Polygenic control of autoimmune peripheral nerve inflammation in rat

Journal of Neuroimmunology 119 Ž2001. 166–174 www.elsevier.comrlocaterjneuroim

Polygenic control of autoimmune peripheral nerve inflammation in rat I. Dahlman a , E. Wallstrom ¨ a, H. Jiao b, H. Luthman b, T. Olsson a, R. Weissert c,) a

b

Neuroimmunology Unit, Center of Molecular Medicine, Karolinska Hospital, S-17176, Stockholm, Sweden Karolinska Institute, Department of Molecular Medicine and Center of Molecular Medicine, Karolinska Hospital, S-17176, Stockholm, Sweden c Department of Neurology, UniÕersity of Tuebingen, Hoppe-Seyler-Strasse 3, D-72076, Tubingen, Germany ¨ Received 14 December 2000; received in revised form 17 May 2001; accepted 16 July 2001

Abstract Experimental autoimmune neuritis ŽEAN. is the principal animal model for Guillain–Barre´ syndrome ŽGBS., an inflammatory disease of the peripheral nervous system. Little is known on the genetic regulation of these diseases. We provide the first genetic linkage analysis of EAN. Susceptibility to EAN in a rat F2 population segregated with high levels of anti-PNM IgG, as well as IgG2b and IgG2c isotype levels, which support that disease genes regulate preferential Th1rTh2 differentiation. Linkage analysis demonstrated co-localization of EAN loci with reported susceptibility loci for experimental arthritis andror encephalomyelitis and a new region on chromosome 17. Further dissection of these loci may disclose disease pathways in GBS. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Autoimmunity; Gene; In vivo animal models; Immunoglobulin; Th1rTh2

1. Introduction Acute inflammatory demyelinating polyradiculoneuropathy, or the Guillain–Barre´ syndrome ŽGBS., is an inflammatory disease of the peripheral nervous system ŽPNS., which is clinically characterized by acute ascending paresis ŽRopper, 1992.. Current treatment modalities have only modest beneficial effects, mortality still occurs and many patients have residual neurological deficits. An autoimmune pathogenesis of GBS is supported by the presence of activated T cells ŽTaylor and Hughes, 1989; Hartung et al., 1990., PNS-specific antibodies ŽWiner et al., 1998; Hartung et al., 1993., and evidence for molecular mimicry between PNS antigens and infectious agents ŽKaldor and Speed, 1984; Yuki et al., 1990, 1993.. There are definite environmental triggers, since GBS is often preceded by infection ŽMishu et al., 1993; Jacobs et al., 1996; Winer et al., 1998.. However, so far, host factors that may determine disease susceptibility have not been found. Although scattered case reports on familial clustering have suggested a genetic component in the disease ŽWilmshurst et al., 1999., there is no unambiguous association with MHC haplotype ŽHillert et al., 1991., or any systematic studies on non-MHC gene influences as for many other organ) Corresponding author. Tel.: q49-7071-298-2141; fax: q49-7071600-137. E-mail address: [email protected] ŽR. Weissert..

specific inflammatory diseases. This can possibly be explained by the low incidence of disease, and consequently, small patient material available for analysis. Host factors may thus be more easily analyzed in proper experimental models for GBS. Experimental autoimmune neuritis ŽEAN. in rats is a model of GBS, which can be induced by active immunization using peripheral nervous system ŽPNS. antigens and ˚ ¨ and Waksman, 1962; Hughes Freund’s adjuvant ŽAstrom . et al., 1981 . Like GBS, EAN is characterized by ascending peripheral paresis with impaired nerve conduction, mononuclear inflammation, demyelination in the PNS, and increased serum levels of antibodies against peripheral nerve myelin ŽPNM. antigens ŽHahn, 1996.. Strain differences in susceptibility to EAN have demonstrated genetic influence on disease susceptibility ŽSteinman et al., 1981; Linington et al., 1986.. Dissection of such strain-dependent genetic factors by linkage analysis is important for a better understanding of the pathogenesis of EAN and the identification of new therapeutic targets ŽJacob et al., 1991.. Such linkage studies have been performed in other organ-specific inflammatory diseases in the rat and there are genome regions described which regulate arthritis ŽRemmers et al., 1996; Lorentzen et al., 1998; Vingsbo-Lundberg et al., 1998. and experimental autoimmune encephalomyelitis ŽEAE. ŽDahlman et al., 1999a,b; Roth et al., 1999; Bergsteinsdottir et al., 2000.. A further rationale for linkage analysis in EAN is to enable comparisons of disease

0165-5728r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 0 1 . 0 0 3 9 5 - 2

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regulating genome regions between EAN, EAE and arthritis due to differences in target organ pathology. Certain gene-regulated pathogenetic pathways may thus be shared, while others may be unique for a particular organ ŽVyse and Todd, 1996.. An overlap in disease susceptibility regions for different diseases may not necessarily imply sharing of disease linked genes, since a particular region can contain several disease-regulating genes ŽTeuscher et al., 1999.. However, further fine genetic and mechanistic dissections may resolve these issues, with important implications for the definition of therapeutic targets in different inflammatory diseases. We now report the first genetic linkage study of EAN by analysis of an F2 intercross between EAN susceptible DA rats ŽLorentzen et al., 1995. and resistant ACI rats. We demonstrate a strong co-segregation between IgG isotype levels and disease. We also identify susceptibility loci on chromosomes 10, 12, 15, 17, and 18; all except the chromosome 17 region have previously been reported in EAE andror arthritis in DA rats.

2. Materials and methods 2.1. Rats DA rat breeding pairs were originally obtained from the Zentralinstitut for Versuchstierzucht ŽHannover, Germany., and ACI rat breeding pairs from Harlan Sprague–Dawley ŽIndianapolis, USA.. DA and ACI rats have identical serological MHC haplotypes. A reciprocal ŽDA = ACI. F2 intercross was established. The rats were routinely tested for specific pathogens according to a health-monitoring program for rats at the National Veterinary Institute in Uppsala, Sweden. The rats were housed in polystyrene cages containing aspen wood shavings and provided food and water ad libitum. 2.2. Induction and clinical assessment of EAN PNM was prepared from bovine peripheral nervous tissue ŽKadlubowski et al., 1980.. Rats, 8–15 weeks of age, were anaesthetized with ether and injected intradermally at the base of the tail with 200 ml inoculum, containing 500 mg bovine PNM in 100 ml 0.9% NaCl mixed with 100 ml incomplete Freund’s adjuvant ŽIFA, Difco, Detroit, MI.. All rats were immunized at the same session. The experiment was approved by the local ethical committee, Stockholms norra forsoksdjuretiska namnd. ¨ ¨ ¨ Rats were blindly examined for signs of EAN and weighed daily from day 7 post-immunization Žp.i.. until sacrifice at day 25 p.i. The clinical scoring was as follows: 0 s no illness, 1 s limp tail, 2 s unsteady gait, slight paraparesis, and 3 s severe paraparesis. No animal developed tetraparesis or breathing disturbance. An index of maximum weight loss was calculated as Žweight at immuniza-

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tion y lowest weight after immunization.rweight at immunization. 2.3. Immunohistochemistry To determine the degree of inflammation in peripheral nerves, MHC class II expression was measured by immunohistochemistry ŽStrigard et al., 1987.. An approximately 10-mm-long segment of the right and left sciatic nerves between the sciatic nitch and the knee was excised and snap-frozen in liquid nitrogen. One cryostat section from each nerve was melted onto microscope slides and incubated with the Ox-6 monoclonal antibody to rat MHC class II RT1.B antigen ŽMcMaster and Williams, 1979.. Ox-6 was purified from culture supernatants of hybridomas ŽWeissert et al., 1998a.. The avidin–biotin peroxidase method was used for staining ŽABC vectastatin Elite kit, Vector lab, Burlingame, CA, USA.. Omission of the primary Ab served as negative control. Sections of peripheral lymphoid tissue served as positive control. Ox-6 stained nerve sections were semiquantitatively scored as follows: 0 s no stained cells, 1 s one to two stained cells, 2 s three to four stained cells, 3 s five to nine stained cells, and 4 s more than 10 stained cells per visual field at a magnification of 200 = . The nerve sections were scored without access to genotype data and a random subset was doublechecked. 2.4. Anti-PNM IgG-subclass determination Sera for antibody determination were sampled on day 12 p.i. by tail-bleeding. We have performed kinetic studies in rat EAE and EAN serum samples, which have shown that at day 12 p.i., isotype switching has occurred and genetic differences affecting isotypes appear strongest. At later time points, differences between strains leveled out. ELISA plates ŽNunc, Roskilde, Denmark. were coated with homogenized PNM suspended in 0.1 M NaHCO 3 Ž2 mgrml. Ž100 mlr well. overnight at 4 8C. Plates were washed with PBSr0.05% Tween 20 and free-binding sites were blocked with 5% skimmed milk in PBSr0.05% Tween 20 for 1 h at room temperature ŽRT.. After washing, diluted serum samples were added and plates were incubated for 1 h at RT. Plates were then washed, and diluted rabbit a-rat IgG, a-rat IgG1, a-rat IgG2a, a-rat IgG2b or a-rat IgG2c ŽNordic, Tilburg, Netherlands. were added and incubated for 1 h at RT. Subsequently, unbound antibodies were removed by washing prior to addition of a peroxidase-conjugated goat-anti-rabbit antiserum ŽNordic. diluted into PBSr0.05% Tween 20 Ž1:10000.. After 30 min, plates were washed thoroughly and bound antibodies were visualized by addition of 3,3X ,5,5X-tetramethylbenzidine ŽTMB; Sigma.. The enzymatic reaction was stopped with 1 M HCl after 15 min incubation in darkness. The optical density was recorded at 450 nm.

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2.5. Genotype analysis Genomic DNA was prepared from tail tips according to a standard protocol ŽLaird et al., 1991.. Microsatellite markers were used for genotyping, using the polymerase chain reaction essentially as previously described ŽJacob et al., 1991., except that primers were end-labeled with 33 PgATP. Primers were obtained from Research Genetics, Huntsville, AL, USA or from GENSET, Paris, France. The PCR products were size-fractionated on 6% polyacrylamide gels and visualized by autoradiography. All genotypes were scored manually and double-checked. The MAPMAKER computer package ŽLander et al., 1987. was employed for assembling the markers into a genetic linkage map of the ŽDA = ACI. F2 intercross. 2.6. Statistical analysis The Pearson chi-square test statistic was used to determine whether the observed genotype distribution at individual markers among the 46 most severely EAN affected rats significantly deviated from the expected DrD:Dr A:ArA frequencies of 0.25:0.5:0.25. The same test was used to analyze if there was a significant difference in observed genotype distribution between affected rats, i.e. with positive clinical score, and non-affected rats. The Kruskal–Wallis test was used to determine whether different genotypes differed with regard to MHC class II expression in the sciatic nerves and, among rats with positive clinical scores, differences in maximum score. The Kaplan–Meier method was used to analyze differences in day of onset between genotypes among rats with positive clinical scores. Linkage analysis of anti-PNM antibodylevels was performed with interval mapping using the MAPMAKERrQTL computer package ŽLander et al., 1987.. Two-tailed tests were used consistently.

3. Results 3.1. Disease characteristics in parental and F2 rats Six out of twelve DA rats Ž50%. developed clinical signs of EAN compared with none out of five ACI rats. In affected rats, there were classical signs of EAN with

Fig. 1. MHC class II expression in sciatic nerves. The percentage ŽDA=ACI. F2 rats with different degrees of MHC class II infiltration in the sciatic nerves are shown separately for rats developing clinical signs of EAN Ž N s 52. and resistant rats Ž N s135., respectively. For procedures see Materials and Methods.

ascending paraparesis, followed by a recovery phase. Of 187 ŽDA = ACI. F2 rats, 52 rats Ž27.5%. displayed monophasic clinical neurological deficits. In the affected F2 rats, the mean day of onset was day 16 ŽSD " 4. and the mean number of consecutive days with positive scores was nine ŽSD " 4. ŽTable 1.. The incidence of EAN in male F2 rats was 28r97 Ž29%. and in female F2 rats 24r90 Ž27%.. The incidence of EAN in the F2 population did not differ between reciprocal crosses. T cells wane from the PNS quite rapidly following the acute paralytic phase, while inflammation-induced MHC class II expression on cells in the endoneurium and infiltrating MHC class II expressing cells persist for a longer time ŽStrigard et al., 1987.. The degree of class II expression at late time points after the acute episode therefore gives an indication of the degree of inflammation in the PNS. MHC class II expression on macrophage-like cells scattered throughout the endoneurium were observed with a concentration close to vessels symmetrically on both sides Ždata not shown.. The degree of MHC class II expression assessed semiquantitatively correlated only partly to degree of neurological deficits. Thus, rats with

Table 1 Disease distribution in parental and F2 ŽDA = ACI. rats

DA ACI F2 ŽDA = ACI. F2 ŽDA = ACI. a

Among affected rats.

Sex

Ž%. Affected

Maximum score Žmedian" range. a

Onset Žm " SD. a

Duration Žm " SD. a

female female female male

50 Ž6r12. 0 Ž0r5. 27 Ž24r90. 29 Ž28r97.

2 Ž1–3. – 2 Ž1–3. 2 Ž1–3.

14.3 " 1.5 – 16.1 " 4.1 16.7 " 3.9

10.0 " 2.7 – 9.0 " 4.1 7.9 " 3.7

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high MHC class II expression were overrepresented among rats displaying clinical EAN signs. However, there were many rats that had displayed clinical signs of EAN and had a low score for class II expression. Furthermore, there were rats with no clinical signs of EAN, which displayed a significant MHC class II expression ŽFig. 1.. Serum anti-PNM antibodies were measured as a third phenotypic out read. Numbers of investigated DA and ACI founder rats were too low to statistically evaluate differences in serum anti-PNM Ig levels between the strains. There was a highly significant difference in serum antiPNM IgG, IgG2a, IgG2b and IgG2c, but not IgG1 levels between ŽDA = ACI. F2 rats with paresis and rats without any clinical signs of EAN ŽFig. 2.. 3.2. Linkage analysis Forty-six rats with the highest cumulative scores, i.e. sum of clinical score for paresis from day 7 until day 25

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p.i., were selected for a systematic genome-wide linkage analysis of EAN phenotypes. These rats were genotyped with 138 markers. The genotype at each marker was determined as DA homozygous ŽDrD., heterozygous ŽDrA. or ACI homozygous ŽArA.. The linkage map and 10 unlinked markers covered 1350 cM with a range of 10 cM on each side for individual markers. We were not able to get a higher chromosomal coverage due to lack of informative markers. We selected markers from the MIT http:rrwaldo.wi.mit.edurratrpublicr. and Wellcome Trust http:rrwww.well.ox.ac.ukr rat – mapping – resourcesr. Ratmap. About 10% of markers tested at random in our laboratory were informative between the DA and ACI strains, and only approximately 65% of the markers that should be informative according to the MIT Ratmap turned out to be informative between our strains. For 13 regions with P - 0.10 for linkage to a clinical phenotype ŽEAN affected, disease onset, or sever-

Fig. 2. A–E. Anti-PNM IgG determination. Sera were sampled from ŽDA = ACI. F2 Ž N s 187. and from parental strain rats ŽACI n s 5; DA n s 12. on day 12 p.i. with PNM. Anti-PNM IgG and the four IgG subclasses were measured. The following serum dilutions were used: IgG 1:2000, IgG1 1:200, IgG2a 1:2000, IgG2b 1:2000 and IgG2c 1:200. Dots represent OD450nm values for individual serum samples. Differences in IgG levels between rats developing clinical signs of EAE Ž N s 52. and resistant rats Ž N s 135. were analyzed with ANOVA Ž2 df .. Ž ) . Indicates P - 1 = 10y8 . For procedures see Materials and Methods.

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ity., all 187 rats were genotyped with additional markers covering these regions. A nominal P-value of less than 5.2 = 10y5 has been reported to represent a 5% genome-wide significance level in most studies of F2 intercrosses ŽLander and Kruglyak, 1995.. When evaluating support for linkage in particular regions, we have considered both nominal P-values and support for linkage in other studies. A nominal P-value of less than 0.01 has been suggested when confirming linkage in independent studies ŽLander and Kruglyak, 1995.. We have not adjusted the threshold due to analysis of multiple phenotypes, since the phenotypes are dependent on one another and this covariation is difficult to quantify.

Among 187 ŽDA = ACI. F2 rats, 11 chromosomal regions with nominal P - 0.05 in support of linkage to clinical phenotypes of EAN, PNS-inflammation, or serum anti-PNM IgG levels were identified ŽTables 2 and 3.. The regions on chromosomes 10, 12, 15, and 18 have previously been shown to be linked to other models of autoimmune diseases in DA rats ŽRemmers et al., 1996; Lorentzen et al., 1998; Vingsbo-Lundberg et al., 1998; Kawahito et al., 1998; Dahlman et al., 1998, 1999a,b; Bergsteinsdottir et al., 2000.. Since they confirmed already published disease loci, the regions can be considered as EAN-linked loci, although none of these regions fulfilled the strict criteria for significant genome-wide linkage in our study

Table 2 Linkage analysis in 187 ŽDA = ACI. F2 rats a RNO b

Marker

cM

Penetrance Ž%. c

2

D2Mgh15 D2Rat77 D2Mit18 D2Rat21 D2Rat16 SCPIIR D3Rat40 D4Rat43 D5Rat41 D6Rat47 D10Rat7 D10Mgh3 D10Rat15 R265 D10Rat55 D10Rat219 D10Rat29 VAMPB D12Rat24 D12Rat23 R1390 D15Rat6 D15Rat13 D15Mgh2 D15Rat23 D17Rat99 D17Rat46 D17Rat36 D17Rat32 D17Mit3 D18Rat5 D18Mit6 D18Mgh4 D18Rat10

3 4 5 6 10

12

15

17

18

a

Score Ž P . e

Onset Ž P . f

0.032 0.024 0.0071 0.024

0.0048 0.0099

RT1.B Ž P . g

Disease allele h

Ž P .d

21.6 10.4 15.5

D 0.036

5.1 12.2

11:35:41 5:16:25

0.0067 0.032 0.030

20.1

42:21:23

0.031

8.0 3.7 2.3 1.2

40:30:10 36:31:12 42:26:11 38:28:5 35:30:08 40:30:12 42:32:10 43:30:13

0.0091 0.042 0.0038 0.0024 0.0096 0.013 0.0047 0.019

0.032

0.0086

3.8 20.6 12.8 2.3

A

D

0.034

1.3 0.0 23.5

0.050 0.0048 0.0026 0.0059 0.036 0.018 0.028 0.012 0.067

D 0.0065 0.036 0.038

0.023 8.2 2.9 2.9 0.1 3.7 4.6 18.4 1.2

A D

0.036 0.016 0.032 0.060 0.037 0.0063 0.0075 0.042 0.065 0.0063 0.014 0.0023 0.017

D

D 0.056

D D

0.058 0.047 0.056 D 0.053

Markers in regions with P - 0.05 in support of linkage to a phenotype are shown. Rat chromosome number. c Percentage of rats developing paresis of all rats with genotype DrD:DrA:ArA. mean " SD of genotypess 178 " 4.5. d The chi-square test was used to analyze differences in genotype frequencies between rats with positive clinical score and non-affected rats. e Among affected rats, differences in maximum score between different genotypes were analyzed using the Kruskal–Wallis test. f Among affected rats, differences in disease onset between different genotypes were analyzed with the Kaplan–Maier method calculating the Wilcoxon test statistic. g Differences in degree of MHC class II RT1.B expression in the sciatic nerves among rats were analyzed with the Kruskal–Wallis test. h D indicates DA allele, A indicates ACI allele. b

I. Dahlman et al.r Journal of Neuroimmunology 119 (2001) 166–174 Table 3 Linkage analysis of serum a-PNM IgG levels Chromosome interval

Phenotype

Subgroup

LOD

D15Rat6–D15Rat13

a-PNM IgG2b

all female founder DA female founder ACI all male female all male female all affected unaffected all affected unaffected all affected unaffected all all all

3.00 3.12 1.23 – 3.02 0.07 2.18 2.62 0.28 2.70 4.19 0.39 2.41 3.03 0.82 3.05 2.45 0.71 2.85 2.28 3.01

a-PNM IgG1

D15Rat23–D15Rat68

D17Rat99–D17Rat40

a-PNM IgG2b

a-PNM IgG

a-PNM IgG2a

a-PNM IgG2b

D17Rat46–D17Mit6

a-PNM IgG a-PNM IgG2a a-PNM IgG2b

ŽLander and Kruglyak, 1995.. The region on chromosome 10 and a new region on chromosome 3 were linked to EAN susceptibility. The region on chromosome 12 was linked to inflammation in sciatic nerves. The chromosome 15 region was linked to disease onset and anti-PNM IgG2b levels. Interestingly, the linkage to IgG2b levels was much stronger in the cross with a DA female founder and among male rats. The region on chromosome 18 was linked to disease onset and severity. A new region showing linkage to EAN phenotypes was identified on chromosome 17. This region showed suggestive linkage to maximum score, disease onset, and serum levels of PNM IgG, IgG2a and

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IgG1. Linkage to IgG levels was genome-wide significant among EAN affected rats, but displayed only weak linkage among unaffected rats ŽTable 3 and Fig. 3.. The marker D20Mit4, located close to or in the MHC, showed no evidence of linkage to EAN susceptibility in all 187 investigated rats.

4. Discussion In the first genetic linkage analysis of EAN in a ŽDA = ACI. F2 population, we have identified susceptibility loci for EAN on rat chromosomes 10, 12, 15, 17 and 18. Furthermore, we have demonstrated that EAN susceptibility segregates with levels of anti-PNM IgG isotype antibodies. In the rat, Th1 cells induce switching to IgG2b and IgG2c ŽGracie and Bradley, 1996.. The higher levels of anti-PNM IgG2b and IgG2c among EAN-susceptible F2 rats therefore support previous observations that EAN is a Th1-mediated disease ŽZhu et al., 1994.. Since we demonstrate genetic linkage of certain chromosomal regions to IgG isotype patterns in relation to disease susceptibility, we suggest that some of the susceptibility genes for EAN regulate Th1rTh2 differentiation. We are currently investigating this hypothesis in congenic strains for identified loci In a wide variety of autoimmune disease models, disease-causing T cells are of the Th1 type, such as experimental autoimmune encephalitis ŽSegal and Shevach, 1996; Weissert et al., 1998b., experimental autoimmune myasthenia gravis ŽBalasa et al., 1993., experimental uveitis ŽCaspi et al., 1996., and experimental type 1 diabetes ŽZekzer et al., 1998.. Genes regulating Th1rTh2 differentiation might therefore predispose for several different

Fig. 3. Anti-PNM IgG LOD score curve for chromosome 17. Sera were sampled from ŽDA = ACI. F2 Ž N s 187. rats on day 12 p.i. with PNM. Anti-PNM IgG levels were measured using a serum dilution of IgG 1:2000. Linkage analysis of anti-PNM levels was performed for EAN Ž N s 52. affected and resistant rats Ž N s 135. separately using MapmakerrQTL with a free mode of inheritance. For procedures see Materials and Methods.

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experimental autoimmune diseases. In agreement with this hypothesis, the identified regions on chromosome 10 ŽRemmers et al., 1996; Lorentzen et al., 1998; Dahlman et al., 1999b., chromosome 12 ŽVingsbo-Lundberg et al., 1998; Dahlman et al., 1999a; Bergsteinsdottir et al., 2000., chromosome 15 ŽKawahito et al., 1998., and chromosome 18 ŽDahlman et al., 1999b. have previously been linked to arthritis or EAE susceptibility in DA rats ŽTable 4.. Our findings confirm that there are chromosomal regions regulating general susceptibility to different autoimmune diseases ŽVyse and Todd, 1996; Dahlman et al., 1998; Becker et al., 1998; Sun et al., 1999.. Recently, Merriman et al., Ž2001. found evidence for association of a human chromosome 18 region and its orthologe on rat and mouse chromosomes to several autoimmune diseases underscoring the great potential for elucidating human disease genes in experimental disease models. In strong support for the usefulness of animal models for the mapping of human susceptibility genes is also the reported orthologeous gene locus in mouse EAE ŽSundvall et al., 1995. and a locus conferring susceptibility in the Finnish multiple sclerosis population ŽKuokkanen et al., 1996.. The reported EAN regions on chromosomes 10 and 12 are orthologeous to human regions that have also been linked to autoimmune disease Žsee comparative map at http:rr www.otsuka.genome.ad.jp r cgi-bin r comparative – home. pl. ŽBecker et al., 1998.. These observations strengthen the relevance of the EAN data for GBS.

Table 4 Comparison of EAN susceptibility loci with other autoimmune disease loci in rats RNO a

Disease modelb

Intercross

10

CIA ŽRemmers et al., 1996. OIA ŽLorentzen et al., 1998. MOG–EAE ŽDahlman et al., 1999a,b. EAN Žthis study. PIA ŽVingsbo-Lundberg et al., 1998. SC–EAE ŽDahlman et al., 1999a,b. CIA ŽWilder et al., 1999. MOG–EAE ŽDahlman et al., 1999a,b. EAU ŽSun et al., 1999. SC–EAE ŽBergsteindottir et al., 2000. EAN Žthis study. AIA ŽKawahito et al., 1998. EAN Žthis study. EAN Žthis study. MOG–EAE ŽDahlman et al., 1999a,b. EAN Žthis study.

DA=F344 DA=LEW.1AV1 DA=ACI DA=ACI DA=E3 DA=BN DA=BN DA=ACI LEW=F344 DA=E3 DA=ACI DA=F344 DA=ACI DA=ACI DA=ACI DA=ACI

12

15 17 18

All studies have been performed in F2 ŽDA=ACI. intercross rats. a Rat chromosome number. b Loci linked to susceptibility or component phenotypes of: AIA s adjuvant-induced arthritis, CIA s collagen-induced arthritis, EAN s experimental autoimmune neuritis, EAUsexperimental autoimmune uveitis, MOG–EAE s myelin–oligodendrocyte-glycoprotein-induced EAE, OIA s oil-induced arthritis, PIA s pristane-induced arthritis, SC– EAEsspinal cord-induced EAE.

Similar to other organ-specific autoimmune disease, the overall genetic regulation of EAN in the F2 generation is compatible with a polygenic threshold model ŽLeiter et al., 1998.. Thus, no locus was necessary for EAN. Genetic heterogeneity was further supported by the modest drop in incidence of EAN from 50% in the DA rats to 25% in the F2 rats. In addition, specific disease-linked loci were dependent on sex andror female founder, demonstrating the importance of genetic and possible epigenetic interactions in genetic regulation of EAN. Besides loci that seem to be involved in susceptibility to many autoimmune diseases, we have found the strongest linkage of EAN phenotypes to a new region on chromosome 17, which, so far, has not been found in other autoimmune disease models. This locus might therefore be specific for regulation of autoimmune disease in the peripheral nervous system. The linkage to antibody levels has subsequently been confirmed by congenic mapping ŽDahlman et al., unpublished.. Currently, we are investigating candidate genes in this region to better define its functional influence on EAN. There was only a weak correlation between the degree of MHC class II expression in sciatic nerves and the degree of neurological deficits in our study. There are several possible reasons for this, for example: Ž1. some rats may have had neurological deficits Žfor example, sensory disturbances. not assessed in the scoring; Ž2. neurological deficits and the degree of inflammation may not reflect converging phenotypes; and Ž3. inflammation may have been present in nerve roots not histologically examined. Interestingly, genomic loci conferring EAN susceptibility showed linkage to either or both phenotypes, suggesting that they may partly be differentially regulated. Our data supports that susceptibility to EAN is regulated by multiple genes, some of which are immunoregulatory and shared between different autoimmune diseases. Identification of susceptibility genes for peripheral autoimmune neuropathy might give clues to the etiology and to critical steps in the pathogenesis of GBS. Although GBS is a self-limiting disease, long-term disability is common. Therefore, the primary objective with further genetic and functional dissection of identified susceptibility loci for EAN is to develop more rational ways to prevent and treat GBS.

Acknowledgements This study has received grant support from the EU biomed 2 program ŽContract No BMH4-97-202., the Swedish Medical Research Council, the Swedish Society for Neurologically Disabled, the AFA Foundation, Petrus and Augusta Hedlunds Stiftelse and the Deutsche Forschungsgemeinschaft ŽWe 1947r1-1 and 2-1..

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