluation of EAE Rats between 8–11 weeks of age were anesthetized with halothane and immunized i.d. at the tail base. We immunized both female and male rats and in total between 17-20 rats per strain. Each rat received 200 ? l inoculum containing 15 ? g rMOG (aa1–125) [44], mixed with 100 ? l IFA (Sigma) and 100 ? l phosphate buffer-saline (Life Technologies). Animals were weighed and clinical signs of disease evaluated from day 7 to day 40 p.i. as follows: 1: tail weakness or tail paralysis; 2: hind leg para-paresis or hemi-paresis; 3: hind leg para-paralysis or hemi-paralysis; 4: tetraplegy, urinary and/or fecal incontinence. If balance disturbance and severe disease was observed for more than 1 day, the rat was sacrificed. The diagnosis of EAE was made only when a rat displayed clinical EAE signs for more than 1 day, and onset was calculated as the first day the clinical signs were observed. A relapsing/remitting disease was defined as a diseases course when the rats had remitted from maximum score 2–4 to 0 or 1, respectively (two scale units), for at least two consecutive days and then relapsed for at least 2 days with maximum score 2–4.

4.4 Histopathology Rats were perfused with buffered paraformaldehyde at day 40 p.i.; brain and spinal cord were dissected and routinely embedded in paraffin. Adjacent serial sections (2–4 mm thick) were cut on a microtome and stained with hematoxylin/eosin and Luxol fast blue to assess inflammation and demyelination, respectively. The inflammatory index was determined from the number of perivascular infiltrates of each animal on an average of 15 complete cross-sections of the spinal cord. The degree of demyelination was evaluated as follows: 0.5: traces of perivascular or subpial demyelination; 1: marked perivascular or subpial demyelination; 2: confluent perivascular or subpial demyelination; 3: massive confluent demyelination (e.g. half of spinal cord, one optic nerve complete); and 4: extensive demyelination (transverse myelitis, half of the cerebellar white matter or more, both optic nerves complete). Scores were determined for brain and spinal cord separately and then added, giving a maximum possible demyelination score of 8 per animal.

4.5 Anti-MOG IgG isotype determination Serum was sampled from each rat day 12 p.i. Anti-MOG IgG, IgG1, IgG2a, IgG2b and IgG2c for each rat was determined with ELISA [11]. ELISA plates (Nunc, Roskilde, Denmark) were coated with 100 ? l rat rMOG (aa1–125) diluted in 0.1 M NaHCO3 pH 8.2 at a concentration of 10 ? g/ml. The coated plates were stored overnight at 4°C. The sera for measuring IgG, IgG2a and IgG2b isotype levels were diluted 1:1,600 and the sera for IgG1 and IgG2c were diluted 1:160. Twofold dilution series were performed in PBS/1% milk powder. Antisera were diluted as follows: IgG, IgG2a and

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IgG2b, 1:2,000; IgG1, 1:1,000; IgG2c, 1:500 (Nordic). Goat anti-rabbit conjugate was diluted 1: 10,000 (Nordic). Bound antibodies were visualized through addition of 3,3’,5,5’tetrametyhlbenzidine (Sigma) and the enzymatic reaction was stopped with l M HCI. Optical density values were read at 450 nm. Each plate had a pooled serum in duplicates (with a high Ab titer) as a standard. This standard serum was diluted in twofold dilution series in eight steps. Arbitrary units were calculated with the standard curve for the standard serum as reference. 4.6 Lymphocyte proliferation assay Lymph nodes from rats immunized with rMOG were dissected day 12 p.i. and cells were prepared [45]. Proliferation experiments were performed in triplicates. Cells were diluted to 2×106 cells/ml media (5% FCS) and plated (100 ? l/well) in a U-bottom 96-well plate (Nunc) and stimulated with 5 ? g/ml MOG (aa1-125) pH 7.2, 5 ? g/ml MOG (aa91-108) pH 7.2, medium alone, or 3 ? g/ml Con A (Sigma) pH 7.2, respectively, and then cultured. The cells were cultured for 72 h at 37°C, 5% CO2. During the last 24 h of culturing, 1 ? Ci [3H]methylthymidine was added per well (Amersham International, GB). The cells were harvested on a filter and thymidine incorporation was measured using a g -counter. 4.7 Enumeration of antigen-specific cells secreting IFN- q Lymph nodes from rat immunized with rMOG were dissected day 12 p.i. Cells were prepared to a final concentration of 4×106 cells/ml and stimulated with 5 ? g/ml MOG (aa1-125) pH 7.2, 5 ? g/ml MOG (aa91-108) pH 7.2, medium alone, or 3 ? g/ml Con A (Sigma) pH 7.2, respectively. An Elispot method was used to enumerate T cells secreting IFN- + after antigen exposure [18–20]. Nitrocellulosebottomed 96-well plates (Millipore, MAHA N45, Bedford, MA) were coated with mAb DB1 (anti-rat IFN- + ; a generous gift from Dr. Peter van der Meide, TNO Primate Centre, Rijswik, The Netherlands). After blocking with Dulbecco’s modified Eagle’s medium containing 5% FCS (Life Technologies), we added triplicates of antigen and 4×105 cells in 200 ? l of culture media per well to the plates and incubated them for 48 h at 37°C in a humidified atmosphere, containing 5% CO2. Secreted and bound IFN- + was visualized with biotinylated mAb DB12 (anti-rat IFN- + , Dr. Peter van der Meide), avidin-biotin peroxidase (Vector Laboratories Inc., Burlingame, CA), and staining with carbazole (Sigma). 4.8 Statistical methods Data were analyzed using the Mann-Whitney U-test. The Fisher’s exact test was used to compare the strains for EAE incidence. All values were calculated using the PC version of JMP 4.0.2 (SAS Institute Inc., NC); p values p 0.05 were considered significant.

Eur. J. Immunol. 2003. 33: 1907–1916 Acknowledgements: We thank Associate Professor Robert Harris for linguistic advice. This study was supported by the Network for Inflammation Research funded by the Swedish Foundation for Strategic Research, and by grants from the Swedish Medical Research Council, the Swedish Rheumatism Association, the Swedish Foundation for Neurologically Disabled, King Gustav V 80th birthday Fund, Åke Wibergs Fund, Ulla and Gustav af Ugglas Fund, Magn. Bergvalls Foundation, Nanna Svartz Fund, Petrus and Augusta Hedlunds Foundation, Alex and Eva Wallström Fund, and the Montel Williams Foundation. J.C.L. is in receipt of a Fellowship from the Swedish Medical Research Council.

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Correspondence: Kristina Becanovic, Neuroimmunology Unit, CMM L8:04, KS, S-171 76 Stockholm, Sweden Fax: +46-8-517-76248 e-mail: Kristina.Becanovic — cmm.ki.se