Polymorphism at the HLA-DQ Locus Determines
Susceptibility to Experimental Autoimmune
Myasthenia Gravis
This information is current as
of June 16, 2017.
Raghavan Raju, Wen-Zhi Zhan, Peter Karachunski, Bianca
Conti-Fine, Gary C. Sieck and Chella David
J Immunol 1998; 160:4169-4174; ;
http://www.jimmunol.org/content/160/9/4169
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References
Polymorphism at the HLA-DQ Locus Determines Susceptibility
to Experimental Autoimmune Myasthenia Gravis1
Raghavan Raju,* Wen-Zhi Zhan,† Peter Karachunski,‡ Bianca Conti-Fine,‡ Gary C. Sieck,†
and Chella David2*
M
yasthenia gravis (MG)3 is an important model for the
study of molecular mechanisms of autoimmunity,
since the target autoantigen is known (nicotinic acetylcholine receptor (AChR)), sequenced, and well characterized
(1). Muscle AChR is formed by four homologous subunits in a
stoichiometry of a2bg(or e)d (1, 2). Anti-AChR CD41 T cells
from MG patients are known to recognize several epitopes on all
muscle AChR subunits (3–10). MG is an Ab-mediated disease, as
Abs have been shown to reduce the number of available muscle
AChRs (Ref. 11 and references therein). CD41 T cells are required
for generation of the high affinity IgG Abs that bind muscle
AChR (12–14).
MHC class II molecules serve the dual function of selection of
specific peptides to be bound and presented to the TCR and of
regulation of TCR specificities during the process of T cell differentiation and maturation in the thymus. Identification of class II
molecules associated with autoimmune diseases helps formulate
specific models of pathogenesis of autoimmune diseases. Specific
class II susceptibility genes have been described in different autoimmune diseases, such as type I diabetes (HLA DR4, DR3, DQ8),
Departments of *Immunology and †Anesthesiology Research, Mayo Clinic, Rochester, MN 55905; and ‡Department of Biochemistry and Pharmacology, University of
Minnesota, St. Paul, MN 55108
Received for publication October 17, 1997. Accepted for publication December
22, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI14764 (to
C.S.D), NS23919 (to B.M.C.-F.), HL34817 (to G.C.S.), and HL37680 (to G.C.S.);
and an Osserman Postdoctoral Fellowship of the Myasthenia Gravis Foundation of
America (to R.R.).
2
Address correspondence and reprint requests to Dr. Chella David, Department of
Immunology, Guggenheim-310, Mayo Clinic, Rochester, MN 55905. E-mail address:
[email protected]
3
Abbreviations used in this paper: MG, myasthenia gravis; AChR, acetylcholine
receptor; EAMG, experimental autoimmune myasthenia gravis; TAChR, Torpedo
acetylcholine receptor.
Copyright © 1998 by The American Association of Immunologists
rheumatoid arthritis (HLA DR4), pemphigus vulgaris (HLA DR4,
DR6), and other diseases (as reviewed in Ref. 15).
HLA linkage in MG as reported in literature varies with ethnic
groups, age, and sex. However, some of the initial epidemiologic
studies demonstrate that HLA B8 and HLA DR3 are linked to MG
in Caucasians (16 –22). One of the first reports that established the
significance of HLA-DQ b gene polymorphism in MG pathogenesis was that of Bell et al. (16). However, the specific role of any
particular HLA-DQ gene in the etiopathogenesis of MG is not as
clear as in other autoimmune diseases such as type I diabetes or
rheumatoid arthritis.
In young Caucasian MG patients, mainly women without thymoma and with high levels of AChR Abs have a high frequency of
the B8 and/or DR3 haplotypes (119 –22). MG was also found to be
positively associated with the DQB1*0604 allele, particularly in
patients with thymoma (23). Horiki et al. (24) reported that combinations of HLA-DPB1 and HLA-DQB1 alleles determine the
susceptibility to early onset MG in Japan. In Jamaicans, MG is
most strongly associated with HLA-B8, HLA B13, and DQ4 and
is negatively associated with HLA-A2. Female MG patients under
30 yr of age at the onset of disease had a significantly higher
frequency of DQB1*03, which includes *0301, *0302, and *0303,
compared with healthy controls (25). In a Swedish study, it was
reported that two different DQ2 haplotypes (DQA1*0501/
DQB1*0201 and DQA1*0201/DQB1*0201) were positively associated with MG (26). Carlsson et al. (27) observed a strong association in Caucasian MG patients of a DR-DQ haplotype
(DR3DR52DQ6) with MG in young females and an association
with DR4-DQ8 in elderly non-DR3 males. A recent study of 79
Swedish patients and 155 unrelated, population-based controls,
found that polymorphic domains on the HLA-DQ molecule are
associated with disease heterogeneity in MG (28). By analyzing
polymorphic domains on HLA-DQ molecules contributing to positive and negative association with MG, they found that a domain
with residues common to DQB1*05 and DQB1*06 alleles is negatively associated with the disease in patients with thymic hyperplasia or an early disease onset.
0022-1767/98/$02.00
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Studies in myasthenia gravis (MG) patients demonstrate that polymorphism at the HLA-DQ locus influences the development of
MG. Several studies using the mouse models also demonstrate the influence of class II molecules, especially the H2-A, which is the
mouse homologue of HLA-DQ, in experimental autoimmune myasthenia gravis (EAMG). We used transgenic mice expressing two
different DQ molecules, DQ8 (DQA1*0301/B1*0302) and DQ6 (DQA1*0103/B1*0601), to evaluate the role of HLA-DQ genes in
MG. These mice do not express endogenous mouse class II molecules since they contain the mutant H2-Ab0 gene. The mice were
immunized with Torpedo acetylcholine receptor, and EAMG was assessed by clinical evaluation and was confirmed by electrophysiology. Clinical scores for EAMG were highest in HLA-DQ8 transgenic mice, whereas the scores of HLA-DQ6 mice rarely
exceeded grade 1. There was no incidence of EAMG in class II-deficient (H2-Ab0) mice. These results demonstrate that polymorphism at the HLA-DQ locus affects the incidence and the severity of EAMG. The manifestation of susceptibility to EAMG in
the context of human class II molecules underscores the important roles of these molecules in the initiation and perpetuation of
EAMG. The Journal of Immunology, 1998, 160: 4169 – 4174.
4170
EAMG IN HLA TRANSGENIC MICE
pancuronium or edrophonium chloride was sufficient to establish the myasthenic nature of muscle weakness, we believed that it would be more
informative if electrophysiologic experiments were also performed (43,
44). Briefly, the right midcostal diaphragm muscle was excised together
with the phrenic nerve and mounted vertically in a glass tissue chamber
containing Ringer’s solution. The solution was aerated with 95% O2/5%
CO2 and maintained at 26°C. The central tendon was attached in series to
a calibrated force transducer, while the rib insertion was clamped to a
micromanipulator. The muscle was stimulated directly by 0.5-ms pulses
using a pair of platinum electrode. Muscle fiber length was adjusted until
maximum isometric twitch force responses were obtained. The phrenic
nerve was stimulated using 0.2-ms duration pulses delivered via a suction
electrode. Neuromuscular transmission failure was assessed at 40 Hz.
Nerve stimulation was presented at 40 Hz in 330-ms duration trains repeated one train per second for a period of 2 min. Every 15 s, direct muscle
stimulation at 40 Hz was superimposed. The force decline during direct
muscle stimulation reflects only the contribution of muscle fatigue, while
the force loss during nerve stimulation reflects the contributions of both
muscle fatigue and neuromuscular transmission failure.
Materials and Methods
Results
Mice
Rationale
Transgenic mice expressing functional HLA-DQ8 (HLA-DQA1*0301/
DQB1*0302) and DQ6 (HLA-DQA1* 0103/DQB1*0601) genes were generated as described previously (33–37). Cosmid clones H11A and X10A
containing the DQA1*0301 and DQB1*0302 genes were provided by Dr.
J. Strominger, while cosmids pAKQ 4116 and pAKQ 056 containing
DQA1*0103 and DQB1*0601 genes, respectively, were gifts from Dr. H.
Inoko. Cosmids pAKQ 4116 and pAKQ 056 were derived from the
AKIBA cell line, linearized by restriction enzyme digestion, and microinjected into (CBA/J 3 B10.M)F2 or (SJL 3 SWR)F2 embryos (34). HLADQ8 and DQ6 transgenic mice were bred into H2b mice lacking Abb expression (the latter hereafter referred to as Ab0 mice) to obtain DQ8.Ab0or
DQ6.Ab0 mice. The mice were all homozygous for the transgene, as demonstrated by the absence of nontransgenic offsprings in the generations
previous to that studied.
The susceptibility of different strains of mice to EAMG is well
characterized. The H2-A molecule is important for the induction of
the disease, and polymorphism at this locus influences the susceptibility to EAMG. MG is an Ab-mediated disease, and Th cells are
important in providing help for the generation of high affinity Abs
by B cells. The presentation of pathogenic epitopes to CD41 T
cells is important in the manifestation and perpetuation of MG
through generation of high affinity Abs. The selection of CD41 T
cells in the thymus is regulated by MHC class II molecules. MHC
class II molecules can present pathogenic epitopes to CD41 T
cells, and polymorphism at this locus should influence such peptide binding and presentation, leading to susceptibility or protection to disease. In humans, the study of the significance of HLA in
autoimmune diseases is limited by the fact that every APC would
express at least three different (DR, DQ, and DP) class II isotypes
along with different class I molecules, making interpretations of
the role of any single molecule difficult. Mice transgenic for the
MHC genes are a useful model to study the role of HLA molecules
in the pathogenesis of MG and other autoimmune diseases. To
understand the influence of polymorphism at human class II locus
in the pathogenesis of MG and EAMG, we used two different HLA
transgenic mice in this study. We used three groups of mice, Ab0.
DQ8 (DQA1*0301/DQB1*0302)., Ab0.DQ6 (DQA1*0103/DQB1*
0601), and Ab0. These mice do not express endogenous class II molecules, since they contain the disrupted H2-Ab gene. Previous studies
have shown that no hybrid molecules (DQbAa or DQaEb) are generated (37). The DQ8 and DQ6 mice differ only in the DQ genes.
Since mice that are deficient in endogenous class II molecules are
resistant to EAMG, the susceptibility of HLA transgenic mice underscores the significance of HLA single class II molecules in the initiation and perpetuation of MG.
Purification of TAChR
TAChR was purified from Torpedo californica (Aquatic Research Consultants, San Pedro, CA) electric organ as postsynaptic membrane fragments that were further enriched for TAChR by alkaline pH treatment (38,
39). The purity of the isolated TAChR was ascertained by SDS-PAGE
(40). TAChR preparations were characterized using 125I-labeled a-bungarotoxin; the purified receptor preparation contained 4 to 6 nmol of a-bungarotoxin binding sites/mg of protein (41).
Induction of the disease
Mice were given three injections (s.c.) of TAChR (20 mg/mice) at 4-wk
intervals. The first injection was given in CFA, and boosters were given in
IFA in a 1:1 ratio. After 12 wk, mice were sacrificed.
Assay of serum anti-AChR Abs
Anti-AChR Abs in the sera were assayed by a radioimmunoprecipitation
assay using 125I-labeled a-bungarotoxin (38).
Disease assessment
Clinical assessment. Muscle weakness was assessed every week in a
blind study (42). Briefly, mice were allowed to grip their paws on cage top
grids, were pulled off the grid by tail consecutively for 25 times for forced
exercise, and were scored as follows: grade 0, there was no weakness at
rest or after exercise; grade 1, normal strength at rest, but weak with chin
on the floor and inability to raise the head after exercise; grade 2, the mice
exhibit grade 1 weakness at rest; and grade 3, moribundity or quadriplegia.
The methods used for the clinical assessment of EAMG are subjective, and
various factors may interfere with them. To confirm that the mouse weakness was of a myasthenic nature, muscle weakness of grade 1 was verified
by pancuronium-sensitized forced exercise tests, which exacerbate myasthenic weakness. Grade 2 weakness was confirmed by the use of tensilon
(edrophonium chloride).
Electrophysiology. Since neuromuscular junction failure is the hallmark
of myasthenia, we measured the neuromuscular transmission failure in a
few DQ8 transgenic mice that showed muscle weakness and in mice that
were normal. Due to practical reasons we were unable to perform this test
in all the mice we studied. Although use of pharmacologic agents such as
EAMG in HLA-DQ8.Ab0 mice
HLA-DQ8 transgenic mice were highly susceptible to EAMG as
demonstrated in Figure 1A. Seven of the 10 mice studied developed EAMG; in six of them the disease score was 2 or more,
demonstrating increased severity of disease in these mice. Clinical
symptoms of EAMG were transient in the mice that had grade 1
disease. EAMG onset in all the mice except one occurred immediately after the second immunization. One mouse developed
EAMG before the third immunization. By the time of the third
immunization four mice (one of them had had transient grade 1
disease earlier) had no disease, and none of these mice showed
symptoms of EAMG even after the third immunization. This is
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In mice the H2b haplotype is strongly associated with susceptibility to experimental autoimmune myasthenia gravis (EAMG)
(17, 29). Other susceptible haplotypes are H2q, H2r, and H2i. The
resistant strains include the H2d, H2k, and H2p haplotypes. In
C57BL6 (H2b) mice, the susceptibility to EAMG was mapped to
the H2-Ab genes (30 –32). The role of H2-Ab in the development
of EAMG is further supported by the finding that a mutation in
Abb at positions 67, 70, and 71 generates the H2bm12 strain that is
not susceptible to EAMG (30, 31).
In this study we investigated the roles of HLA-DQA1*0301/
DQB1*0302 (DQ8) and HLA-DQA1* 0103/DQB1*0601 (DQ6)
in the pathogenesis of MG by using mice transgenic to the respective HLA class II molecule in the absence of endogenous mouse
class II molecules. The mice were immunized with Torpedo acetylcholine receptor (TAChR) to examine the effect of polymorphism at the HLA DQ locus on their susceptibility for EAMG.
The Journal of Immunology
4171
FIGURE 2. TAChR Ab levels. TAChR Ab levels were estimated in five
mice in each group at three different time points (days 0, 20, and day 45)
and expressed as micromolar concentrations of BGTx binding sites.
Absence of EAMG in class II knockout mice
Discussion
FIGURE 1. EAMG in mice immunized with TAChR. Arrows on the
x-axis show the days immunized. Score: grade 0, no weakness at rest or
after exercise (25 consecutive paw grips on cage top steel grids); grade 1,
normal strength at rest, but weak with chin on the floor and inability to raise
the head after exercise; grade 2, grade 1 weakness at rest; grade 3, moribundity, dehydration, or quadriplegia. * indicates that the mouse was killed
or died. A horizontal line with a blunt end denotes death of the mouse due
to reasons other than EAMG. A, HLA DQ8 transgenic mice; B, HLA DQ6
transgenic mice; C, Ab0 (MHC class II-deficient) mice.
consistent with the Ab titers in these mice, which remained practically unchanged after the second immunization (Fig. 2). Figure 3,
A and B, shows the repetitive nerve stimulation test of a normal
HLA-DQ8 transgenic mouse compared with that of a mouse that
showed grade 3 clinical symptoms of EAMG. Figure 3, A and B,
clearly demonstrates marked differences in the forces evoked by
muscle stimulation vs nerve stimulation, reflecting extensive neuromuscular transmission failure.
EAMG in HLA-DQ6.Ab0 mice
HLA-DQ6 transgenic mice showed only a moderate susceptibility
to EAMG (Fig. 1B). Six of the nine mice in this group developed
EAMG, but five of these six mice had very modest symptoms
(grade 1), and the other one had grade 2 symptoms. Most of the
mice that had grade 1 EAMG did not show persistence of the
clinical symptoms, which were transient. The anti-TAChR Ab titer
was lower in this group than in the DQ8 transgenic mice (Fig. 2).
The titer of the anti-TAChR Abs remained same at 3 and 7 wk. In
this group we did not observe any clinical signs of muscle weakness before the second immunization. However, after the second
immunization, transient symptoms continued to occur until termination of the experiment at 12 wk.
The clinical symptoms in HLA-DQ8 transgenic mice immunized
with TAChR were very severe compared with those in HLA-DQ6
transgenic mice. None of the Ab0 mice developed clinical EAMG.
This strongly points out the influence of polymorphism at the human class II locus in the development of MG. The influence of
H2-A gene polymorphism on susceptibility to EAMG in the mice
is well studied (29 –32). However, our knowledge of the effect of
HLA gene polymorphism in humans is limited, probably due to the
low incidence of MG as well as the MHC heterogeneity among
individuals. Our model, in which a human class II gene is introduced in isolation in the absence of the endogenous mouse class II
genes, is a valuable tool in addressing the immunogenetics of
human MG.
The only MHC class II genes functional in these mice are the
human DQ8 and DQ6 molecules, respectively. The H2-Abb
knockout mice express H2-Aab and H2-Ebb in the cytoplasm, but
these two chains do not combine to form functional heterodimers
expressed on the cell surface (36). Also, surface expression of
heterodimers formed by H2-Aab and DQ b-chains was not observed in either DQ8.Ab0 or DQ6.Ab0 transgenic mice (34, 37).
Previous reports from our laboratory have demonstrated an HLADQ8-restricted T cell response in DQ8.Ab0 mice and an HLADQ6-restricted T cell response in DQ6.Ab0 mice (37, 45, 46). The
functional significance of HLA-DQ8 molecules in DQ8.Ab0 mice
is underscored by the finding that these mice were highly susceptible to collagen-induced arthritis (37).
The present study compares the susceptibility of mice transgenic
to HLA-DQ8 and HLA-DQ6 molecules to assess the differential
susceptibility to EAMG due to polymorphism at the DQ locus in
an experimental system. Our observation of zero incidence of
EAMG in class II-deficient mice is similar to that reported in a
previous study that concluded that functional class II molecules
and CD41 T cells are essential for the development of EAMG and
rule out any pathogenic effector role for MHC class I-restricted
CD81 T cells, gd TCR-bearing cells, or NK cells, which are intact
in the MHC class II mutant mice (32).
Previous studies using bm12 mutant mice concluded that polymorphism at the H2-A locus (homologous to HLA-DQ) strongly
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The Ab0 mice did not show any clinical signs of muscle weakness.
Among the 10 mice in this group, one developed weakness in one
of the rear limbs. The clinical symptom in this mice was not
changed after administration of either pancuronium or edrophonium chloride. We concluded that this could not be due to EAMG
and hence deleted this mouse from the study.
4172
EAMG IN HLA TRANSGENIC MICE
influences EAMG susceptibility (31, 47– 49). C57BL6 mice were
susceptible to TAChR-induced EAMG, while C57BL6bm12 mice
were resistant to the disease (231, 47– 49). The Abm12 mutation
drastically affected the epitope repertoire of murine CD41 T cells
sensitized to Torpedo AChR (30). Such changes in the epitope
repertoire and the lack of recognition of the otherwise immunodominant CD41 T cell epitope were proposed as likely reasons for
the disease resistance of bm12 mutant mice (30). In another study
it was observed that the expression of the Abb:Aak transpair in
C57BL10 transgenic mice suppressed the cellular and humoral autoimmune responses to AChR and reduced the incidence of
muscle weakness and its associated abnormal electrophysiologic
response (50).
However, such stringent genetic studies with single variant genetic elements are impossible to perform with human subjects due
to the considerable polymorphism at the HLA locus and the presence of multiple class II and class I molecules on the cell surface.
This had been a major limitation for the study of HLA restriction
of different autoimmune diseases in which in vitro experiments
using blocking Abs and HLA-identical APCs were the only limited
options to understand the roles of individual HLA genes in the
disease pathogenesis. Such limitations are overcome by transgenic
technology, by the introduction of a specific HLA transgene into a
mouse enabling study of the function of these molecules in isolation. The obvious drawback is the fact that these human MHC
molecules select mouse T cells, and hence the pathogenic epitopes
are recognized in the context of the mouse T cells. Also, in the
presence of endogenous mouse class II molecules, there is a potential problem of preferential interaction of these molecules with
mouse CD4 molecules, leading to the selection of predominantly
mouse class II-restricted T cells. This was overcome by crossing
the HLA transgenic mice to class II knockout mice. In our HLA
class II transgenic mice that do not express the endogenous mouse
class II molecules, we found that normal levels of CD41 T cells
are restored, confirming that HLA class II molecules can efficiently
interact with mouse CD4 molecule and TCRs (37).
EAMG was severe in HLA-DQ8 transgenic mice and very moderate in HLA-DQ6 transgenic mice (Table I). This shows that the
HLA-DQ8 haplotype might be contributing to a serious predisposition to MG, whereas the role of HLA-DQ6 may be limited. The
manifestation of the disease is underscored by the drastic reduction
in nerve conduction, as shown by the repetitive nerve stimulation
Table I. Summary of EAMG scores of mice studied in each group
Gradesa
Mice
DQ8.Ab
DQ6.Ab0
Ab0
0
a
b
0
1
2
3
Maximum Grade
of Severityb
n
3
3
9
1
5
0
3
1
0
3
0
0
2.3
1.1
0
10
9
9
Mice were scored as described in Materials and Methods.
Mean of the scores for grades 1 to 3.
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FIGURE 3. Repetitive nerve stimulation test in HLA DQ8 transgenic mice. A, Control mice not immunized with TAChR; B, mice immunized with
TAChR and reached grade 3 weakness. For experimental details, see Materials and Methods.
The Journal of Immunology
Acknowledgments
We thank Drs. Jack Strominger and Hidetoshi Inoko for providing the
HLA-DQ8 and DQ6 cosmids, Drs. Christophe Benoist and Diane Mathis
for the Ab0 mice, and Drs. Paul Zhou, Shen Cheng, and Jeanine Baisch for
generating the transgenic mice used in this study. The assistance of Michelle Smart in screening the mice is appreciated. Thanks are also due to Julie
Hanson and her crew for the breeding and maintenance of the mice used in
this study.
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experiment presented in Figure 3. We conducted this experiment in
four normal DQ8 mice and did not observe more than a 10%
decline in the initial force ratio between tetanic nerve stimulation
vs direct muscle stimulation (data not shown). The weakness observed in the TAChR-immunized HLA transgenic mice should
therefore be due to severe neuromuscular junction failure correlating with the clinical score.
The necessity of class II molecules and hence the CD41 T cells
in MG has been consistently demonstrated by different laboratories
(32, 51). This finding corroborates the evidence that MG is an
Ab-mediated disease, and the help of CD41 T cells is required for
the production of high affinity pathogenic Abs. The role of CD81
T cells in EAMG is less clear. One study used b2m-deficient mice,
which do not express functional class I molecules on the cell surface and are deficient in CD81 T cells, and found that the mice
were susceptible to EAMG. This suggested that CD81 T cells are
not necessary for the development of EAMG. However, another
study found that CD81 T cell deficiency caused resistance to
EAMG and proposed that both CD41 and CD81 T cells are
needed for the development of EAMG, perhaps due to a CD41CD81 T cell collaboration in the pathogenesis of the disease (51).
Nevertheless, both studies confirmed the important role of class II
molecules in the disease process. The present study verifies the
importance of class II molecules in human MG and demonstrates
that HLA-DQ8 could be one of the predisposing haplotypes for the
development of MG. As the role of class I and hence CD81 T cells
in EAMG is a matter of controversy, it is essential to study its role
in HLA class II transgenic mice either using class I-deficient mice
or using strains in which different class I transgenes are introduced
into the HLA class II transgenic animals.
This is the first report to directly demonstrate the role of any
particular HLA molecule in isolation in the modulation of MG in
vivo in an experimental system. This humanized mouse model is
an excellent resource toward better understanding the immunogenetic basis of MG as well and to develop treatment and prophylactic strategies for MG.
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