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*
Department of Neuroimmunology, Max-Planck Institute for Neurobiology, Martinsried, Germany;
Institute of Neurology, University of Vienna, Vienna, Austria; and
Clinic of Abdominal and Transplantation Surgery, Department of Surgery, Medical School Hanover, Hannover, Germany
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism by which MHC genes modulate susceptibility to autoimmune diseases is generally discussed in terms of the effects of class I and II MHC alleles on the selection, activation, or effector function of the T cell repertoire (2, 5, 6). However, this interpretation is derived almost solely from EAE models in which disease is T cell mediated (2) and is independent of the B cell response (7). In contrast, human diseases such as multiple sclerosis (MS) are generally far more complex in that both cellular and humoral immune effector mechanisms are involved in disease pathogenesis (8, 9, 10, 11, 12).
This complex immunopathology is reproduced in rats with EAE induced by active immunization with the myelin oligodendrocyte glycoprotein (MOG) (13, 14, 15). MOG is a unique myelin autoantigen in that it induces both an encephalitogenic T cell response and a demyelinating autoantibody response in rodents and primates. In the LEW rat, the T cell response to MOG is only weakly encephalitogenic (16), and disease induction exhibits an absolute requirement for the MOG-specific autoantibody response (13, 16). The formation of demyelinating lesions depends on synergy between the MOG-specific T cell and autoantibody responses (13, 16). The T cell response initiates a subclinical inflammatory reaction in the CNS, disrupting the blood-brain barrier and allowing Ab to enter the CNS compartment. Activation of the complement cascade by MOG-specific Ab bound to the myelin surface then initiates demyelination while at the same time enhancing the local inflammatory response through the production of proinflammatory factors.
These observations led us to suspect that genetic susceptibility to MOG-induced EAE may be independent of MHC effects on the T cell response. We therefore investigated the susceptibility of the BN rat to MOG-induced EAE, given that this rat strain exhibits a generalized resistance to many purely T cell-mediated autoimmune diseases (1, 17, 18) but is susceptible to autoantibody-mediated diseases (19, 20). Surprisingly, despite its pronounced resistance to other models of EAE (1, 3), active immunization with the extracellular Ig-V-like domain of MOG (13) in CFA induced a lethal demyelinating disease of the CNS. We demonstrate that clinical disease was mediated by a high titer MOG-specific autoantibody response that overwhelmed the protective capacity of the BN genotype that suppresses disease induction by purely T cell-mediated effector mechanisms. Analysis of congenic strains either carrying the BN MHC haplotype (RT1n) on the LEW genetic background (LEW.1N) or the MHC haplotype of LEW (RT1l) on the BN background (BN.1L) confirmed that the severity and clinical course of MOG-induced EAE were modulated by an MHC or MHC-linked effect on the anti-MOG Ab response, rather than a direct effect on the pathogenicity of the encephalitogenic T cell response. This response pattern is determined by genes located within both the centromeric part of the MHC including RT1.A–RT1.C and the RT1.M region located telomeric of RT1.C, as demonstrated by the intra-MHC recombinant LEW.1R38 rat strain. MOG-induced EAE is therefore an Ab-mediated, T cell-dependent autoimmune disease of the nervous system in which the B cell rather than the T cell response can determine disease susceptibility. Moreover, both MHC and MHC-linked effects influence this B cell response and thereby modulate disease penetrance and severity. These observations suggest that the functional significance of disease-associated MHC alleles in MS should be reassessed in the context of the MOG-specific Ab response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BN (RT1n) rats (120–200 g) were obtained from Charles River (Sulzfeld, Germany), congenic LEW.1N (RT1n) and BN.1L (RT1l) rats were provided by Prof. H. J. Hedrich (Medizinsche Hochschule Hannover, Germany), and LEW rats were obtained from the animal facility of the Max-Planck Institute for Biochemistry (Martinsried, Germany). For the BN.1L strain, a small deletion in the RT1.C region has previously been demonstrated; therefore its MHC haplotype is also classified as RT1lm3 (21). The LEW.1R38 (RT1r38) strain has been developed from the LxB17L recombinant inbred strain (22) by serial back-crossing on the LEW background and is maintained in the rat colony of one of the authors (K.W.).
CFA, IFA, and heat-killed Mycobacterium tuberculosis (H37Ra)
were purchased from Difco (Detroit, MI). Purified protein derivative
was purchased from Statens Serum Institut, Copenhagen, Denmark.
Recombinant protein (MOG) corresponding to the Ig-domain (Igd) of rat
MOG (aa 1–125) was expressed in Escherichia coli and
purified to homogeneity (13). The purified protein
dissolved in 6 M urea was dialyzed against 20 mM sodium acetate buffer
(pH 3.0) to obtain a soluble preparation that was stored frozen at
-20°C. The following murine mAbs were used: W3/25 (CD4); OX22
(CD45RC); R73 (rat 
ß TCR); W3/25 was purified by protein G
affinity chromatography from the culture supernatants of the
appropriate hybridoma cell lines. OX22 and R73 were purchased from
Camon, Wiesbaden, Germany.
Immunization protocols
Rats were immunized s.c. at the base of the tail with 50 µg MOG emulsified in CFA containing 225 µg of heat-killed M. tuberculosis (H37Ra) in a total volume of 100 µl. Animals were weighed and examined daily for clinical signs of EAE that were scored on the following scale: 0.5, partial loss of tail tone; 1, complete tail atony; 2, hind limb weakness; 3, hind limb paralysis; 4, moribund; 5, dead.
Generation of T cell lines and adoptive transfer experiments
MOG-specific T cell lines were generated as described previously (13). Briefly, the draining lymph nodes were removed 10 days postimmunization (dpi), and a single-cell suspension was cultured for 72 h at a concentration of 107 cells/ml in DMEM supplemented with glutamine, penicillin-streptomycin, sodium pyruvate, essential amino acids (Life Technologies), and 1% rat serum in the presence of the selecting Ag (20 µg/ml). T cell blasts were then isolated by density gradient centrifugation and propagated for a further 5–10 days in medium containing 15% supernatant of MLA cells (T cell growth factor (23)). Ag-specific T cell lines were subsequently maintained by cycles of Ag-specific restimulation using irradiated (5000 rad) syngeneic thymus cells as APCs, followed by expansion in T cell growth factor.
Adoptive transfer experiments were performed using freshly activated T cell blasts suspended in a volume of 1 ml DMEM. T cells were injected into the tail vein of rats anesthetized with ether. In some experiments, rats were subsequently injected i.v. with 100 mg/kg rat IgG purified by sodium sulfate precipitation from the pooled sera of rats immunized 11 days earlier with either CFA-buffer or CFA-MOG.
Enzyme-linked immunosorbent assay
Blood was collected at the times stated in the text by cardiac puncture immediately before perfusion. After clotting at 4°C, serum was collected by centrifugation and stored at -20°C. ELISA was performed with polystyrene 96-well PVC plates (Costar, Cambridge, MA) coated with 5 µg/ml Ag (3 h, 37°C) in 50 mM carbonate-bicarbonate buffer, pH 9.6. The plates were washed with PBS (0.05% Tween 20, Sigma, Deisenhofen, Germany) and blocked with 1% BSA in PBS (pH 7.4) overnight at 4°C. After washing with PBS-Tween, 100 µl of serial serum dilutions in PBS were incubated for 4 h at 37°C. Total anti-MOG levels were determined directly using 100 µl peroxidase-conjugated rat IgG and IgM-specific goat Ab (1:4000). Isotype-specific anti-MOG Ab levels were determined using 1:4000 dilutions of a panel of mouse mAbs specific for rat IgM, IgG1, Ig2a (Serotec, Oxford, U.K.) followed by a mouse-specific peroxidase conjugate (1:8000 in PBS, Dianova, Hamburg, Germany). To determine the levels of IgE, 2% dried milk powder in PBS was used as a blocking agent. Furthermore, the samples and a goat serum specific for rat IgE (1:5000) (Dunn, Asbach, Germany) as well as a HRP-conjugated donkey anti-goat serum (1:2000, Dianova) were diluted in PBS containing 0.1% milk powder. All plates were developed with o-phenylenediamine dihydrochloride (Sigma), the reaction was stopped with 3 M HCl, and optical density was determined at 490 nm.
Cytokine, proliferation, and RT-PCR assays
Proliferation assays were performed in flat-bottom 96-well
tissue culture plates in a total volume of 200 µl using either 5
x 105 lymph node cells (LNCs) or 2 x
104 T cells plus 5 x
105 syngeneic, irradiated (5000 rad) thymus cells
as APCs. Ag-specific proliferation was assessed by the incorporation of
[3H]thymidine (10 µCi/well) during the final
16 h of a 72-h culture period using a Packard Matrix 96 Direct ß
counter. After 72 h of production of IFN-
, TNF-, IL-10,
and IL-4 was determined with commercial ELISA assays (Laboserv,
Staufenberg, Germany) in cell culture supernatants.
IL-4 mRNA was analyzed in LNCs after 24 or 72 h of in vitro culture in the presence or absence of Ag. Total RNA was extracted from 5 x 106 cells, and 1 µg was reversely transcribed into cDNA (Superscript, Life Technologies, Gaithersburg, MD). Fifty nanograms of cDNA served as a template in PCR (annealing, 60°C, 45 s; elongation, 72°C, 45 s; denaturation, 94°C, 30 s; 35 cycles). The primers were designed to amplify fragments of 700 bp for ß-actin and 177 bp for IL-4. ß-Actin forward, 5'-TGC TAG GAG CCA GGG CAG TAA TC-3'; ß-actin reverse, 5'-TAC AAT GAG CTG CGT GTG GCC-3'. IL-4, commercially available primer-pair (Laboserv, Staufenberg, Germany).
Histopathological analysis
Histological evaluation was performed on paraformaldehyde-fixed, paraffin-embedded sections of brains, and spinal cords were sampled at various time points of disease. Paraffin sections were stained with hematoxylin-eosin, Luxol fast blue, and Bielschowsky silver impregnation to assess inflammation, demyelination, and axonal pathology. In adjacent serial sections, immunohistochemistry was performed with Abs against following targets: macrophages/activated microglia (ED1; Serotec), T-cells (W3/13; Sera-Lab, Crawley Down, U.K.), C9 (24), rat Ig (biotinylated anti-rat, Amersham, Little Chalfont, U.K.). Bound primary Ab was detected with a biotin-avidin technique previously described in detail (25). Control sections were incubated in the absence of primary Ab or with nonimmune rabbit serum.
The following histopathological parameters were evaluated: 1) inflammatory index: the mean number of perivascular inflammatory infiltrates derived from an average of 15 complete cross-sections of the spinal cord of an individual animal; 2) demyelination: demyelination in the spinal cord according to the following grading system: traces of perivascular or subpial demyelination (0.5), marked perivascular or subpial demyelination (1), confluent perivascular or subpial demyelination (2), massive confluent demyelination (e.g., one-half the cross-section of spinal cord) (3), extensive demyelination (transverse myelitis) (4).
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Results |
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Despite their resistance to EAE induced by active immunization
with either myelin basic protein (MBP) or spinal cord tissue (1, 3, 4), we observed that BN rats were highly susceptible to EAE
induced by sensitization with rMOG in CFA (Table I
). Immunization with 50 µg MOG
resulted in severe clinical disease, which progressed rapidly with
virtually all animals reaching a clinical score of 
3 within 48 h
of onset. In comparison, the incidence of MOG-induced disease was lower
and disease onset and progression were delayed in the
"EAE-susceptible" LEW rat (Table I).
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, Table II). The demyelinating lesions increased
in size between days 10 and 14 postimmunization (pi) and were
associated with the local deposition of C9 and IgG (Fig. 1D). The cellular infiltrate associated with these lesions
contained large numbers of polymorphonuclear cells (PMN), together with
fewer macrophages and T cells (Fig. 1, A, B,
E, and F). Most striking, and atypical for rat
EAE, was the presence of many eosinophils in the inflammatory
infiltrate, which accounted for 15–20% of inflammatory cells invading
the CNS (Fig. 1, A and B). In contrast,
ED1+ macrophages were the dominant cell type in
the cellular infiltrate in the LEW rat, no eosinophils were present,
and only a small number of PMNs were observed in the demyelinating
lesions (Fig. 2A,
B, D, and E; Table II).
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The pathogenicity of the autoimmune response to MOG in the BN rat was surprising because this rat strain is resistant to a wide range of Th1 T cell-mediated autoimmune diseases of which EAE is the prototypic example. However, the presence of large numbers of PMNs and eosinophils in the CNS was reminiscent of the histopathology induced by autoaggressive Th2, rather than Th1 T cell responses in the mouse (26), raising the possibility that a Th2 T cell response to MOG was responsible for initiating clinical disease. We therefore analyzed the primary response to MOG to define the involvement of Th2 and Th1 T cell responses in disease pathogenesis.
Both BN and LEW LNCs proliferate significantly in response to MOG in
vitro (Table III), and the Ag-specific
induction of mRNA transcripts for IL-4 was detected by RT-PCR in BN,
but not LEW LNC cultures (Fig. 3).
However, this was not accompanied by the secretion of detectable
amounts of IL-4 into the culture supernatants, as determined with a
commercially available ELISA kit for rat IL-4 (sensitivity, 12 pg/ml,
Table III). In contrast, both rat strains mounted a significant
Th1-like response to MOG, as demonstrated by the secretion of IFN-
in response to MOG in vitro, accompanied by similar amounts of the
proinflammatory cytokine TNF- (Table III). It therefore appears
unlikely that a Th2-like response to MOG played a significant role in
disease induction after immunization with MOG in CFA in the BN
rat.
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to IL-10 secretion in BN and LEW were 3.9
and 9.5, respectively (Table III
). This difference in the cytokine
profile was maintained in short term MOG-specific T cell lines (TCLs,
Table IV) composed of a dominant
population (> 85–95%) of CD4+,
CD45R0-, and ß TCR+ T
cells (data not presented). LEW-derived TCLs secreted large amounts of
IFN- (>100,000 pg/ml) and substantially less IL-10 (8200 pg/ml),
whereas BN MOG-specific TCLs secreted lower but similar amounts of
these two cytokines (IFN-, 5100 pg/ml; IL-10, 4200 pg/ml) (Table IV). No mRNA transcripts for IL-4 were detected in either BN or LEW
TCLs (data not presented). However, despite these marked phenotypic
differences neither LEW nor BN TCLs were able to mediate severe
clinical EAE by adoptive transfer. Even at cell doses of
107 T line cells per recipient, the freshly
activated TCLs induced only a mild, subclinical inflammatory response
in the CNS (Table IV). These observations indicate that the
CD4+ T cell response to MOG, irrespective of its
cytokine profile, is not directly responsible for the clinical disease
induced in either BN or LEW rats by active immunization with MOG
in CFA.
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The pathogenicity of MOG-specific autoantibodies is well
established (13, 27), yet their functional significance in
the pathogenesis of EAE is normally discussed in terms of demyelination
rather than a primary mechanism responsible for disease induction.
However, analysis of BN and LEW rats revealed an association between
the kinetics/intensity of the MOG-specific autoantibody response and
the timing of disease onset and course. In BN rats, the anti-MOG Ab
developed rapidly between days 5 and 10 pi to reach a titer of between
1:20,000 and 1:30,000 by disease onset. This response was maintained
until day 14 pi which because of the severity of disease was the last
time point from which representative samples were available (Fig. 4a). In contrast, in LEW rats
the MOG-specific Ab response was delayed and had reached a titer of
only 
1:1,000 by day 10 pi (Fig. 4a). At this time, LEW
rats exhibit no clinical or histopathological signs of EAE. However, by
the time LEW rats developed clinical signs of EAE (days 14 to 17 pi;
see Table I), the Ab titer had increased to 1:5,000–1:10,000. By 10
dpi, both strains had mounted a significant T cell response to MOG in
the draining lymph nodes, despite this 20- to 30-fold difference in the
intensity of the MOG-specific Ab response. Moreover, in both strains,
Th1 (IgG2b (Fig. 4f)) and Th2 (IgG1 (Fig. 4d) and
IgE (Fig. 4e))-associated Ig isotypes were affected to
similar extents, indicating that there was no selective enhancement
of the Th2-like response to MOG in BN.
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), combination with the MOG-reactive IgG fraction
induced clinical disease and CNS demyelination in all recipients (Figs. 5 and 6)
but without duplicating the eosinophilic infiltrate observed in
actively immunized BN rats.
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The above results suggest that the ability of MOG to induce clinical EAE in BN depended on the induction of a high titer MOG-specific autoantibody response, rather than the primary T cell response to this autoantigen. As the MHC can modulate Ab responses, we extended this study to investigate the susceptibility of two reciprocal MHC congenic rat strains to MOG-induced EAE. The LEW.1N strain carries the RT1n haplotype of BN on the LEW background, the BN.1L strain the RT1l haplotype of LEW on the BN background.
The RT1n haplotype clearly segregated with
early disease onset, an acute fulminant disease course (Table I), and a
high titer Ab response to MOG (Fig. 4). In contrast, the
RT1l haplotype was associated with later
onset of disease, decreased susceptibility, and a delayed rise in
anti-MOG Ab titers. This MHC-associated effect on the kinetics of
anti-MOG Ab responses in LEW and BN affected both Th1- and
Th2-associated Ig isotypes equally (Fig. 4, g and
h). The dominant role of the autoantibody response in
determining differences in disease onset/severity in LEW and BN strains
was supported by analysis of the T cell response to MOG in the MHC
congenic rat strains. Similar to the situation in the parental strains,
the MOG-specific T cell responses exhibited a Th0/Th1 cytokine
phenotype synthesizing large amounts of IL-10 and IFN-, but no
detectable IL-4 (Tables III and IV) and were unable to mediate severe
clinical EAE by adoptive transfer (Table IV). These observations
confirm the importance of the MOG-specific Ab response in determining
the clinical course and time of onset of MOG-induced EAE in the rat,
but using only the MHC congenic strains LEW.1N and BN.1L we were unable
to further define the MHC genes involved in this effect.
Intriguingly, the MOG gene is itself located within the
telomeric region of the MHC (28, 29), raising the
possibility that MOG polymorphisms (22) may
themselves influence the autoantibody response. To exclude this
possibility, we took advantage of the rat strain LEW.1R38. The
RT1r38 haplotype represents an intra-MHC
recombination between LEW and BN and was identified in mapping
studies of RT1.M class I genes representing homologues of
the H-2 M genes of the mouse (22). The recombination
breakpoint in r38 defines the RT1.M region
representing the most telomeric part of the rat MHC. Demonstration of a
RFLP of the MOG gene in LEW and BN allowed mapping of this
gene to the RT1 complex with the LEW.1N strain and, more
specifically, to the RT1.M region with the r38
haplotype (Fig. 7) (22).
Surprisingly, we observed that active immunization of LEW.1R38 rats
resulted in a fulminant disease course that was similar to that seen in
LEW.1N rather than LEW (Table I) although disease onset was delayed by
2 days (Table I). Also, histopathological changes in LEW.1R38 were
similar to those in LEW.1N in that inflammatory infiltrates were
characterized by large numbers of PMNs. The r38 haplotype
therefore enhances disease susceptibility even in the presence of
virtually the entire RT1 complex (regions
RT1.A–RT1.C) of the RT1l
haplotype associated with low susceptibility to MOG-EAE. Once again,
this effect does not seem to be directly related to the T cell
response, because we could not detect any differences to either the
parental LEW strain or LEW.1N in the primary T cell response to MOG
(Table III). However, the r38 haplotype did influence the
MOG-specific B cell response. The intra-MHC recombinant rats mounted a
B cell response with intermediary kinetics to MOG as compared with LEW
and LEW.1N (Fig. 8). This indicates that
the anti-MOG Ab response is influenced by genes located within
regions both centromeric and telomeric of the recombination breakpoint
in r38. Again, in all strains disease onset was concomitant
with the development of a significant anti-MOG response that
reached comparable titers at the endpoint of analysis (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have associated the resistance of BN to MBP-induced
EAE with TNF- polymorphisms (30), a robust HPA axis
response (31), defects in Ag processing (32),
and differential expression of class II molecules (33).
More recently, it was shown that BN but not LEW rats are susceptible to
Th2-dependent autoimmune diseases induced by treatment with either gold
salts or HgCl2. The adoptive transfer of
autoreactive Th2 T cell lines into CD8-depleted BN but not LEW
recipients induces an autoimmune syndrome characterized by polyclonal B
cell activation, increased titers of autoAbs, proteinuria, and
glomerular nephritis (20). Conversely, LEW rats are
resistant to HgCl2-induced autoimmune disease,
and treatment with HgCl2 will suppress EAE in
this strain (34). In the context of EAE, Th1 and Th2 T
cell responses are seen as being mutually antagonistic by virtue of the
reciprocal regulatory effects of Th1 (IFN-, IL-12)- and Th2 (IL-4,
IL-5, IL-10)-associated cytokines (35). The adoptive
transfer of neuroantigen-specific Th2 T cell lines or clones (26, 36) induces a subclinical eosinophilic infiltrate in the CNS of
normal recipients, whereas in immunodeficient hosts these T cells
mediate clinical disease associated with a PMN infiltrate
(37).
The observation that the cellular infiltrate in MOG-induced EAE in BN contained large numbers of polymorphonuclear leukocytes (PMNs) and eosinophils was initially taken to indicate that a Th2 T cell response was involved in disease pathogenesis. However, we were unable to demonstrate the secretion of the classical Th2 cytokine, IL-4 by either primary LNC cultures or short term MOG-specific T cell lines following antigenic stimulation in vitro. Moreover despite the presence of IL-4 mRNA transcripts in BN LNCs we were unable to demonstrate a selective enhancement of IgE, relative to other Ig isotype responses to MOG in BN as compared with LEW, LEW.1N, and BN.1L. These observations suggest that disease in BN is independent of the induction of a substantial Th2 T cell response to MOG, and at present the molecular basis for the eosinophilic infiltrate seen in BN is unclear. This pathology was only observed in the context of the BN genotype, and preliminary studies have failed to demonstrate the local expression of either IL-5 or eotaxin expression in the CNS, although these molecules play a critical role in recruitment of eosinophils into other organs (38, 39). However, the large numbers of PMNs found in the lesions may reflect the acuteness of the fulminant Ab-driven disease in BN, LEW.1N and LEW.1R38 rats, given that similar infiltrates have previously been demonstrated in acute cotransfer paradigms of EAE (40, 41).
Although the primary encephalitogenic Th1 CD4+ T
cell response is necessary for disease induction, we were able to
demonstrate that clinical expression of disease is mediated by Abs
rather than T cells. Indeed despite large differences in their ability
to synthesize IFN- none of the MOG-specific TCLs derived from any of
the four strains studied were able to induce severe clinical EAE by
adoptive transfer. This was noted previously for LEW rats (13, 16) and now appears to be an Ag, rather than strain related
phenomenon. Interestingly, not only MOG-specific but also MBP and P2
protein-specific TCLs can be derived from Ag primed BN donors that are
pathogenic in vivo (18, 42) indicating that protection
from actively induced disease is not maintained by deletional
mechanisms. This finding is concordant with the observation that the
resistance of BN to MBP-induced EAE can be overcome by using carbonyl
iron as the adjuvant (43).
The subclinical T cell response to MOG is essential for the induction of clinical EAE as it is responsible for transiently disrupting the blood brain barrier (44), which in BN occurs in the presence of a very high titer autoantibody response. Ab entering the CNS binds to the myelin sheath and activates the complement cascade (45) which may directly lead to myelin destruction (24, 46). The local production of C3a and C5a then initiates an increasing spiral of inflammation, further BBB dysfunction, and demyelination (24, 44). In the presence of an established high titer Ab response to MOG this results in a catastrophic cycle of increasing CNS inflammation and demyelination that cannot be controlled by counterinflammatory mechanisms, such as the up-regulation of complement inhibitory proteins within the CNS compartment (47). These observations are clinically relevant as there is an increasing body of evidence implicating Ab and complement mediated mechanisms in the immunopathogenesis of demyelination in MS (9, 10). Moreover, in a subgroup of patients with acute fulminate disease, demyelination is associated with a PMN/eosinophilic infiltrate together with complement and Ig deposition similar to that seen in MOG-induced EAE in the BN rat (H. Lassmann, personal communication).
Comparison of the kinetics of the Ab response in LEW and BN and the corresponding MHC congenics support this concept in that disease onset/severity correlated with the serum anti-MOG Ab titer, and these effects were associated with the MHC; the RT1n haplotype was associated with severe early onset disease and a rapid and high titer Ab response to MOG, whereas in the context of the RT1l haplotype both the Ab response and disease onset were delayed. Adoptive transfer experiments also demonstrated that neither the background genotype nor MHC haplotype grossly influenced the pathogenicity of MOG-specific TCLs in this strain combination. Although MHC genes are well known to influence the B cell response, we did not anticipate this effect on the kinetics and magnitude of the anti-MOG Ab response would be sufficient to induce clinical EAE in the BN rat. The MHC genes responsible for this effect remain to be identified but it should be noted that there is a partial Ag specificity as the RT1n haplotype supports a high Ab response to MOG (this study) and OVA (48, 49), but not MBP (50) or PPD (A. Stefferl, unpublished observation). This may implicate a role for class II MHC genes as they support the Ag/peptide specific expansion of CD4+ T cells that can provide B cell help.
However, the results obtained with the intraMHC recombinant LEW.1R38 rat indicate that two distinct regions of the MHC influence the Ab response and thereby disease severity in MOG induced EAE. In line with previous studies demonstrating a major effect of the class II region (RT1.B/D) in a set of recombinants between parental haplotypes RT1a and RT1u there is clear evidence for an effect of the segment spanning the RT1.A–RT1.C region that includes RT1.B/D (51). A new and unexpected finding was the additional strong effect of the telomeric RT1.M region. Interestingly, the gene encoding the target Ag MOG itself is located within this region (28, 29) and is known to be polymorphic between LEW and BN (22), making it a prime candidate for this effect. However, a sequence analysis of MOG-Igd cDNA has not revealed any differences between the strains used in this study (data not shown). This does not exclude genetic differences outside of the coding region that may influence the expression or turnover of MOG, and hence its antigenic properties. We are currently investigating this possibility further. It should also be noted that apart from MOG, other genes contained in this gene segment of the r38 recombinant haplotype may also influence disease susceptibility to EAE and/or the Ab response to MOG.
This study demonstrates that both MHC and MHC-linked effects can influence disease penetrance and severity via the B cell response in MOG-EAE, a model that closely resembles MS (52). In view of recent reports implicating anti-MOG Abs in the pathogenesis of MS (53) the functional significance of MHC-associated susceptibility genes should be re-assessed in the context of the MOG–specific B cell response in MS.
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Footnotes |
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2 A.S., U.B., and M.S. contributed equally to this publication.
3 Address correspondence and reprint requests to Dr. C. Linington, Department of Neuroimmunology, Max-Planck Institute for Neurobiology, Am Klopferspitz 18a, 82152 Martinsried, Germany. E-mail address:
4 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; BN, Brown Norway; LEW, Lewis; MS, mutiple sclerosis; MOG, myelin oligodendrocyte glycoprotein; MBP, myelin basic protein; RT1, MHC of the rat; MBP, myelin basic protein; pi, postimmunization; dpi, days pi; PMNs, polymorphonuclear leukocytes; LNCs, lymph node cells; TCLs, T cell lines.
Received for publication June 25, 1998. Accepted for publication April 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression by astrocytes from experimental allergic encephalomyelitis- susceptible and -resistant rat strains. J. Exp. Med. 173:801.This article has been cited by other articles:
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 H. R. Kim, E. Y. Kim, J. Cerny, and K. D. Moudgil Antibody Responses to Mycobacterial and Self Heat Shock Protein 65 in Autoimmune Arthritis: Epitope Specificity and Implication in Pathogenesis J. Immunol., November 15, 2006; 177(10): 6634 - 6641. [Abstract] [Full Text] [PDF]
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 R. Gold, C. Linington, and H. Lassmann Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research Brain, August 1, 2006; 129(8): 1953 - 1971. [Abstract] [Full Text] [PDF]
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N. Kawakami, F. Odoardi, T. Ziemssen, M. Bradl, T. Ritter, O. Neuhaus, H. Lassmann, H. Wekerle, and A. Flugel Autoimmune CD4+ T Cell Memory: Lifelong Persistence of Encephalitogenic T Cell Clones in Healthy Immune Repertoires J. Immunol., July 1, 2005; 175(1): 69 - 81. [Abstract] [Full Text] [PDF]
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T. Ishizu, M. Osoegawa, F.-J. Mei, H. Kikuchi, M. Tanaka, Y. Takakura, M. Minohara, H. Murai, F. Mihara, T. Taniwaki, et al. Intrathecal activation of the IL-17/IL-8 axis in opticospinal multiple sclerosis Brain, May 1, 2005; 128(5): 988 - 1002. [Abstract] [Full Text] [PDF]
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M. A. Staykova, J. T. Paridaen, W. B. Cowden, and D. O. Willenborg Nitric Oxide Contributes to Resistance of the Brown Norway Rat to Experimental Autoimmune Encephalomyelitis Am. J. Pathol., January 1, 2005; 166(1): 147 - 157. [Abstract] [Full Text] [PDF]
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H. Sakuma, K. Kohyama, I.-K. Park, A. Miyakoshi, N. Tanuma, and Y. Matsumoto Clinicopathological study of a myelin oligodendrocyte glycoprotein-induced demyelinating disease in LEW.1AV1 rats Brain, October 1, 2004; 127(10): 2201 - 2213. [Abstract] [Full Text] [PDF]
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P. Hurt, L. Walter, R. Sudbrak, S. Klages, I. Muller, T. Shiina, H. Inoko, H. Lehrach, E. Gunther, R. Reinhardt, et al. The Genomic Sequence and Comparative Analysis of the Rat Major Histocompatibility Complex Genome Res., April 1, 2004; 14(4): 631 - 639. [Abstract] [Full Text] [PDF]
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M. Kerschensteiner, C. Stadelmann, B. S. Buddeberg, D. Merkler, F. M. Bareyre, D. C. Anthony, C. Linington, W. Bruck, and M. E. Schwab Targeting Experimental Autoimmune Encephalomyelitis Lesions to a Predetermined Axonal Tract System Allows for Refined Behavioral Testing in an Animal Model of Multiple Sclerosis Am. J. Pathol., April 1, 2004; 164(4): 1455 - 1469. [Abstract] [Full Text] [PDF]
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 R. Diem, M. Hobom, K. Maier, R. Weissert, M. K. Storch, R. Meyer, and M. Bahr Methylprednisolone Increases Neuronal Apoptosis during Autoimmune CNS Inflammation by Inhibition of an Endogenous Neuroprotective Pathway J. Neurosci., August 6, 2003; 23(18): 6993 - 7000. [Abstract] [Full Text] [PDF]
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 T. Berger, P. Rubner, F. Schautzer, R. Egg, H. Ulmer, I. Mayringer, E. Dilitz, F. Deisenhammer, and M. Reindl Antimyelin Antibodies as a Predictor of Clinically Definite Multiple Sclerosis after a First Demyelinating Event N. Engl. J. Med., July 10, 2003; 349(2): 139 - 145. [Abstract] [Full Text] [PDF]
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C. Bourquin, A. Schubart, S. Tobollik, I. Mather, S. Ogg, R. Liblau, and C. Linington Selective Unresponsiveness to Conformational B Cell Epitopes of the Myelin Oligodendrocyte Glycoprotein in H-2b Mice J. Immunol., July 1, 2003; 171(1): 455 - 461. [Abstract] [Full Text] [PDF]
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A. Lobell, R. Weissert, S. Eltayeb, K. L. de Graaf, J. Wefer, M. K. Storch, H. Lassmann, H. Wigzell, and T. Olsson Suppressive DNA Vaccination in Myelin Oligodendrocyte Glycoprotein Peptide-Induced Experimental Autoimmune Encephalomyelitis Involves a T1-Biased Immune Response J. Immunol., February 15, 2003; 170(4): 1806 - 1813. [Abstract] [Full Text] [PDF]
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K. Becanovic, E. Wallstrom, B. Kornek, A. Glaser, K. W. Broman, I. Dahlman, P. Olofsson, R. Holmdahl, H. Luthman, H. Lassmann, et al. New Loci Regulating Rat Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis J. Immunol., January 15, 2003; 170(2): 1062 - 1069. [Abstract] [Full Text] [PDF]
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R. Gold and C. Linington Devic's disease: bridging the gap between laboratory and clinic Brain, July 1, 2002; 125(7): 1425 - 1427. [Full Text] [PDF]
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 J. L. McQualter, R. Darwiche, C. Ewing, M. Onuki, T. W. Kay, J. A. Hamilton, H. H. Reid, and C. C.A. Bernard Granulocyte Macrophage Colony-Stimulating Factor: A New Putative Therapeutic Target in Multiple Sclerosis J. Exp. Med., October 1, 2001; 194(7): 873 - 882. [Abstract] [Full Text] [PDF]
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R. Meyer, R. Weissert, R. Diem, M. K. Storch, K. L. de Graaf, B. Kramer, and M. Bahr Acute Neuronal Apoptosis in a Rat Model of Multiple Sclerosis J. Neurosci., August 15, 2001; 21(16): 6214 - 6220. [Abstract] [Full Text] [PDF]
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 A. Stefferl, M. K. Storch, C. Linington, C. Stadelmann, H. Lassmann, T. Pohl, F. Holsboer, F. J. H. Tilders, and J. M. H. M. Reul Disease Progression in Chronic Relapsing Experimental Allergic Encephalomyelitis Is Associated with Reduced Inflammation-Driven Production of Corticosterone Endocrinology, August 1, 2001; 142(8): 3616 - 3624. [Abstract] [Full Text] [PDF]
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R. Weissert, K. L. de Graaf, M. K. Storch, S. Barth, C. Linington, H. Lassmann, and T. Olsson MHC Class II-Regulated Central Nervous System Autoaggression and T Cell Responses in Peripheral Lymphoid Tissues Are Dissociated in Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis J. Immunol., June 15, 2001; 166(12): 7588 - 7599. [Abstract] [Full Text] [PDF]
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C. L. Keech, A. D. Farris, D. Beroukas, T. P. Gordon, and J. McCluskey Cognate T Cell Help Is Sufficient to Trigger Anti-Nuclear Autoantibodies in Naive Mice J. Immunol., May 1, 2001; 166(9): 5826 - 5834. [Abstract] [Full Text] [PDF]
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S. Ioannidu, L. Walter, R. Dressel, and E. Gunther Physical Map and Expression Profile of Genes of the Telomeric Class I Gene Region of the Rat MHC J. Immunol., March 15, 2001; 166(6): 3957 - 3965. [Abstract] [Full Text] [PDF]
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 T. Furuya, J. L. Salstrom, S. McCall-Vining, G. W. Cannon, B. Joe, E. F. Remmers, M. M. Griffiths, and R. L. Wilder Genetic dissection of a rat model for rheumatoid arthritis: significant gender influences on autosomal modifier loci Hum. Mol. Genet., September 1, 2000; 9(15): 2241 - 2250. [Abstract] [Full Text] [PDF]
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A. Stefferl, A. Schubart, M. Storch2, A. Amini, I. Mather, H. Lassmann, and C. Linington Butyrophilin, a Milk Protein, Modulates the Encephalitogenic T Cell Response to Myelin Oligodendrocyte Glycoprotein in Experimental Autoimmune Encephalomyelitis J. Immunol., September 1, 2000; 165(5): 2859 - 2865. [Abstract] [Full Text] [PDF]
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P. S. Heeger, T. Forsthuber, C. Shive, E. Biekert, C. Genain, H. H. Hofstetter, A. Karulin, and P. V. Lehmann Revisiting Tolerance Induced by Autoantigen in Incomplete Freund's Adjuvant J. Immunol., June 1, 2000; 164(11): 5771 - 5781. [Abstract] [Full Text] [PDF]
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Y.-H. Lou, K.-K. Park, S. Agersborg, P. Alard, and K. S. K. Tung Retargeting T Cell-Mediated Inflammation: A New Perspective on Autoantibody Action J. Immunol., May 15, 2000; 164(10): 5251 - 5257. [Abstract] [Full Text] [PDF]
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) and LEW (
) rats, and titers were 
) or CFA-immunized controls (
). Rats
receiving BN-derived MOG-specific Abs developed paralysis and weight
loss, whereas animals receiving control Abs remained clinically
healthy.
) had the
highest titers of anti-MOG Abs on day 10 pi, followed by
intermediate levels in the LEW.1R38 (•) and low levels in the LEW
(