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Circulating Immune Complexes Augment
Severity of Antibody-Mediated Myasthenia
Gravis in Hypogammaglobulinemic RIIIS/J
Mice
Erdem Tüzün, Benjamin G. Scott, Huan Yang, Bo Wu,
Elzbieta Goluszko, Michelle Guigneaux, Stephen Higgs and
Premkumar Christadoss
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 172:5743-5752; ;
doi: 10.4049/jimmunol.172.9.5743
http://www.jimmunol.org/content/172/9/5743
The Journal of Immunology
Circulating Immune Complexes Augment Severity of
Antibody-Mediated Myasthenia Gravis in
Hypogammaglobulinemic RIIIS/J Mice1
Erdem Tüzün,* Benjamin G. Scott,* Huan Yang,* Bo Wu,2‡ Elzbieta Goluszko,3*
Michelle Guigneaux,§ Stephen Higgs,† and Premkumar Christadoss4*
E
xperimental autoimmune myasthenia gravis (EAMG)5 is
a T cell-dependent, Ab-mediated autoimmune disease of
the neuromuscular junction (NMJ), which closely mimics
human myasthenia gravis (MG) in its clinical and immunopathological manifestations (1). The autoantibodies to acetylcholine receptor (AChR) induce NMJ destruction and the loss of AChR in
MG and EAMG. The production of Abs is dependent on MHC
class II-restricted CD4⫹ T cell help (1–3). However, for the activation of AChR-specific CD4⫹ cells and production of anti-AChR
Departments of *Microbiology and Immunology and †Pathology, University of Texas
Medical Branch, Galveston, TX 77555; ‡Department of Neurology, University of
Texas Southwestern Medical Center, Dallas, TX 75390; and §Sealy Center for Molecular Sciences, University of Texas Medical Branch Genomics Core, Galveston, TX
77555
Received for publication December 8, 2003. Accepted for publication February
5, 2004.
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 study is supported by NIHR21A1049995, Muscular Dystrophy Association,
and Advanced Technology Program of the Texas Higher Education Coordinating
Board. E.T. is a Myasthenia Gravis Foundation Osserman/Sosin/McClure postdoctoral fellow. B.G.S. is a National Institutes of Health Immunology and Mucosal Defense Training Grant Fellow. H.Y. is a Muscular Dystrophy Association Neuromuscular Disease Research Career award recipient.
2
Current address: Department of Psychiatry, John Peter Smith Hospital of Tarrant
County, Fort Worth, TX 76104.
3
Current address: Department of General Surgery, University of Texas Medical
Branch, Galveston, TX 77555-0722.
4
Address correspondence and reprint requests to Dr. Premkumar Christadoss, Department of Microbiology and Immunology, University of Texas Medical Branch,
301 University Boulevard, Galveston, TX 77555-1070. E-mail address:
[email protected]
5
Abbreviations used in this paper: EAMG, experimental autoimmune myasthenia
gravis; AChR, acetylcholine receptor; C1q-CIC, C1q-conjugated circulating immune
complex; CIA, collagen-induced arthritis; EAE, experimental allergic encephalomyelitis; KO, knockout; MAC, membrane attack complex; MG, myasthenia gravis;
NMJ, neuromuscular junction; PNA, peanut agglutinin.
Copyright © 2004 by The American Association of Immunologists, Inc.
Abs, B7-CD28 and CD40-CD40L costimulatory molecule interactions are required (4, 5). Besides these costimulatory molecules,
proinflammatory cytokines TNF-␣, IL-6, IL-18, and IL-1␤ contribute to the activation of AChR-specific T and B cells and play
a critical role in EAMG pathogenesis (6 –10). One important
mechanism by which anti-AChR Abs induce EAMG is the activation of a complement-mediated NMJ destruction. Proposed
mechanism is largely based on experiments that revealed that
IgG, C3, and membrane attack complexes (MAC) are colocalized in the NMJ of patients with MG (11, 12), and C5 gene
knockout (KO) mice are highly resistant to EAMG (13). Moreover, we have recently shown that the classical complement
pathway is responsible for C3 and MAC deposition at the NMJ,
and is therefore essential for the development of EAMG in
C57BL/6J (B6) mice (14).
We previously used the RIIIS/J mouse strain, which demonstrates increased EAMG incidence and develops more severe
EAMG than the well-established EAMG-susceptible B6 strain
(15). RIIIS/J mice, which have the largest known genomic deletion
in TCR V␤ gene (13 of 21 V␤ genes) (16), are resistant to both
experimental allergic encephalomyelitis (EAE) (17–19) and collagen-induced arthritis (CIA) (20 –22). However, they are highly
susceptible to EAMG, displaying more severe EAMG than B6
mice and the H-2-identical B10.RIII (H-2r) mice, suggesting that
in RIIIS/J mice, non-MHC genes or factors influence disease severity. To study the non-MHC factors contributing to augmented
EAMG severity in RIIIS/J mice, we delineated the role of deleted
V␤ genes and analyzed the genetic, cellular, and immunopathological differences between RIIIS/J and B10.RIII mice. The increased severity of EAMG in hypogammaglobulinemic RIIIS/J
mice turned out to be primarily associated with increased amounts
of lymph node CD4⫹ T cells and C1q-conjugated circulating immune complex (C1q-CIC) levels.
0022-1767/04/$02.00
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Experimental autoimmune myasthenia gravis (EAMG) is severe in RIIIS/J mice, despite a significant B cell immunodeficiency and
a massive TCR V␤ gene deletion. Severity of EAMG in RIIIS/J mice is greater than MHC-identical (H-2r) B10.RIII mice,
suggesting the influence of non-MHC genes as an EAMG-potentiating factor in this strain. To delineate the role of deleted TCR
V␤ genes in RIIIS/J mice, we obtained (RIIIS/J ⴛ B10.RIII)F1 (V␤b/c) ⴛ RIIIS/J (V␤c) backcross mice using Mendelian genetic
methods and immunized them with acetylcholine receptor. EAMG susceptibility was not elevated in mice with V␤c genotype
having 70% V␤ gene deletion. Next, we performed microarray analysis on 12,488 spleen cDNAs obtained from spleens of naive
RIIIS/J and B10.RIII mice. In RIIIS/J mice, 263 cDNAs were overexpressed and 303 cDNAs were underexpressed greater than
2-fold, compared with B10.RIII mice. TCR gene expression was augmented, whereas NK receptor, C1q, and C3 gene expressions
were diminished in RIIIS/J mice. RIIIS/J mice also had increased lymph node T cell counts, elevated serum anti-AChR Ab levels,
and serum C3 and C1q-conjugated circulating immune complex levels. A direct correlation between increased serum C1qconjugated circulating immune complex levels and disease severity was observed in RIIIS/J mice. The Journal of Immunology,
2004, 172: 5743–5752.
5744
Materials and Methods
AChR and mice
AChR was purified from the electric organ of Torpedo californica by an
␣-neurotoxin affinity column (23). Seven- to 8-wk-old RIIIS/J, B10.RIII,
and B6 mice were purchased from The Jackson Laboratory (Bar Harbor,
ME). All animals were housed in the viral Ab-free barrier facility at the
University of Texas Medical Branch and maintained according to the Institutional Animal Care and Use Committee Guidelines.
Induction and clinical evaluation of EAMG
RIA for anti-muscle AChR Ab
The Ab response to mouse muscle AChR was measured by RIA, as previously
described (23). The anti-AChR Ab levels were expressed in nanomoles of
␣-bungarotoxin binding sites bound per liter of serum for individual mice.
ELISA for IgG isotypes
Affinity-purified mouse AChR (0.5 ␮g/ml) was coated onto a 96-well microtiter plate (Dynatech Immulon 2; Dynatech Laboratories, Chantilly,
VA) with 0.1 M carbonate bicarbonate buffer (pH 9.6) overnight at 4°C.
The plates were blocked with 2% BSA in PBS at room temperature for 30
min. Serum samples were diluted 1/1000 in PBS/0.05% Tween 20 and
incubated at 37°C for 90 min. After four washes, HRP-conjugated goat
anti-mouse IgM and IgG isotypes (IgG, IgG1, IgG2b IgG2c, and IgG3
(Caltag Laboratories, San Francisco, CA)) diluted 1/500 –1000 in PBS/
0.05% Tween 20 were added and incubated at 37°C for 90 min. Subsequently, ABTS (indicator) solution in 0.1 M citric buffer, pH 4.3, in the
presence of H2O2 was added, and color was allowed to develop at room
temperature in the dark. Absorbance values were measured at a wavelength
of 405 nm, using a Molecular Devices (Sunnyvale, CA) EMAX microplate
reader, and the results were expressed as OD values.
ELISA for serum total IgG levels
ELISA was performed in the same manner as mentioned above, except that
the plates were coated with goat anti-mouse IgG (Chemicon International,
Temecula, CA) with 0.1 M carbonate bicarbonate buffer (pH 9.6) overnight
at 4°C. After incubation with serum samples, HRP-conjugated goat antimouse IgG Ab (Caltag Laboratories), directed to a different determinant
than the primary coating Ab, was added in 1/1000 dilution in PBS/0.05%
Tween.
DNA microarray analysis of gene expression
mRNA was extracted from five spleens each of RIIIS/J and B10.RIII mice.
Spleens were pooled for each strain and then probed using one chip each
for RIIIS/J and B10.RIII mice. Briefly, spleen cells were transferred to
Eppendorf tubes, and 0.2 ml of chloroform was added. Tubes were mixed
vigorously, incubated for 15 min at room temperature, and centrifuged at
12,000 rpm for 15 min at 4°C. The upper aqueous layer was removed, 0.5
ml of 50% isopropanol was added, and final mixture was incubated at room
temperature for 10 min. After centrifugation at 12,000 rpm for 10 min at
4°C, samples were washed in 1 ml of 75% ethanol and centrifuged again
at 7,500 rpm for 5 min at 4°C. Pellets were dissolved in 100 ␮l of diethylpyrocarbonate-treated water (Sigma-Aldrich, St. Louis, MO).
MGU74Av2 mouse array was obtained from Affymetrix (Santa Clara,
CA). The microarray covered 12,488 probes. First-strand cDNA synthesis
was performed using total RNA (10 –25 ␮g), a T7-(dT)24 oligomer, and
SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Secondstrand synthesis converted the cDNA into a dsDNA template for use in an
in vitro transcription reaction. The cRNA or target RNAs were labeled with
biotin during the in vitro transcription reaction. Biotin-labeled target RNAs
were fragmented to a mean size of 200 bases to facilitate their hybridization to probe sequences on the gene chip array. Hybridization of gene chip
array was performed at 45°C for 16 h in 0.1 M MES, pH 6.6, 1 M sodium
chloride, 0.02 M EDTA, and 0.01% Tween 20. Array was washed using
both nonstringent (1 M NaCl, 25°C) and stringent (1 M NaCl, 50°C) conditions before staining with PE streptavidin (10 ␮g/ml final). Gene chip
array was scanned using a 2500 Gene Array Scanner and analyzed using
the Microarray Suite 5.0 software (both Affymetrix).
The p values for detection of individual genes were calculated, and
absent calls (detection p value ⬎0.06) were removed. Before comparison
analysis, a global normalization method was used to correct variations and
normalize intensity levels. The comparison analysis was performed by Wilcoxon signed rank test to examine the hybridization intensity data from one
gene chip and compare that with another gene chip. Relative changes in the
expression level of each target RNA were noted. Among the significantly
changed RNA levels, those with at least 2-fold increased or decreased
average RNA levels were taken into consideration.
Flow cytometry analysis
Single-cell suspensions of lymph node cells were incubated for 30 min
with one of the following anti-mouse Abs: PE-conjugated anti-CD4 and
anti-CD19; FITC-conjugated anti-CD5 and anti-CD8 (all from BD Biosciences, San Diego, CA). PE- or FITC-conjugated rat isotypes were used
for controls. Cells were washed twice and then were fixed with 2% paraformaldehyde and analyzed with a BD FACScan (BD Biosciences).
Immunohistochemical staining for germinal centers
Four-micrometer-thick sections of 10% formalin-fixed and paraffin-embedded spleens were prepared. Sections were deparaffinized and rehydrated. Endogenous peroxidase activity was quenched with 3% H2O2 in
methanol. Sections were blocked for nonspecific binding with normal goat
serum diluted 1/20 in DAKO (Carpinteria, CA) Ab diluent for 15 min. Ag
retrieval was done with DAKO Target Retrieval Solution in steam for 20
min. Sections were then cooled down on the bench top for 20 min, rinsed
two to three times with distilled water, and transferred to TBS. Slides were
then incubated for 30 min with peanut agglutinin (PNA)-biotin (Vector
Laboratories, Burlingame, CA) diluted 1/250 in DAKO Ab diluents and
washed, followed by a second incubation with streptavidin-HRP for the
LSAB2 system (KO675). Bound conjugates were visualized with DAKO
liquid diaminobenzidine substrate-chromagen for 5 min when a brown
color for PNA-positive cells was obtained. For all staining steps, a DAKO
Autostainer was used. Slides were counterstained for 2 min with Mayer’s
modified hematoxylin diluted 1/5 in distilled water.
Immunohistochemical and immunofluorescence staining for B
cell markers
Seven-micrometer-thick spleen sections were frozen in liquid nitrogen, and
stored at ⫺80°C. Slides were allowed to air dry and then were fixed in cold
acetone. Endogenous peroxidase activity and nonspecific binding were
blocked with 3% H2O2 in methanol and 20% FBS in PBS, respectively.
After washing with PBS, the sections were incubated overnight at 4°C in
the presence of rat anti-mouse B220 or rat anti-mouse IgM Abs (both from
BD PharMingen, San Diego, CA) (Abs diluted 1/400), followed by biotinconjugated goat anti-rat IgG Ab (Kirkegaard & Perry Laboratories, Gaithersburg, MD) (diluted 1/500), avidin-HRP (BD PharMingen (diluted
1/500), and DAKO liquid diaminobenzidine substrate-chromogen for development of a brown color. For detection of CD19⫹ B cells on the spleen
tissue, frozen sections were incubated with FITC-conjugated rat antimouse CD19 Ab (BD PharMingen) (diluted 1/500) overnight at 4°C and
then viewed in a fluorescence microscope (Olympus IX-70). Digital pictures were taken with a DP-11 digital camera.
Measurement of serum C3 and C1q-CIC levels by ELISA
Ninety-six-well microtiter plates (Dynex-Immulon 2; Dynatech Laboratories) were covered with goat Abs to mouse C3 (ICN Biomedicals/Cappel,
Aurora, OH) in 0.1 M sodium carbonate buffer (pH 8.2) overnight at 4°C.
The plates were then blocked with 2% BSA in PBS at room temperature for
30 min. Diluted (1/10 in PBS-0.05% Tween 20) serum samples (30 ␮l)
were added and incubated at 37°C for 90 min. After four washes, HRPconjugated goat anti-mouse C3 complement (ICN Biomedicals/Cappel),
diluted 1/500 in PBS/0.05% Tween 20, was added and incubated at 37°C
for 90 min. Subsequently, ABTS substrate solution in 0.1 M citric buffer
(pH 4.3) in the presence of H2O2 was added, and color was allowed to
develop at room temperature in the dark. Plates were read at a wavelength
of 405 nm using a Dynatech ELISA reader, and the results were expressed
as OD values. Serum C1q-CIC levels were determined with the same principle, with the difference that the plate was covered with goat Abs to
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For in vivo studies, all mice were anesthetized and immunized with 20 ␮g
of AChR emulsified in CFA (Difco, Detroit, MI) s.c. at four sites (two hind
footpads and shoulders) on day 0. All of the mice were boosted with 20 ␮g
of AChR in CFA s.c. at four sites on the back on day 30 (second immunization). For clinical examination, mice were left for 3 min on a flat
platform and were observed for signs of EAMG. Clinical muscle weakness
was graded, as follows: grade 0, mouse with normal posture, muscle
strength, and mobility; grade 1, normal at rest, with muscle weakness characteristically shown by a hunchback posture, restricted mobility, and difficulty to raise the head after exercise, consisting of 20 –30 paw grips on
cage top grid; grade 2, mouse showed grade 1 symptoms without exercise
during observation period on flat platform; grade 3, dehydrated and moribund with grade 2 weakness; and grade 4, dead.
CIC IN EXPERIMENTAL MYASTHENIA
The Journal of Immunology
5745
Results
AChR-immunized RIIIS/J mice revealed increased incidence,
severity, and earlier onset of EAMG compared with H-2compatible B10.RIII mice
RIIIS/J mice exhibited an enhanced anti-AChR Ab response
following AChR immunization, despite B cell deficiency and
lower total serum IgG levels
FIGURE 1. Kinetics of severity and accumulated clinical incidence of
EAMG for RIIIS/J, RIIIS/J ⫻ B6, B10.RIII, and B6 mice. RIIIS/J mice had
significantly higher average clinical grades as compared with other strains
of mice using Mann-Whitney U test (all p values ⬍0.01) (A). The clinical
incidence values of RIIIS/J, F1, and B10.RIII mice were significantly
higher than those of B6 mice using Fisher’s exact test (all p values ⬍0.001)
(B). RIIIS/J mice also had earlier onset of disease, with 20% of mice
developing EAMG before first immunization (day 28). The data for each
mouse strain represent the highest EAMG incidence obtained in either one
of three experiments (numbers of RIIIS/J, F1, B10.RIII, and B6 mice are
10, 8, 10, and 14, respectively). Bars indicate SEs.
human C1q (Sigma-Aldrich) and HRP-conjugated goat anti-mouse IgG
(Caltag Laboratories) was used as a secondary Ab.
Statistical analysis
To determine the significance of the observed results, three statistical tests
were used. Incidences of clinical EAMG were compared using the Fisher’s
exact test, clinical scores were compared using Mann-Whitney U test, and
all other parameters were compared using Student’s t test.
Sera from individual mice were collected before immunization and
45 days after the first (15 days after the second) immunization with
AChR in CFA. The anti-AChR IgG Ab response was measured by
RIA and ELISA, and total serum IgG levels were detected by
ELISA. Both RIIIS/J and F1 mice had significantly lower total
serum IgG levels before and after immunization (Fig. 2A), and
significantly higher serum anti-AChR Ab levels in response to
AChR immunization (Fig. 2B), as compared with B10.RIII and B6
mice. These results indicate that the B cells of RIIIS/J mice may be
responding vigorously to AChR immunization with a very restricted TCR V␤ repertoire due to the deletion of 70% of V␤ genes
and possibly a restricted Ig repertoire because of partial B cell
deficiency (Fig. 3). Furthermore, the B cell deficiency and reduced
total serum IgG were not corrected in (RIIIS/J ⫻ B6)F1 progeny.
A kinetic analysis of serum anti-AChR Ab levels (Fig. 2C) revealed that RIIIS/J mice have a lower IgM response as compared
with B10.RIII mice throughout the experiment, and they lack the
initial IgM peak observed in B10.RIII mice. These findings indicate that the higher serum anti-AChR IgG levels (Fig. 2B) of
Table I. Severe EAMG in RIIIS/J mice compared with B10.RIII and B6 mice
Clinical Grades %
Strains
Grade 1
Grade 2
Grade 3
p Values for Grade 3
(compared with RIIIS/J)a
RIIIS/J
B10.RIII
B6
RIIIS/J ⫻ B6
10
20
7.1
50
50
80
78.6
37.5
40
0
7.1
12.5
⬍0.0001
⬍0.0001
⬍0.0001
a
b
Average Days of
Onset after
First Immunization
p Values for
Onset of Disease
(compared with RIIIS/J)b
35.2
35.6
37.9
44.3
0.3899
0.0444
0.0004
Disease incidences were compared using Fisher’s exact probability test.
Average values of days between first immunization and onset of disease were compared using Student’s t test.
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To induce EAMG, RIIIS/J, B10.RIII, B6, and (RIIIS/J ⫻ B6)F1
(F1) mice were immunized with AChR in CFA. Among all strains
tested, RIIIS/J mice had significantly higher average clinical
grades (Fig. 1), and the ratio of mice with severe (grade 3) EAMG
was remarkably higher than that of other strains (Table I). One
hundred percent of RIIIS/J, B10.RIII, and F1 mice developed
EAMG, whereas the incidence of clinical EAMG was 92.8% in B6
mice (Fig. 1). Moreover, RIIIS/J mice developed disease significantly
earlier than F1 and B6 mice (Table I), and 20% of RIIIS/J mice
developed the clinical signs of EAMG before the second AChR
immunization. MHC class II molecule is essential for the development of EAMG (2), and therefore, we immunized mouse strains with
identical MHC class II alleles, I-Ar (RIIIS/J and B10.RIII mice), and
also EAMG-susceptible B6 (I-Ab) mice. The significantly increased
severity of clinical EAMG observed in RIIIS/J mice despite sharing
the MHC class II allele I-Ar with B10.RIII mice suggests that
non-MHC genes or factors influence disease severity in RIIIS/J mice.
The high incidence and severity of EAMG in RIIIS/J mice make them
an excellent new preclinical model for MG. The presence of EAMGsusceptible H-2b and H-2r alleles in the F1 mice did not augment
disease, suggesting that H-2r homozygosity and/or homozygosity in
the non-H-2 gene(s) conferred increased EAMG susceptibility (as
observed in RIIIS/J mice).
5746
RIIIS/J mice 2 wk after second immunization might be associated
with augmented class switching from IgM to IgG, as compared
with B10.RIII mice, starting from 5 days following the second
AChR immunization. Additionally, RIIIS/J mice had lower serum
anti-AChR IgG1, but significantly higher serum anti-AChR IgG2b
and IgG3 levels as compared with B10.RIII mice.
CIC IN EXPERIMENTAL MYASTHENIA
TCR V␤ gene deletion did not contribute to increased EAMG
severity of RIIIS/J mice
Initially, we hypothesized that V␤ gene deletion might have been
contributing to increased EAMG susceptibility due to the deletion
of T cells bearing specific V␤ genes involved in the regulation of
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FIGURE 2. Total serum IgG and anti-AChR Ab kinetic analysis in B10.RIII and RIIIS/J mice. Comparisons of A, total serum IgG vs B, anti-muscle
AChR Ab levels of all mouse strains before and 45 days after the first AChR immunization and kinetics of serum levels of anti-AChR IgM and IgG isotypes
(C). Asterisks on the top of the bars in A and B indicate p values for the comparisons between a specified strain of mice and B10.RIII mice separately for
before- and after- immunization values, and in C, between RIIIS/J and B10.RIII mice for each time point. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001 using
Student’s t test. Bars indicate SEs. One representation of three independent experiments.
The Journal of Immunology
autoimmunity (15). In a previous study, we used congenic mice
expressing V␤a, V␤b, and V␤c in the B10 background. The incidence of EAMG was increased in V␤c-bearing B10.TCRc mice,
suggesting the possible linkage of V␤c genotype to EAMG susceptibility (15, 24). To confirm the role of V␤c genotype in
EAMG, we performed Mendelian backcross genetics by making
an F1 generation between RIIIS/J (V␤c) and B10.RIII (V␤b) mice
and backcrossing them to RIIIS/J mice. According to the Mendelian genetics, the backcross segregated 1:1 V␤c and V␤b/c genotypes. The V␤c and V␤b/c mice were immunized with AChR and
were screened for clinical EAMG. There was no statistically significant difference between the EAMG incidences of mice with
V␤c and V␤b/c genotypes. Thus, the Mendelian genetic analysis
ruled out the influence of the V␤c genotype in RIIIS/J mice as an
EAMG susceptibility contributing factor (Table II).
Augmented TCR and diminished NK receptor and complement
gene expression of RIIIS/J mice in DNA microarray analysis
Table II. EAMG incidence in mice with V␤c and V␤b/c genotype
derived from (RIIIS/J ⫻ B10.RIII)F1 ⫻ RIIIS/J backcross
V␤
Genotype
V␤c
V␤b/c
Number of AChRImmunized Mice
11
12
EAMG Grade Grade
Incidence
1
2
(%)
(%)
(%)
9.1
33.3
0
25
9.1
8.3
p Value for Total
Incidence
0.13
be due to a compensatory increase in T cell numbers and/or TCR
expression due to the deletion of 70% of V␤ genes. NK cell receptor and CD63 (monocyte-macrophage marker) genes were expressed less in RIIIS/J mice as compared with B10.RIII mice, suggesting that RIIIS/J mice might have a phagocytic cell deficiency.
Interestingly, two complement proteins, C1q and C3, that are
known to be important for EAMG induction (14) were underexpressed, and the complement inhibitor DAF-2 was overexpressed
in RIIIS/J mice. There is a large amount of data regarding the
significant role of proinflammatory cytokines in EAMG (6 –10).
However, neither microarray analysis nor cytokine measurements
of serum samples or supernatants of AChR or immunodominant
peptide ␣146 –162-stimulated lymph node cells of AChR-immunized RIIIS/J mice revealed any significant alteration for any of the
cytokines (IL-2, IL-6, IL-10, IL-18, and IFN-␥) tested (data not
shown), suggesting that increased or decreased production of these
cytokines between RIIIS/J and B10.RIII mice might not be associated with increased EAMG severity of RIIIS/J mice. However,
TNF superfamily, member 7 (CD27) expression was increased
more than 2-fold in RIIIS/J mice. Overall, the major identifiable
susceptibility factor seemed to be increased TCR expression in
RIIIS/J mice. The increased TCR expression correlates with augmented lymph node T cell counts in RIIIS/J mice (Fig. 3). Additionally, production of genes that might potentially be related with
EAMG due to their importance in isotype switching (protein tyrosine phosphatase receptors and CD27), Ag presentation (cathepsin E), and cytokine network (IL-11R and IFN-induced genes) was
also enhanced in RIIIS/J mice. Microarray data are freely available
on the website Array Express, accession number: E-MEXP-86.
RIIIS/J mice had increased T cells and reduced B cells in their
lymph nodes before and following AChR immunization
FIGURE 3. FACS analysis of lymph node T and B cell counts in unimmunized and AChR-immunized mice, estimated by FACS analysis. Results are expressed as the individual percentage of positive cells for each
marker. Percentages at the top of bars indicate the ratio of difference between RIIIS/J and B10.RIII mice for the specified markers. Results obtained for individual mice of each strain were compared using Student’s t
test. ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001; ⴱⴱⴱⴱ, p ⬍ 0.0001; ⴱⴱⴱⴱⴱ, p ⬍ 0.00001
(between RIIIS/J and other strains); and bars indicate SEs. Data represent
one of three independent experiments.
T and B cell ratios of the lymph nodes of RIIIS/J and B10.RIII
mice were estimated by detecting the expression of CD4, CD5,
CD8, and CD19 molecules in the lymph node cells using flow
cytometry analysis. As illustrated in Fig. 3, both unimmunized and
immunized RIIIS/J and F1 mice had significantly increased
amounts of T cells (CD4, CD5, and CD8⫹ cells) and decreased
levels of B cells (CD19⫹ cells) as compared with B10.RIII and B6
mice, suggesting that this cell profile is an inborn characteristic of
RIIIS/J mice and is only intensified, but not induced following
AChR immunization. Not only the percentages of CD4⫹, CD8⫹ T
cells, and CD19⫹ B cells, but also their absolute numbers were
significantly augmented and diminished, respectively (data not
shown). Therefore, increased TCR gene expression obtained in
microarray data is more likely to be the consequence of increased
T cell numbers rather than increased receptor expression or of both
factors. Moreover, the elevated ratio of CD8⫹ T cells in F1 mice
(with a moderate EAMG severity) was comparable to that of
RIIIS/J mice, whereas RIIIS/J mice had exceedingly higher numbers of CD4⫹ T cells, suggesting that augmented CD4⫹ cell population and TCR gene expression could have contributed to heightened cellular and humoral immune response to AChR (i.e., IgG
isotype switching (25)) and disease in RIIIS/J mice. It is very
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To identify the genetic determinants associated with increased
EAMG severity in RIIIS/J mice, DNA microarray analysis was
performed using the spleens of adult unimmunized RIIIS/J and
B10.RIII mice. Among 12,488 cDNAs analyzed by microarray
analysis, 263 were overexpressed and 303 were underexpressed
greater than 2-fold in RIIIS/J mice as compared with B10.RIII
mice. The most notable finding was the higher expression of TCR
genes in RIIIS/J mice as compared with B10.RIII mice. This could
5747
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CIC IN EXPERIMENTAL MYASTHENIA
interesting that RIIIS/J mice have ⬃50% less CD19⫹ B cells as
compared with B10.RIII and B6 mice. Despite the reduction in the
lymph node B cells before and after immunization with AChR in
CFA, the serum anti-AChR Ab levels were very high in RIIIS/J
mice. Therefore, RIIIS/J mice are not deficient in self (AChR)reactive B cells and rather have a higher frequency.
Spleen cells of RIIIS/J mice revealed suppressed surface
markers for B cells at maturation stages and decreased amounts
of germinal centers
To further characterize the role of B cells in the secondary lymphoid organs of RIIIS/J mice, spleens and lymph nodes of AChRimmunized and unimmunized RIIIS/J, B10.RIII, and F1 mice were
collected and flash frozen. The frozen sections were tested by immunohistochemistry or immunofluorescence for the presence of
pro-B cell (B220, CD19) and immature B cell (IgM) markers and
also germinal centers by PNA staining on paraffin-embedded sections. RIIIS/J mice revealed lower intensity of staining for all
markers tested as compared with B10.RIII and B6 mice (Fig. 4),
suggesting that B cells are deficient as a result of deficient production from the very early stages of maturation due to a not yet
known mechanism. The reason for stronger B220 staining in Fig.
4 might be due to the fact that this molecule is also found on the
surfaces of dendritic cells and NK cells. In contrast, LPS-induced
B cell and Con A-induced T cell responses of spleen cells of
RIIIS/J mice indicated that both B and T cells are capable of duly
responding to mitogen stimulation (data not shown). PNA staining
revealed an extraordinary circular staining pattern around the usual
anatomical location for germinal centers in both RIIIS/J and
B10.RIII mice, suggesting either different germinal center struc-
ture for these strains of mice or deficient B cell activation by T
cells (Fig. 4). Nevertheless, in these mice, the average number of
germinal centers was significantly lower than that of B6 mice (data
not shown). Results obtained from lymph nodes of mice with orwithout AChR immunization and from spleens of mice without
AChR immunization were highly comparable to the results obtained from spleen sections of AChR-immunized mice (identical
germinal center architecture, but decreased staining intensity for
all markers tested in unimmunized mice’s lymph node and spleen
sections).
RIIIS/J mice had remarkably higher serum C3 and C1q-CIC
levels after AChR immunization, despite lower serum levels of
C3 and C1q-CIC before AChR immunization
As supportive data to the molecular analysis experiments, naive
RIIIS/J mice had diminished serum C3 and C1q-CIC levels as
compared with B6 mice (although this difference did not attain
statistical significance). However, AChR immunization significantly augmented the production of both serum C1q-CIC (Fig. 5A)
and C3 (Fig. 5B), particularly in RIIIS/J mice. Moreover, there was
a significant correlation between clinical scores of RIIIS/J mice
and their serum C1q-CIC ( p ⫽ 0.0004 and r ⫽ 0.9698) levels (but
not C3 levels, tested by Spearman’s method for nonparametric
correlations) (Fig. 5C). Given the reported importance of C3 in
isotype switching, we suggest that the high C3 levels of F1 and
RIIIS/J mice could have contributed to the high serum anti-AChR
IgG levels in these two strains. In this study, we observed that: 1)
serum C1q-CIC levels are augmented in AChR-immunized RIIIS/J
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FIGURE 4. Immunohistochemistry showing reduced numbers of B cells and germinal centers in the
splenic tissue of RIIIS/J mice. Spleens were isolated
at day 45 after second AChR immunization and immunohistochemistry was performed by using PNA
as the marker for germinal centers or indicated B
cell surface markers for different maturation levels
of B cells. For markers B220, IgM, and PNA, dark
areas represent positively stained sections of germinal centers for each examined marker, and light areas represent nonstained sections, whereas for
marker CD19, dark areas represent nonstained and
bright regions represent positively stained sections
of the germinal centers. Excessively bright sections
belong to the blood vessels. Original magnification,
⫻100. One representation of 5 ⫻ 3 spleen sections
for each strain. Only spleen sections are presented
here as representative of all tissue samples tested.
The Journal of Immunology
5749
mice; 2) there is a direct correlation between clinical EAMG severity and C1q-CIC levels in RIIIS/J mice; and 3) mouse strains
with higher clinical severity have higher serum C1q-CIC levels.
These data implicate that C1q-CICs play an essential role in the
pathogenesis of EAMG and thus constitute the first evidence for
association between serum C1q-CIC levels and clinical severity
of EAMG.
Discussion
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Inbred mouse strains with well-defined immune deficiencies or
augmented immune functions enable the characterization of resistance or susceptibility factors of individual immunological diseases. RIIIS/J mice constitute a special example in this respect due
to an immunodeficiency manifested as deletion of ⬃70% of TCR
V␤ genes (16), low B cell counts in the peritoneal cavity (26),
spleen (19) and lymph nodes (Fig. 3), low levels of serum IgM
(26), low Ab responses to T cell-dependent or -independent Ags
(27), and yet by their augmented immunological functions including increased spleen T cell frequencies (19), elevated complement
activation capacity (28), and serum C3 and C1q-CIC levels in our
studies following AChR immunization (Fig. 5). Similarly, it is also
noteworthy that they are resistant to T cell-dependent EAE (17–19)
and CIA (20 –22), while being highly susceptible to Ab- and complement-mediated EAMG, as reported in this work. Being highly susceptible to EAMG, while having a B cell immunodeficiency, makes
this mouse strain a potential candidate to study the association between autoimmune diseases and hypogammaglobulinemia.
There are interesting studies attempting to link the resistance of
RIIIS/J mice to EAE to certain genetic loci (17, 19). Moreover,
genetic linkage analysis suggested that the major loci controlling
CIA in crosses between RIIIS/J and B10.RIII mice were located
outside the MHC region (20), and similarly the gene(s) influencing
the severity of EAMG in RIIIS/J mice appears to be located outside of the MHC region. It is also plausible that rather than gene(s),
other defective immunological factors in RIIIS/J mice could have
contributed to increased disease severity.
TCR V␤ gene deletion does not contribute to RIIIS/J disease
severity
RIIIS/J mice (TCR V␤c haplotype) have a genomic deletion of
⬃130 kb of DNA encoding 13 V␤ genes (V␤ 5.2, 8.3, 5.1, 8.2, 5.3,
8.1, 13, 12, 11, 9, 6, 15, and 17) of the 21 known V␤ genes (16).
Therefore, it is conceivable that T cells bearing some of these V␤
molecules could have been involved in the regulation/suppression
of the autoimmune response to AChR, and thus played a role in
EAMG susceptibility of RIIIS/J mice. However, mice with V␤c
haplotype in F1 ⫻ RIIIS/J backcross failed to reveal an increased
EAMG susceptibility. In accordance with these results, previous
studies have shown that the use of multiple V␤ genes is required
in EAMG pathogenesis (29, 30), and the absence of some of the
V␤ genes does not cause a significant change in EAMG induction.
Although TCR V␤6 and V␤8 have been shown to be preferentially
used during the in vivo response to AChR (29, 30), neither depletion of V␤6⫹ T cells nor the genetic deficiency of V␤6 gene can
prevent the immune response to AChR or clinical development of
EAMG (30). Moreover, V␤8 transgenic mice do not reveal increased EAMG susceptibility or serum anti-AChR Ab levels (30).
Our results might also suggest that unlike myelin and collagen
Ags, AChR T cell epitope(s) appears to interact with multiple TCR
V␤ repertoire and can induce an immune response even in the
absence of a large group of V␤ genes. It has also been suggested
that autoimmune diseases might have been encountered in the
course of primary immunodeficiencies due to the abnormal han-
FIGURE 5. Serum C1q-CIC (A) and C3 (B) levels of all strains of mice
before and after AChR immunization. In RIIIS/J mice, serum C1q-CIC
levels are correlated with the clinical severity of EAMG (C). ⴱ, p ⬍ 0.05;
ⴱⴱ, p ⬍ 0.01 using Student’s t test; bars indicate SEs. One representation
of three independent experiments.
dling of certain Ags (i.e., superantigens) (31), and AChR might be
acting as a superantigen in RIIIS/J mice.
Elevated TCR gene expression and CD4⫹ T cells
The most significant factor revealed in microarray gene analysis
was the elevated TCR expression in the spleen tissues of RIIIS/J
5750
CIC IN EXPERIMENTAL MYASTHENIA
FIGURE 6. Hypothetical illustration to depict the chain of events leading to increased EAMG severity in
RIIIS/J mice.
clearance and the elimination of autoreactive B cells (34). Although MG is not frequently observed in B cell-immunodeficient
cases, several Ab-dependent, complement-mediated autoimmune
diseases such as MG (i.e., hemolytic anemia, idiopathic thrombocytopenic purpura, autoimmune glomerulonephritis) are detected
in these patients (31). RIIIS/J mice might thus constitute a good
model for research on B cell immunodeficiency syndromes (e.g.,
common variable immunodeficiency), in which hypogammaglobulinemia and complement-mediated autoimmune diseases occur together (31). Augmented EAMG in partial B cell-deficient RIIIS/J
mice suggests that partial B cell deletion alone should not be attempted in the clinical trial of MG, and rather specific depletion of
anti-AChR-producing B cells should be tried.
B cell immunodeficiency and autoimmunity in RIIIS/J mice
TCR V␤ gene deletion suppresses cell-mediated autoimmunity,
and B cell deficiency augments B cell-mediated autoimmunity
By using both immunohistochemistry/immunofluorescence methods and FACS analysis on the secondary lymphoid tissues of mice
(Figs. 3 and 4), we showed that RIIIS/J mice have lower B cell
counts, which is consistent with lower serum IgM levels (Fig. 2C)
observed in this strain. PNA staining is used for detection of germinal centers, and in cases of disturbed T cell-mediated B cell
activation, PNA staining of splenic tissue may be significantly reduced (32, 33). This might be indicative of the fact that both
RIIIS/J and B10.RIII mice have a problem with T cell-dependent
B cell activation. However, underexpression of B cell markers for
earlier B cell maturation stages (CD19, B220) might suggest that
B cell deficiency of RIIIS/J mice might not be only due to deficient
activation, but also due to decreased B cell production. Increased
elimination of B cells by either apoptosis or necrosis still remains
a possibility. However, FACS analysis on the lymph node cells
obtained from AChR-immunized RIIIS/J and B10.RIII mice did
not reveal any increase in necrotic (14.28 vs 10.45%) or apoptotic
(37.32 vs 36.13%) cells (assessed by 7-amino actinomycin D and
annexin-V staining, respectively) in RIIIS/J mice as compared
with B10.RIII mice. A deficiency of B cells has been proposed to
cause autoimmune diseases due to a consequential decrease of
complement receptors, which are important for immune complex
RIIIS/J mice are capable of producing high amounts of anti-AChR
Abs, despite their lower serum total IgG levels, which might be a
consequence of the restricted TCR V␤ and/or Ig repertoire, and
could lead to defective tolerance induction to self Ag and thus
emergence of self-reactive T and B cells. However, RIIIS/J mice
are not highly susceptible to other induced autoimmune diseases
such as EAE and CIA. These two diseases are predominantly mediated by T cells, while EAMG is an Ab- and complement-mediated disease. We can argue that the deficiency of B cells and possibly restricted Ig repertoire could augment Ab-mediated diseases,
while the restricted TCR V␤ repertoire could suppress the cellmediated diseases such as EAE or CIA due to the deletion of
specific V␤⫹ cells involved in the infiltration of the target tissues,
myelin in EAE, and synovium in CIA. Another important topic
about Abs is how RIIIS/J mice can produce increased amounts of
anti-AChR IgGs, while their anti-AChR IgM levels are lower than
other strains. In Fig. 2C, RIIIS/J mice are shown to make a less
intense and a quicker peak IgM response as compared with
B10.RIII mice, and it is also seen that at the same time, their IgM
levels are decreasing, while their serum IgG levels are rising in a
significantly steady state manner, eventually exceeding the IgG
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mice, presumably reflecting higher spleen T cell counts, as shown
in a previous report (19). Although our studies showed both increased CD4⫹ and CD8⫹ T cells, the latter subtype of cells does
not seem to be an important part of increased EAMG severity,
because F1 mice also had relatively increased CD8⫹ T cells in their
spleens. Plausibly, increased CD4⫹ T cells might have been inducing increased EAMG severity in RIIIS/J mice by also increasing production of cytokines relevant to EAMG induction. However, in several experiments, we could neither show any difference
in cytokine levels in serum samples or supernatants of AChRexposed lymph node cells of RIIIS/J mice (data not shown), nor
any alteration in the expression of any of the cytokines tested in
microarray analysis.
The Journal of Immunology
C3, C1q-CIC, and EAMG
It is remarkable that C3, which seems to be produced to a smaller
degree in RIIIS/J mice before immunization (as revealed by both
ELISA studies and microarray analysis), achieves a giant leap in
production after immunization and serum C3 levels of RIIIS/J
mice surpass that of other strains (Fig. 5). C3 and other complement components are known to be highly important for EAMG
induction and pathogenesis. We have previously shown that C3
(14) and C5 KO mice (13) are highly resistant to EAMG induction
by AChR immunization. However, it is difficult to attribute increased EAMG severity to serum C3 levels, because F1 mice,
which have moderate disease severity, have equivalent serum C3
levels. Our previous unpublished studies have shown that serum
C3 levels are not strictly associated with EAMG incidence and
severity, and highly EAMG-resistant strains may have serum C3
levels comparable to those of EAMG-susceptible B6 mice. Therefore, serum C3, possibly, is not the most significant or sole contributor of increased EAMG susceptibility in RIIIS/J mice, although it is rather plausible that it plays a very important role in
EAMG pathogenesis. It is notable that two strains (RIIIS/J and F1)
with higher serum C3 levels also have higher serum anti-AChR
levels. These high Ab levels might have been accomplished by
isotype switching enhancing effect of C3. C3 is known to have two
major roles in enhancing Ab production: 1) binding to CD35/21
receptors of B cells and dendritic cells in the secondary lymphoid
organs together with the Ag, and 2) assisting isotype switching. In
the absence of C3, B cells undergo apoptosis because of inadequate Ag stimulation (35, 36). To our knowledge, previous studies
performed to explore these mechanisms in more depth have been
performed either using C3 KO mice or immunizing mice with
C3-conjugated Ags (37, 38). However, both attempts either eliminate or activate both mechanisms. Reminiscent of C3 KO mice,
RIIIS/J mice have low lymph node and spleen B cell numbers. Our
results show that with lower B cell numbers and a less intense
primary immune response, but increased serum C3 levels, RIIIS/J
mice are still capable of producing high AChR-specific IgG levels,
which might be due to enhancement of isotype-switching effect by
C3 or other yet unknown molecules. In this respect, CD27, which
was overexpressed in microarray analysis, might be of particular
interest for future studies, because this molecule is known to be
important for isotype switching.
Elevated serum C1q-CIC levels of RIIIS/J mice might be a better explanation for increased EAMG severity because serum C1qCIC levels are higher in RIIIS/J mice than all other three strains
(Fig. 5). Increased serum C1q-CIC levels might be increasing disease severity by increasing the efficiency and magnitude of Ab-Ag
interactions at the NMJ. We recently showed that the classical
complement pathway is crucial for induction of EAMG, and C4
KO mice are highly resistant to EAMG (14). Elevated immune
complex levels might also be facilitating better complement activation, increased C3 and MAC production at the NMJ, and thus
more intense NMJ destruction. To our knowledge, this is the first
study showing an association between serum C1q-CIC levels and
clinical severity of EAMG. We predict that serum C1q-CIC measurement in human MG patients could predict clinical severity and
response to treatment.
Concluding remarks
Based on studies that RIIIS/J mice are resistant to certain autoimmune diseases while being highly susceptible to EAMG, we propose the below hypothesis: the increased EAMG severity of
RIIIS/J mice might be due to increased amounts of CD4⫹ T cells
and increased complement activation capacity. Despite lower B
cell counts, AChR triggers a very strong immune response in
RIIIS/J mice, and B cells of RIIIS/J mice are capable of producing
high amounts of anti-AChR IgGs. These Abs conjugate with C1q
to form C1q-CICs, which can potentially activate T cells (39),
explaining significant increase in CD4⫹ cells in RIIIS/J mice after
AChR immunization. Activation of the complement cascade by
C1q increases the production of C3, and C3 further increases Ab
production. This vicious circle between T cells, C3, and IgGs (Fig.
6), combined with a highly active complement system, stimulates
the production of higher amounts of immune complexes and C3,
which eventually destroy NMJ and thus increase EAMG severity.
We believe that our findings and further studies testing the abovementioned predictions might aid in understanding mechanisms underlying the pathogenesis of autoimmune diseases observed during
the course of B cell immunodeficiency syndromes.
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