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*
Institut National de la Santé et de la Recherche Médicale, Unit 28, Institut Fédératif de Recherche 30, and Université Paul Sabatier, Hôpital Purpan, Toulouse, France; Departments of
Immunology and
Neurology, University of Maastricht, Maastricht, The Netherlands; and
§
Unité de Neurobiologie Moléculaire, Institut Pasteur, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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than BN lymph node cells, but
expressed less IL-4 mRNA. IgG1 and IgG2b anti-AChR isotype
predominated in BN and LEW rats, respectively, confirming the dichotomy
of the immune response observed between the two strains. Furthermore,
although IL-12 administration or IFN- neutralization strongly
influenced the Th1/Th2 balance in BN rats, it did not affect the
disease outcome. These data demonstrate that a Th1-dominated immune
response is not necessarily associated with disease severity in EAMG,
not only in rats with disparate MHC haplotype but also in the same rat
strain, and suggest that in a situation where complement-fixing Ab can
be generated as a consequence of either Th1- or Th2-mediated T cell
help, deviation of the immune response will not be an adequate strategy
to prevent this Ab-mediated autoimmune disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental autoimmune myasthenia gravis (EAMG) can be induced in susceptible mouse and rat strains by immunization with AChR from the electric organs of Torpedo (tAChR) emulsified in CFA. Animals develop a T cell-dependent Ab response against tAChR that cross-reacts with their own receptor, resulting in a neuromuscular disease with clinical symptoms resembling human MG (1). It is now well accepted that weakness and fatigability of the voluntary muscles, which are the hallmarks of MG, are due to autoantibody-mediated destruction of AChR at the neuromuscular junctions (6, 10). Several mechanisms may account for the pathogenicity of anti-AChR Abs. They include complement-mediated destruction of the postsynaptic membrane and accelerated internalization and degradation of functional AChR (10, 11, 12, 13). Although susceptibility to this disease has been mapped to the I-A locus in the mouse (14, 15, 16), there is evidence in rats (17) and in mice (13) that non-MHC-linked genes may control resistance and susceptibility to EAMG.
CD4+ T cells play a central role in the induction and
regulation of the immune response and have been shown to be
phenotypically and functionally heterogeneous in rats (18, 19), mice
(20), and humans (21). In the rat, CD4+ T cells can be
subdivided into two major subsets based on their different lymphokine
production patterns (19). Th1 cells, which produce IL-2 and IFN-,
can transfer cell-mediated immunity. These cells also induce
preferentially the synthesis of Abs expressing the IgG2b isotype (22, 23). Conversely, Th2 cells produce IL-4 and cause B cell proliferation
and differentiation, eliciting mainly IgG1 and IgE Ab production.
Polarized Th1 responses have been initially implicated in the
pathogenesis of many organ-specific autoimmune diseases (24, 25).
However, recent studies have shown that Th2 cells can also mediate this
type of disease when transferred into immunodeficient hosts (26, 27).
Concerning the role of Th cell subsets in EAMG, several recent studies
in mice have demonstrated a crucial role for IFN- and IL-12, but not
for IL-4, in the pathogenesis of EAMG (28, 29, 30, 31). However, the role of
these cytokines in the development of MG symptoms in humans and rats
has not yet been clarified.
Since EAMG is mediated by T cell-dependent, complement-fixing Ab
response (13, 14, 32, 33), the requirement for IL-12 and IFN- in
mice could be explained by the fact that the generation of
complement-fixing IgG subclasses in this species is dependent on
Th1-mediated T cell help (30). Conversely, in the rat, both Th1 and Th2
cells can help B cells to make complement-fixing Abs (34, 35).
Therefore, we postulated that EAMG in rats, unlike that in mice, could
occur in the context of a polarized Th1- or Th2-type immune response.
To test this hypothesis, we analyzed the relative contributions of
CD4+ T cell subsets in the development of rat EAMG by 1)
comparing the polarization of the immune response and the development
of EAMG between Lewis (LEW) and Brown-Norway (BN) rats, which differ
markedly in their susceptibility to develop either Th1- or Th2-mediated
autoimmune manifestations (36); and 2) manipulating the Th1/Th2 balance
by administration of IL-12 or anti-IFN- mAb in the same rat
strain (BN). The results obtained show that a Th1-dominated immune
response is not necessarily associated with disease severity, not only
in rats with disparate MHC haplotype but also in rats of the same
strain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to 10-wk-old LEW or BN rats of either sex were used in this study. These animals were obtained from the Centre d’Elevage R. Janvier (Le Genest St. Isle, France) and were maintained in our animal house facility in specific pathogen-free conditions. In individual experiments, all animals were of the same sex.
Antibodies
Anti-IFN- mAbs, DB1 and DB12, were provided by Dr. P. H.
Van der Meide (37). Isotype-specific mouse anti-rat mAbs, MARG1–2
(anti-rat IgG1), MARG2a-1 (anti-rat IgG2a), and MARG2b-3
(anti-rat IgG2b), were obtained from LO/IMEX (University of
Louvain, Brussels, Belgium) and were biotinylated.
Peroxidase-conjugated goat anti-rat IgG was a gift from E. Druet
(Institut National de la Santé et de la Recherche Médicale,
Unit 28, Toulouse, France).
Induction and clinical scoring of EAMG
tAChR was purified from electric organs of Tordepo marmorata by affinity chromatography. Briefly, the AChR-rich membranes were prepared as described by Saitoh et al. (38) and then treated at pH 11 as previously described (39). Alkaline-treated membranes were incubated with 2-ME for 2 min at a final concentration of 3 mM and then solubilized by the addition of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) buffer (5 mM Na2PO4, 1 mM EDTA, 100 mM NaCl (pH 7.2), and 0.4% CHAPS). The supernatant was applied to a bromoacetylcholine affinity column and eluted with 50 mM carbamylcholine. Carbamylcholine was then removed by repeated concentrations using Centripreps (Millipore, Saint Quentin-Yvelines, France) cutting at 50 kDa. To induce active EAMG, rats were immunized with 20 or 2 µg of tAChR emulsified in CFA (Difco, Detroit, MI) in a total volume of 100 µl, injected in the hind footpads. Control rats received an equal volume of PBS in CFA. The severity of clinical signs of disease in EAMG was scored by measuring muscular weakness. The muscle strength of the animals was assessed by their ability to grasp and lift repeatedly a 300-g rack from the table while suspended manually by the base of the tail for 30 s. Clinical scoring was based on the presence of tremor, hunched posture, muscle strength, and fatigue. Animals were scored daily for clinical signs of disease on a scale from 0–3 depending on severity: 0, normal; 1, no abnormalities before testing, but reduced strength at the end; 2, clinical signs present before testing, i.e., tremor, head down, hunched posture, and weak grip; and 3, severe clinical signs present before testing, no grip, and moribund (40, 41).
IL-12 and anti-IFN- treatment
Mouse rIL-12 was a gift from Dr. D. H. Presky (Hoffmann-La
Roche, Nutley, NJ) and was diluted in PBS containing 1% homologous rat
serum to give a final concentration of 2 µg/ml. BN rats were
immunized with 2 µg of tAChR in CFA on day 0 in the hind footpads and
injected i.p. once a day with 2 µg/rat of rIL-12 from days 0–5.
Control rats received the same volume of PBS-1% homologous serum.
Concerning the anti-IFN- treatment, BN rats were immunized with
2 µg of tAChR in CFA and injected i.p. with 5 mg of DB1 mAb on day 0
and with 3 mg of DB1 mAb on days 3, 5, 7, 10, 12, and 14 following
immunization.
Measurement of muscle AChR content
The concentration of AChR present in the total body musculature
was measured in muscle detergent extract by RIA as previously described
(42). Briefly, the frozen carcasses were homogenized, and
membrane-bound proteins were extracted with PBS containing 2% Triton
X-100 (Sigma, St. Louis, MO). An aliquot of 250 µl of each extract
was labeled with 2 x 10-9 M 125I-labeled
-bungarotoxin, incubated overnight with an excess of rat
anti-AChR IgG, and precipitated by goat anti-rat IgG. The
concentration of AChR in muscle was expressed as picomoles of
125I--bungarotoxin precipitated per 100 g muscle,
and the percentage of rat AChR loss in test rat carcasses was
calculated by comparison with that in control animals.
RIA for serum anti-rat AChR Abs
The concentration of Abs reactive with rat AChR was determined
in individual sera by RIA as previously described (42). Briefly, rat
AChR was extracted from denervated rat muscle and labeled with 2
x 10-9 M 125I--bungarotoxin (ICN
Pharmaceuticals, Orsay, France). A dilution range of serum samples was
incubated overnight with 200 µl of labeled rat AChR. Ab-AChR
complexes were precipitated by adding an excess of goat anti-rat
IgG Abs. The radioactivity of the complexes was measured in a gamma
counter. Values of 125I--bungarotoxin-AChR pelleted in
the presence of normal rat serum were subtracted from the assay values.
Corrections for interassay variability were made based on serial
dilutions of an EAMG standard control serum pool tested in each assay.
The Ab titers were expressed as moles of
125I--bungarotoxin binding sites precipitated per liter
of serum.
ELISA for serum anti-tAChR Ab titers and isotypes
For detection of tAChR-specific Abs, a standard ELISA technique
was applied. Briefly, microtiters plates (Falcon 3012, Becton Dickinson
Labware, Oxnard, CA) were coated overnight at 4°C with 0.5 µg/ml of
tAChR in PBS. Bound IgG1, IgG2a, and IgG2b anti-tAChR were revealed
using biotinylated mouse anti-rat 1, 2a, and 2b mAbs
respectively. The bound biotinylated mAbs were revealed by addition of
preformed streptavidin-biotin-peroxidase complexes (Amersham, Slough,
U.K.) for 60 min at room temperature. For total IgG measurement, sera
were incubated with peroxidase-conjugated sheep anti-rat IgG. The
plates were washed and incubated with the developing substrate,
3,3'-5,5'-tetramethylbenzidine (Fluka Chemie, Buchs, Switzerland). The
reaction was stopped by adding 50 µl/well of 2 N
H2SO4, and absorbance was read at 450 nm using
an automated microplate ELISA reader (Emax, Molecular Devices,
Sunnyvale, CA). Each serum was tested in duplicate and was assessed at
four different dilutions. Sera were titrated by comparison with a
reference curve built with a pool of sera from LEW and BN rats
immunized with tAChR, and results are expressed as the Ab concentration
in arbitrary units (AU)/ml. This pool was standardized for each
anti-AChR Ab isotype tested; 1 AU corresponds to an absorbance
value at 450 nm of 0.5. For each isotype the standard values were as
follows: IgG1, 2000 AU/ml; IgG2a, 8000 AU/ml; and IgG2b, 4000 AU/ml.
Proliferative response
Popliteal and para-aortic lymph node cells, collected on days 10–12 after immunization with 20 or 2 µg of tAChR, were stimulated with different concentrations of tAChR in 96-well culture plates (Costar, Cambridge, MA). Culture medium was RPMI 1640 (Life Technologies, Cergy Pontoise, France) containing 10% FCS, 1% pyruvate, 1% nonessential amino acids, 1% L-glutamine, 1% penicillin-streptomycin, and 2 x 10-5 M 2-ME. Proliferation was measured by the degree of [3H]thymidine uptake during the last 18 h of a 72-h culture period, and results were expressed as mean counts per minute of triplicate cultures.
Cytokine assays
At various times throughout the culture, supernatants were
removed and stored at -20°C for cytokine determination, cells were
harvested following 24- or 48-h stimulation, and RNA were purified for
analysis of lymphokine gene expression by RT-PCR. IL-2 production was
assessed by measuring the proliferation of the CTLL-2 cell line as
previously described (43). Briefly, culture supernatants were added to
2 x 104 CTLL-2 and incubated for 18 h at 37°C.
The cells were then pulsed with 0.5 µCi of
[3H]thymidine for 6 h, and incorporation of
radiolabel was measured by direct counting using an automated beta
plate counter (Matrix 9600, Packard, Meriden, CT). Results were
calculated from a standard curve constructed using a commercial
preparation of human IL-2 (Boehringer Mannheim, Mannheim, Germany) and
expressed as units per milliliter of IL-2. IFN- protein in the
supernatant was measured by specific ELISA. Ninety-six-well plates were
coated overnight at 4°C with 5 µg/ml of an anti-rat IFN- mAb
(DB1). Serial dilutions of tissue culture supernatant (100 µl/well)
followed by biotinylated DB12, an anti-rat IFN- mAb, were
sequentially incubated for 2 h at room temperature, separated by
three washes. The bound biotinylated Abs were revealed by an additional
60-min incubation with alkaline phosphatase-conjugated streptavidin
(Jackson ImmunoResearch Laboratories, Avondale, PA). The assay was
developed by adding the enzyme substrate 4-nitrophenylphosphate
disodium (Sigma) at 1 mg/ml in diethanolamine buffer, pH 9.6, for 90
min at room temperature. The absorbance was measured at 405 nm using an
automated microplate ELISA reader (Emax, Molecular Devices, Sunnyvale,
CA). Values were expressed as units per milliliter of IFN- derived
from a standard curve constructed using rat recombinant IFN- (a gift
from Dr. P. Van der Meide, TNO, Rijswijk, The Netherlands).
IL-4 mRNA detection
Total cellular RNA was isolated from 3–5 x 106 stimulated or unstimulated lymphocytes using the TRIzol procedure (Life Technologies). RNA was reverse transcribed to cDNA in a final volume of 40 µl as previously described (19). For semiquantitative PCR analysis of cytokine mRNA levels, a series of 3-fold dilutions of the cDNA (six dilutions for each sample) were amplified in a 50-µl reaction volume as previously described (19). Reactions were performed in a DNA thermal cycler (model 9600, Perkin-Elmer, Norwalk, CT) for the indicated number of cycles. Each cycle consisted of 93°C for 1 min, 60°C for 1 min, and 72°C for 1 min, using 35 cycles for IL-4 and 20 cycles for ß-actin. Following amplification, 10 µl of the amplified product was separated by electrophoresis on 2% agarose minigels and visualized by ethidium bromide staining. Photographs of gels were digitized, and densitometric analysis of the bands was performed using the Gel Analyst program (ICONIX, Paris, France). Results were expressed as AU and represent the ratio of the intensity of the band for IL-4 to the intensity of the band for ß-actin x 100. Primers used were as follows: ß-actin sense primer, 5'-ATG CCA TCC TGC GTC TGG ACC TGG C-3'; ß-actin antisense primer, 5'-AGC ATT TGC GGT GCA CGA TGG C-3'; IL-4 sense primer, 5'-TGA TGG GTC TCA GCC CCC ACC TTG C-3'; and IL-4 antisense primer, 5'-CTT TCA GTG TTG TGA GCG TGG ACT C-3' (19). These primers were designed to amplify cDNA fragments, representing mature mRNA transcripts of 607 bp for ß-actin and 378 bp for IL-4 cDNA.
Statistical analysis
Results are expressed as the mean ± SD, and overall differences between variables were evaluated by the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Susceptibility to disease induction and disease symptoms vary
considerably among different strains of rats: Wistar Furth are
resistant, while Fisher and Wistar Munich are highly susceptible to
EAMG (17). Interestingly, BN and LEW rats have been shown to exhibit an
intermediate phenotype, and they reproducibly develop the disease with
similar symptoms (17, 44). Indeed, the results in Table I
show that following single immunization
with 2 or 20 µg of tAChR/rat in CFA, disease incidence and onset were
similar in the two strains. Clinical symptoms appeared 5–9 wk after
tAChR immunization, were indistinguishable between LEW and BN rats, and
were characterized by moderate to severe muscular weakness.
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A, a single
immunization with 2 µg of tAChR in CFA resulted in a significant
reduction in muscle AChR content in both LEW and BN rats. The AChR loss
was similar in both strains and corresponded to a 50–60% reduction
compared with that in normal rats or control animals immunized with CFA
alone.
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B,
anti-rat AChR autoantibodies were detectable in both LEW and BN
rats from days 9–31 following immunization with tAChR. Although
variability occurred among individuals, no significant difference in
autoantibody titer was observed between the two strains for the three
time points tested. Since complement plays a central role in the
pathogenesis of EAMG (32, 33), we next compared the presence of
complement components C5b-9 (membrane attack complex) in muscle biopsy
cryosections from tAChR-immunized LEW and BN rats. The
immunohistochemical staining revealed comparable deposition of
complement components C5b-9 that coincided with the localization of
AChR (data not shown). This indicates that immunopathogenic mechanisms
are operational at the level of the target organ in both strains. Taken
together, these data confirm that according to several clinical and
biochemical criteria, LEW and BN rats are susceptible to the same
degree to EAMG induced by a single immunization with tAChR. Analysis of tAChR-specific T cell response in LEW and BN rats
To analyze the mechanisms involved in the generation of a
pathogenic immune response in EAMG, we analyzed the polarization of the
CD4+ T cell response in both LEW and BN rats. The results
in Fig. 2A show that immune
lymph node cells from tAChR-immunized LEW and BN rats proliferated
equally well in response to tAChR in vitro. Proliferation was also
identical when lower doses of Ag were used in vitro (not shown).
Although, tAChR appears to be strongly immunogenic in both strains, the
analysis of the Th1-associated cytokines in culture supernatants
revealed strong phenotypic differences between LEW and BN T cells. As
shown in Fig. 2, LEW Ag-specific T cells produced larger amounts of
IL-2 and IFN- than BN T cells. This difference was evident for all
Ag doses (Fig. 2, B and C) or all time points
tested (not shown). We also examined IL-4 mRNA synthesis by RT-PCR in
immune lymph node cells restimulated in vitro with the indicated amount
of tAChR. As shown in Fig. 2D, Ag-specific IL-4 mRNA
expression was much higher in BN than in LEW T cells.
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show that, as
for tAChR, although mycobacterium-specific T cells proliferated
strongly and equally well in vitro, the production of the
Th1-associated cytokines was much more pronounced in LEW than in BN
lymph node cells. Reciprocally, after stimulation with PPD in vitro, BN
T cells expressed 3–4 times more mRNA for IL-4 than LEW T cells (data
not shown).
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, while no difference was
observed in the kinetics of the anti-tAChR IgG response,
Ag-specific IgG2b and IgG1 were significantly up-regulated in LEW and
BN rats, respectively. IgG2a production was similar in both strains.
The same results were obtained when LEW and BN rats were immunized with
20 µg instead of 2 µg/rat of tAChR (data not shown). Thus, the
isotype profile of IgG correlates with the polarization of the T cell
response assessed by cytokine production, Th1-type in LEW and Th2-type
in BN rats.
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Using two different strains of rats, we showed that EAMG may occur in the context of either a Th1- or a Th2-dominated response. However, it may be possible that a particular polarization of the immune response is required for the induction of EAMG depending on the genetic background and/or the MHC haplotype. Therefore, we tested whether skewing the immune response from a Th2 to a Th1 phenotype in the same strain would influence disease outcome. For this purpose, BN rats were injected i.p. with recombinant mouse IL-12 at the time of immunization with tAChR in CFA. In these animals, we analyzed the effect of IL-12 administration on the clinical signs of EAMG and on the polarization of the T and B cell responses. The kinetics of muscle rat AChR loss before clinical onset of EAMG was also measured.
The data in Table II, obtained from three
independent experiments, show that IL-12 administration to
tAChR-immunized BN rats induced transient clinical symptoms of EAMG in
30% (6 of 20 rats) of the animals. This acute form of EAMG started
between days 9 and 14 and lasted for 2–5 days. In contrast, during
this period the control BN rats injected only with IL-12 or tAChR did
not show any clinical symptoms of disease. To show that these clinical
signs of acute EAMG were due to rat AChR loss, IL-12-treated
(n = 5, including the two with clinical symptoms of
EAMG) and control (n = 5) rats were killed on day
10 after tAChR immunization, and their carcasses were assayed for AChR
content. The results obtained show that the two animals with clinical
EAMG had marked muscle AChR loss (53 and 40%; data not shown in Fig. 5A). The remaining three
IL-12-treated BN rats, although they did not show any clinical sign of
acute EAMG, exhibited a low but significant (p <
0.01) AChR loss compared with control animals (Fig. 5A).
At this time point, regardless of treatment with IL-12, none of the 10
BN rats had detectable circulating anti-rat AChR autoantibodies in
their sera (Fig. 5B).
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). To assess whether IL-12 administration
had an effect on AChR loss before the appearance of clinical symptoms
of chronic EAMG, three and five rats per group were killed on days 15
and 28, respectively. A strong and equivalent reduction in muscle-AChR
content (40–50% loss) was found in both tAChR-immunized BN rats
treated or not with IL-12 (Fig. 5A). In addition,
anti-self-AChR Ab titers were not significantly different between
the two groups for all time points tested (Fig. 5B). The
remaining seven rats in each group were monitored for clinical symptoms
for a period of 3 mo. On day 20 after tAChR immunization one rat in the
IL-12-treated group died. From days 28–51 postimmunization, five of
six IL-12-treated and five of seven control immunized rats developed
progressive and severe clinical muscle weakness (mean maximum score,
2.2 ± 0.8 vs 1.9 ± 0.5; Table II).
We next analyzed the effect of IL-12 treatment on the polarization of T
and B cell responses. Popliteal and para-aortic lymph nodes from
tAChR-immunized BN rats, injected or not with IL-12, were removed 15
days later, and the immune lymph node cells were cultured with several
concentrations of tAChR. As shown in Fig. 6, immune lymph node cells from
IL-12-treated rats have an increased proliferative response to tAChR
compared with control animals (Fig. 6A). Although this
up-regulation was moderate, it was observed in two of three independent
experiments. Analysis of Ag-specific cytokine production in culture
supernatants demonstrated that in IL-12-treated BN rats, tAChR-specific
T cells were now skewed toward the Th1 phenotype, as indicated by the
up-regulation of IL-2 (Fig. 6B) and IFN- (Fig. 6, C and D) synthesis. Similar results were
obtained in two other independent experiments (data not shown).
However, the IL-4 mRNA synthesis in tAChR-stimulated lymph node cells
was not significantly affected in IL-12-treated compared with control
BN rats (data not shown). Lymph node cells from rats injected with
IL-12 only neither proliferated nor produced cytokines in the presence
of tAChR in vitro (data not shown). To further analyze the effect of
IL-12 on the polarization of the immune response in vivo, we next
examined the isotype profile of anti-tAChR IgG. As shown in Fig. 7, IL-12 administration significantly
increased the total Ab response to tAChR. Analysis of Ag-specific IgG
isotypes demonstrated that IgG2b Abs were markedly up-regulated, while
IgG1 and IgG2a subclasses were profoundly reduced in IL-12-treated BN
rats. These differences lasted for at least 3 mo after tAChR
immunization (data not shown).
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Neutralization of endogenous IFN- in BN rats does not influence
disease outcome
To examine the role of residual IFN- production in the
development of EAMG in BN rats we blocked the endogenous production of
this cytokine by administering the DB1, anti-IFN- mAb. BN rats
were immunized with tAChR in CFA and repeatedly injected with DB1 mAb
for 2 wk. This protocol has been shown to prevent the induction of
arthritis in rats (45). We first tested the effect of this treatment on
IgG2b and IgG1 tAChR-specific Ab production as indicators of Th1- and
Th2-like responses, respectively. The results in Fig. 8 show that anti-IFN- treatment
has no significant effect on the total IgG response to tAChR. Analysis
of Ag-specific IgG isotypes demonstrated a significant increase in IgG1
(p = 0.02 on day 30; p = 0.04
on day 40) and IgG2a (p = 0.04 on day 10;
p = 0.006 on days 20 and 60) responses concomitant with
a significant decrease in IgG2b anti-tAChR Abs
(p = 0.02 on day 20; p = 0.01
on day 30; p = 0.02 on day 40) in
anti-IFN--treated BN rats (Fig. 8). We next analyzed the outcome
of EAMG between control and anti-IFN--treated BN rats. The
results obtained show that this treatment does not affect the severity
or the incidence of disease (Table II). In conclusion, although we
cannot exclude that Th1 cells persist in anti-IFN--treated BN
rats, our data show that the neutralization of endogenous IFN-
induced in vivo a shift in the isotype profile of AChR-specific Abs
without affecting the clinical signs of EAMG.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2, and more IL-4 mRNA compared with LEW lymph node
cells. Second, the production of IgG1- and IgG2b-specific Abs was
higher in BN and LEW rats, respectively, suggesting that Th2- as well
as Th1-dependent autoantibodies were pathogenic. Third, IL-12 treatment
skewed the immune response in BN rats toward a Th1-dominated phenotype,
whereas it has minor effects on clinical and biochemical parameters of
the disease. This treatment had a strong inhibitory effect not only on
IgG1, but also on IgG2a, Ab production. As expected, IgG2b synthesis
was markedly increased in IL-12-treated BN rats (22). Finally, blocking
endogenous IFN- in BN rats resulted in a further polarization of the
isotype profile toward a Th2 phenotype without affecting the outcome of
EAMG. Taken together, our data demonstrate that a Th1-dominated immune
response is not necessarily associated with EAMG outcome not only in
rats with disparate MHC haplotype but also in the same rat strain.
In agreement with our present work, it has been suggested that both
AChR-reactive Th1 and Th2 cells could be involved in rat EAMG (47, 48, 49, 50, 51).
Several studies in humans showed that AChR-specific CD4 T cells from MG
patients could secrete either both Th1-type (IFN-) and Th2-type
(IL-4) cytokines (52, 53) or only Th1-type cytokines (54, 55, 56, 57). However,
in none of these studies has it been clarified which type of cytokines
was essential for the pathogenesis of the disease. By contrast, in the
mouse several studies have established that IFN- plays a pivotal
role in EAMG. First, the localized expression of the IFN- transgene
in the neuromuscular junction induces an MG-like syndrome (28). Second,
IFN- knockout mice, but not IL-4-deficient mice, are resistant to
EAMG (29, 31). Third, IL-12 administration in B6 mice results in an
increased Th1-dependent Ab response and accelerated disease onset,
whereas IL-12-deficient mice are less susceptible to disease induction
(30). Finally, tolerance procedures in mice were effective in
preventing EAMG through induction of Th1 unresponsiveness that was
associated with an up-regulation of Th2 cytokine synthesis (58). Taken
together, it seems now quite clear, at least in mice, that the
pathogenesis of this Ab-mediated disease is associated with a polarized
Th1-type response.
Several explanations may account for the apparent discrepancy between
our conclusions in rats and those reported in the mouse model. Rats are
much more susceptible to EAMG induction than mice. Indeed, we could
readily and reproducibly induce clinical EAMG in rat by a single s.c.
immunization with 2 µg of tAChR. In contrast, higher Ag doses
(10-fold) administered at different sites followed by subsequent
challenges are usually required to induce MG-like symptoms in the mouse
(29). It is therefore not surprising that the strong immunization
regimen required in C57BL/6 mice probably introduces a bias favoring a
role for Th1-type and/or proinflammatory cytokines in the pathogenesis
of the autoimmune process. Alternatively, differences in the effector
function of Ab subclasses could also explain the different requirement
for a Th1-dependent response between the two species. The role of
complement in the pathogenesis of EAMG has been clearly established in
rats (32, 33) and mice (13, 14). Unlike those in the mouse, both Th1-
and Th2-associated rat IgG subclasses are capable of binding complement
(34, 35). As a consequence, while in mice the generation of pathogenic
Abs will necessarily require the induction of the IFN--dependent
IgG2a subclass, the situation might be dramatically different in other
species in which complement-fixing Ab are equally distributed among
Th1- and Th2-dependent isotypes. In agreement with this explanation, it
has been shown that in IL-12-deficient mice resistant to EAMG, serum
anti-AChR Abs were predominately of the IgG1 isotype, and this
subclass was mainly found deposited at the neuromuscular junctions
(30). In humans, a predominance of complement-binding Ig, IgG1, and
IgG3 has been reported in sera from MG patients (59), but there is no
good evidence that the generation of complement-fixing IgG subclasses
in humans is Th1 or IFN- dependent (60). In the rat it has been
shown that passive transfer of rat IgG1, IgG2a, or IgG2b anti-AChR
were all capable to various extents of inducing acute EAMG (61).
However, it has also been reported in rats that the Ab specificity was
more important than the IgG subclass for induction of acute EAMG by
transfer of anti-AChR Abs (62).
It has been described that EAMG in rats could be biphasic when, in
addition to tAChR in CFA, these animals received Bordetella
pertussis (42). An acute phase of EAMG occurred about 7 days after
tAChR immunization that was self limited and characterized by
macrophage infiltration of the motor end plates (63). Rats recovered 5
days later and appeared clinically normal, but developed the chronic
phase of disease 4–5 wk after immunization (42). In our hands, LEW and
BN rats develop chronic EAMG 5–9 wk after tAChR challenge. In
contrast, the acute phase of the disease was observed in six of 20
IL-12-treated BN rats during the second week after tAChR immunization.
We showed in two of these animals a significant muscle AChR loss
without detectable circulating autoantibodies, indicating that cellular
mechanisms might be at play at this earlier time point. However, we
cannot rule out a possible role for autoantibodies, since the overall
humoral response to Torpedo receptor was exacerbated following IL-12
administration, probably as a consequence of the strong up-regulation
of the IFN--dependent IgG2b subclass.
A propensity to develop EAMG in different rat and mouse strains has been shown to be dependent on MHC- or non-MHC-linked genetic predispositions (13, 14, 15, 16, 17). In rats it has been suggested that modified anti-AChR Ab clonotype expression (64) and quantitative differences in cytokine patterns may account for the different susceptibilities among rat strains (51). However, these studies involved rat strains that had differences not only in the genetic background but also in the MHC haplotype. Diversity in the MHC locus could also be responsible for the observed difference between LEW and BN rats. The immunodominant epitopes of tAChR-reactive CD4 T cells are different between the two strains (65) and therefore trigger different T cell repertoires. This could result in different phenotype acquisition, as has been shown in the mouse in response to human collagen IV (66). Alternatively, it has been shown that the resistance of the BN strain to mount a Th1-mediated autoimmune disease (e.g., experimental allergic encephalomyelitis) and its susceptibility to develop Th2-mediated autoimmune disorders (e.g., gold salt-induced autoimmunity) are controlled at least in part by the same locus on chromosome 10 (67, 68). This region is homologous to mouse chromosome 11, which contains a cluster of genes important for T cell differentiation, including IL-4, IL-5, IL-3, IFN regulatory factor-1, and Tpm-1 (69). Therefore, a genetic polymorphism in this region, rather than differences in the MHC haplotype, could be responsible for the differential polarization of CD4+ T cells observed between the two strains in the present study.
In conclusion, the demonstration that the occurrence of EAMG in BN and
LEW rats is associated with an opposite profile of cytokine production
in Ag-specific T cells indicates that an increased frequency of
IFN--producing cells in vivo is not a prerequisite for the
development of this Ab-mediated autoimmune disease. This is further
supported by the finding that the manipulation of Th1/Th2 balance in BN
rats by IL-12 administration or IFN- neutralization did not
influence the disease outcome. These data suggest that in a situation
where complement-fixing Abs can be generated with either Th1- or
Th2-mediated T cell help, deviation of the immune response will not be
an adequate strategy to prevent this Ab-mediated autoimmune disease,
not only in rats but also possibly in humans.
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Acknowledgments |
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Abs and IFN-. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Abdelhadi Saoudi, Institut National de la Santé et de la Recherche Médicale, Unit 28, place du Dr. Baylac, 31059 Toulouse Cedex, France. E-mail address:
3 Abbreviations used in this paper: MG, myasthenia gravis; AChR, nicotinic acetylcholine receptor; EAMG, experimental autoimmune MG; tAChR, AChR purified from Torpedo marmorata electric organs; LEW, Lewis; BN, Brown-Norway; AU, arbitrary units; PPD, purified protein derivative of tuberculin.
Received for publication May 15, 1998. Accepted for publication April 7, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and BN (
) rats were immunized with 2
µg of tAChR in CFA. The age-matched control rats received PBS/CFA
(
). The anti-rat AChR Ab titers were measured on days 0, 9, 22,
and 31 after immunization by RIA and are expressed as nanomoles of
125I-
cpm).
Supernatants were analyzed for the presence of IL-2 (B)
and IFN-
) with IL-12. The IgG, IgG2a, IgG1, and IgG2b
anti-tAChR Ab titers were measured on days 11, 18, and 28 after
immunization by ELISA and are expressed in AU as described in Fig. 4