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Departments of Biochemistry, Molecular Biology and Biophysics, and Pharmacology, University of Minnesota, Minneapolis, MN 55455
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit peptides, but also to several mouse
AChR subunit peptides. These results suggest that in B6 mice,
regulatory mechanisms that involve IL-4 contribute to preventing the
development of a chronic Ab-mediated autoimmune response to the
AChR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental MG (EMG) is a model of MG induced in a variety of animals by one or more injections of AChR in adjuvants (1, 2). Mouse EMG is especially useful to examine the immune mechanisms that cause the myasthenic symptoms, and the roles of different cytokines in the modulation of the anti-AChR response (2). The AChR used for EMG induction can be from a different species, because the conserved AChR structure permits interspecies cross-reactivity of the anti-AChR Ab; the easily purified Torpedo AChR (TAChR) is the most commonly used (1, 2).
C57BL/6 (B6) mice immunized with TAChR develop EMG frequently (1, 2). However, their EMG is not self-maintaining as is human MG, and it has limited duration; mice that do not die recover within a few weeks after the end of immunization treatment (3). After TAChR immunization, B6 mice have anti-TAChR Ab that cross-react with mouse muscle AChR and are the cause of the myasthenic weakness (2); however, their CD4+ T cells do not cross-react with mouse AChR sequences (4, 5), indicating that anti-muscle AChR autoantibodies are generated with the help of CD4+ T cells specific for xenogenic TAChR sequences. These findings suggest that in B6 mice, even when the mouse neuromuscular junction is damaged by the action of the anti-AChR Ab and complement, the resulting inflammatory reaction does not cause sensitization of self-reactive CD4+ T cells and does not trigger the development of a truly autoimmune, self-maintaining response.
IL-4 and TGF-
may have a protective function in mouse EMG
(6, 7, 8). In this study we have examined whether TAChR
immunization in the absence of IL-4 leads to sensitization of
self-reactive anti-AChR CD4+ T cells and
development of a chronic autoimmune form of EMG.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B6 mice genetically deficient in IL-4 (IL-4-/-) and wild-type B6 (WT) mice from The Jackson Laboratory (Bar Harbor, ME) were bred at the animal facility of the University of Minnesota.
Antigens
We purified TAChR as we described previously (4, 5). For cell cultures, we diluted TAChR in RPMI 1640 as needed and sterilized it by UV irradiation. For immunizations, we diluted it to 0.5 mg/ml in PBS. We stored the TAChR at -80°C.
We described previously the synthetic peptides we used (4, 5). They spanned the sequences of the subunits of TAChR and
of mouse AChR and were 
20 residues long; their sequences overlapped
by 2–12 residues.
Immunization
We immunized WT and IL-4-/- mice by s.c. injections, along the back and at the base of the tail, of 30 µg of TAChR in 100 µl of PBS, emulsified in an equal volume of CFA. We killed the mice 2, 4, 8, 16, 24, 32, 40, 48, and 88 wk after the immunization to test the proliferative response of their CD4+ splenocytes.
Evaluation of the clinical symptoms of EMG
Every 2–4 wk, starting on the day before the immunization, we
measured the mouse strength with a forced exercise test sensitized by
pancuronium bromide (0.03 mg/kg i.p.), as we described previously
(7, 8, 9, 10). The mice hang from a grid, and we measure the
time it takes the mouse to fall three times (holding time). The
myasthenic nature of any weakness revealed by a shortened holding time
is verified by administering the cholinesterase inhibitor edrophonium
chloride (Reversol; Organon, West Orange, NJ), which immediately
increases the holding time of mice with EMG. The test gives a
quantitative and reproducible assessment of mouse weakness. Naive
IL-4-/- and WT mice had identical holding times
(8). The average holding time of 285 naive WT mice was
11.4 ± 1.55 min (9). We considered myasthenic the
mice with holding times of 6.75 min (the average holding time of normal
mice minus 3 SD) or less. Among the myasthenic mice, we considered
severely sick those with holding times of <5.2 min (the average
holding time of normal mice minus 4 SD). We used the programs PROC
ANOVA and PROC GLM (SAS Institute, Cary, NC) to determine the
significance of the difference between the strength over time of the
mice in the different groups. We used the following model:
response = treatment + E(mouse) + week + treatment x week,
where response is the holding time in minutes, treatment is the
presence (or absence) of the IL-4 gene, time is the time of the
individual test, and E(mouse) is the degrees of freedom (a function of
the number of mice in the groups analyzed). We considered a difference
to be significant when p 
0.05.
Assay of serum anti-TAChR and anti-mouse AChR
We measured the anti-TAChR Ab in sera obtained after each
clinical testing by a radioimmunoprecipitation assay that we described
previously (11), using Triton X-100-solubilized TAChR
labeled by the binding of 125I-labeled -bungarotoxin
(125I-labeled BTX) and a polyclonal Ab against mouse Ig.
We measured the concentration of Ab that cross-reacted with mouse
muscle AChR by a modification of the anti-TAChR Ab assay, using a
Triton X-100 extract of muscle from naive B6 mice as the source of
AChR, at a concentration of 1 nM BTX binding sites, labeled by
overnight incubation with 2 nM 125I-labeled BTX
(10). We used 0.2 pmol of AChR/sample and usually
triplicate or quadruplicate aliquots for each serum dilution. We set up
precipitation curves using increasing amounts of serum (0.2–5 µl).
After overnight incubation the AChR-Ab complexes were precipitated by
adding 20 µl of Zysorbin (Zymed Laboratories, San Francisco,
CA)/well, followed by incubation for 2 h. All incubations were
performed at 4°C. Ab concentrations are expressed as nanomolar
concentrations of precipitated 125I-labeled BTX binding
sites.
FACS analysis
We used single-fluorescence flow cytometry and FITC-conjugated rat anti-mouse CD40 and hamster anti-mouse CD80 mAb (BD PharMingen, San Diego, CA) to examine the expression of CD40 and CD80 (B7.1) on splenocytes of IL-4-/- and WT mice, naive or immunized with 30 µg of TAChR/CFA or PBS/CFA and euthanized 10 days after the immunization. In some experiments we used splenocytes cultured for 48 h with 2.5 µg/ml of TAChR.
Proliferation assay
We tested T cell sensitization to the TAChR and subunit
peptides using 5- day proliferation assays and pooled splenocytes from
two or three identically treated mice. We used either the total
splenocytes or the splenocytes depleted in CD8+
cells (CD4+ splenocytes) using rat anti-mouse
CD8+ Ab (BD PharMingen) and paramagnetic beads
covalently attached to goat anti-rat IgG (Polyscience, Warrington,
PA). We tested the proliferative response of cells using triplicate or
quadruplicate wells (100,000–200,000 cells/well) and the appropriate
selection among the following stimulants: PHA (10 µg/ml;
Sigma-Aldrich, St. Louis, MO), TAChR (0.5–5 µg/ml), and individual
TAChR or mouse AChR subunit peptides (10 µg/ml). Two sets of
control wells were cultured without any Ag or with a 20-residue peptide
synthesized by the same method as the AChR peptides and unrelated to
the AChR sequence. We determined the rate of cell proliferation from
the incorporation of [3H]thymidine, measured by
liquid scintillation. We assessed the significance of the difference of
the average [3H]thymidine incorporation of
cultures stimulated with a given Ag and that of the control wells,
using two-tailed Student’s t test and the program Excel
(Microsoft Corp., Redmond, WA). When an Ag induced a significant
(p < 0.05) increase in cell proliferation, we
calculated the stimulation index (SI; the ratio between the cpm of a
culture in the presence of the Ag and the average basal proliferation
of the same cells). The use of SI normalized results and allowed
comparison of experiments conducted at different times, with
different mice.
Solid phase ELISPOT assay and ELISA of IFN-
secretion
We used an ELISPOT assay to determine the number of
CD4+ T cells that produced IFN- (as a
representative Th1 effector cytokine) in response to the presence of
TAChR in CD4+ splenocytes from TAChR-immunized
IL-4-/- and WT mice, euthanized 2 or 4 wk after
the immunization. We cultured the cells in 96-well ELISPOT plates
(300,000 cells/well) coated with anti-mouse IFN- mAb (BD
PharMingen), and stimulated them with 2.5 µg/ml of TAChR for 48
h. We used a biotin-conjugated secondary Ab and streptavidin-conjugated
HRP (Vector Laboratories, Burlingame, CA) as the revealing system. The
development solution contained 800 µl of 100 mg of 3-amino-9-ethyl
carbazole in 10 ml of N,N-dimethylformamide, 24
ml of 0.2 M acetic acid, 0.2 M sodium acetate (pH 5.0), and 12 µl of
30% H2O2. We also
determined the concentration of IFN- in the supernatant of
CD4+ splenocytes from TAChR-immunized
IL-4-/- and WT mice euthanized 4 wk after the
immunization, cultured for 48 h in the presence or the absence of
2.5 µg/ml of TAChR.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We determined the time course of EMG in two independent groups of
IL-4-/- mice (n = 5 and
n = 10, respectively) and a control group of WT mice
(n = 10). We measured the mouse strength usually every
2–4 wk for 30 wk after TAChR immunization (Fig. 1
). The IL-4-/-
mouse groups yielded similar results; 8 wk after TAChR immunization
most mice had EMG (Fig. 1, A and B). The number
of affected mice was stable or increased during the observation period;
by wk 30 most IL-4-/- mice in both groups (80
and 100%, respectively) had EMG, which was usually severe (Fig. 1, A and B, 
). Only a few WT mice
developed EMG (Fig. 1C). The frequency of EMG among WT mice
was maximal between 8 and 16 wk after TAChR immunization, then
decreased. From wk 26 onward only one WT mouse had myasthenic weakness,
which was severe. The severity of the muscle weakness was higher in the
IL-4-/- groups than in the WT group; the
difference was statistically significant after wk 16.
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). EMG symptoms were more frequent and
severe in IL-4-/- than in WT mice; the
difference in severity was statistically significant from wk 8
onward.
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The clinical findings reported in Fig. 1
suggested that
IL-4-/- mice, in contrast to WT mice, had
developed a self-sustaining autoimmune response to the autologous AChR.
To verify this hypothesis we measured the concentration of
anti-mouse AChR Ab in the sera of the
IL-4-/- and WT mice used for the experiments
reported in Figs. 1 and 2. Fig. 3 shows
the anti-mouse AChR Ab concentrations of all sera we tested,
obtained at different times after TAChR immunization, as indicated
below the plot. We did not obtain sera from all groups for each time
point. Also, some mice in each group died of EMG or were sacrificed at
different times to examine the anti-AChR sensitization and the
repertoire of their CD4+ T cells. Thus, the
number of sera available varied for the different time points.
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) which were in a concentration range lower than that observed for the
mouse AChR Ab-positive sera from IL-4-/-
mice. Anti-TAChR Ab in IL-4-/- and WT mouse sera
The results of the anti-mouse AChR Ab assay and those of the
clinical testing might have been caused by ineffective sensitization of
the WT mice to the TAChR. To exclude this possibility we measured the
anti-TAChR Ab concentration in the sera of the
IL-4-/- and WT mice used for all the
experiments reported in Figs. 1 and 2. Fig. 4 summarizes the results we obtained.
Both IL-4-/- and WT mice had vigorous and
comparable Ab responses to the TAChR. The serum anti-TAChR Ab
concentrations in IL-4-/- mice were highest 4
wk after TAChR immunization and declined slowly during the next 20 wk.
The anti-TAChR Ab were virtually undetectable 28 and 72 wk after
the immunization, in contrast with the substantial concentration of
anti-mouse AChR Ab in the same sera (Fig. 3). The serum
concentration of anti-TAChR Ab of WT mice remained stable during
the first 20 wk after the immunization. We did not collect sera from WT
mice at later time points.
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Previous studies have suggested that removal of the
CD8+ cells from the splenocytes of
TAChR-immunized WT mice yielded specific proliferative responses to
TAChR and AChR peptides that were more robust and reproducible than
those of total splenocytes (5, 12). In the experiments
conducted 2 wk after TAChR immunization we tested the TAChR-specific
proliferative responses of both total and CD4+
splenocytes. CD4+ splenocytes from both WT and
IL-4-/- mice usually had stronger responses to
TAChR than total splenocytes (Fig. 5A). We used only
CD4+ splenocytes for the experiments conducted 4
wk or more after immunization.
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B reports
the average SI obtained in each experiment for cultures exposed to the
TAChR concentration that induced the highest response. TAChR-induced
responses of CD4+ splenocytes of
IL-4-/- mice were always significantly higher
(p < 0.001) than those of WT mice. Sequence regions of the TAChR recognized by T cells in IL-4-/- and WT mice
Even after intense TAChR immunization (e.g., multiple injections
in CFA) the CD4+ T cells of both WT and
IL-4-/- mice recognize primarily the TAChR
subunit and a dominant epitope within residues 146–169 (4, 5, 13). We examined the epitope repertoire of the
CD4+ cells sensitized by the single TAChR/CFA
injection we used, by measuring the proliferative response of
CD4+ splenocytes from WT and
IL-4-/- mice to the individual overlapping
synthetic peptides spanning the TAChR subunit sequence. To detect
whether the repertoire changed over time, we tested the peptide-induced
proliferative responses from mice sacrificed at different times after
TAChR immunization (2–88 wk for WT mice, 2–48 wk for
IL-4-/- mice). Usually, we conducted one
experiment for each time point. For some time points we conducted two
experiments that yielded consistent results. In one experiment a low
yield of CD4+ splenocytes did not allow the
testing of every peptide. Fig. 6
illustrates the results of typical experiments; the scattering of the
replicates and the background level are representative of those
obtained in all experiments. For the experiments conducted 2 wk after
TAChR immunization, we used both total and CD4+
splenocytes, which responded to the same peptides, although the
responses of the CD4+ splenocytes were usually
more robust (Fig. 7).
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summarizes the results we obtained
in the different experiments, expressed as SI. Because of the
propensity of short peptides to induce proliferative responses of
cross-reactive T cells sensitized to different Ag, we disregarded the
responses that, albeit significant, yielded SI lower than 3. The
CD4+ splenocytes of WT mice always responded to
one or both the peptides spanning the immunodominant sequence
146–169 (Fig. 8A). At 8 wk they recognized only this
immunodominant region, whereas at 2, 16, and 32 wk their repertoire
included several other peptides. The previously described
(4) subdominant epitopes 181–200 and 360–378 were
recognized occasionally. At 88 wk the CD4+
splenocytes of WT mice recognized only the peptides spanning the
immunodominant region 146–169 and the subdominant epitope
181–200. The CD4+ splenocytes of
IL-4-/- mice responded to more numerous TAChR
peptides and more strongly than WT mice (Fig. 8B). They
recognized the sequence region 146–169 most strongly and
consistently, but they recognized a number of other peptides,
especially 16–40 wk after TAChR immunization. They recognized most
frequently and strongly two clusters of peptides. One included the
immunodominant region 146–169 and its flanking peptide sequences
134–153 and 165–184, the subdominant peptide epitope
181–200, and the overlapping peptide 197–217. A second cluster
was located at the carboxyl terminus of the subunit, from residues
346 to the carboxyl terminus. Other frequently recognized peptides
spanned residues 43–80, 111–126, and 276–295. At 48
wk the IL-4-/- CD4+ splenocytes had a
modest response only to the immunodominant sequence region 146–169
and peptide 111–126; we cannot conclude at this time whether this
late reduction of the CD4+ T cell response to
TAChR epitopes is characteristic of IL-4-/-
mice or was due to an abnormally low response in these experiments.
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Previous studies concluded that, at least during the first 16 wk
of the anti-TAChR response, the CD4+ T cells
of B6 mice were not sensitized to mouse AChR epitopes, but only to
TAChR epitopes (1, 2). We determined the proliferative
response of CD4+ splenocytes from WT and
IL-4-/- mice at different times after the
TAChR immunization, to overlapping synthetic peptides spanning the
sequence of the mouse AChR subunit. We tested the response at 2 and
8 wk after the TAChR immunization and then approximately every 8 wk for
a total of 32 wk for WT mice and 52 wk for
IL-4-/- mice. We retested the responses of both
strains 88 wk after TAChR immunization. Usually, we conducted one
experiment for each time point. In the few cases where we conducted two
experiments they yielded consistent results. For the experiments
performed 2 wk after TAChR immunization we used both total and
CD4+-depleted splenocytes; neither population
recognized any mouse peptide. Fig. 9
reports the results of experiments with CD4+
splenocytes from WT and IL-4-/- mice, which are
representative of the scattering of the replicates and the background
levels obtained in all experiments.
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summarizes the results we
obtained, expressed as SI. We have disregarded the proliferative
responses that yielded an SI <3. The CD4+
splenocytes of WT mice (Fig. 10A) did not recognize any
mouse peptide sequence during the first 16 wk after TAChR immunization.
After 24 wk they had a modest response to peptide 216–235. At later
times they recognized, with modest intensity, a few other peptide
sequences (32–51, 216–235, and 277–296 at 32 wk, and the
peptides spanning the sequence regions 51–70 and 76–122 at 88
wk). The CD4+ splenocytes of
IL-4-/- mice started recognizing mouse AChR
peptides 8 wk after TAChR immunization, and they recognized a much
richer repertoire of peptides than WT mice. Their proliferative
responses to the mouse peptides were frequently vigorous (SI of
6–9 and higher). The peptide-induced responses were most diverse and
intense during the first 24 wk. Thereafter the
CD4+ splenocytes recognized a more limited
peptide repertoire, and their responses were less intense. Peptide
216–235 was recognized most frequently and strongly. Other peptides
were recognized in two or three experiments (the overlapping peptides
23–41 and 32–51, 188–137, 277–296 and 402–421).
Peptides recognized only once usually overlapped or flanked peptides
recognized more frequently. At 52 and 88 wk the
CD4+ splenocytes of
IL-4-/- mice recognized only peptides
216–235 and 118–137.
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To test whether the more robust sensitization and diverse CD4+ repertoire of IL-4-/- than WT mice was related to an enhanced costimulatory function of the APC in IL-4-/- mice, we analyzed by FACS the expression of CD40 and CD80 costimulatory molecules in WT and IL-4-/- mice. We used total splenocytes from naive mice and from mice injected with TAChR/CFA or PBS/CFA. Also we determined CD40 and CD80 expression after culturing the splenocytes with TAChR. We did not find any difference between the two strains in the percentage of cells that expressed either of these markers (data not shown).
TAChR-specific CD4+ cells secreting IFN- in
IL-4-/- and WT mice
To assess whether the increased susceptibility to EMG of
IL-4-/- mice is due to an enhanced function of
Th1 cells, we determined by ELISPOT assay the frequency of
TAChR-specific, IFN--secreting cells in the
CD4+ splenocytes of WT and
IL-4-/- mice 2 and 4 wk after TAChR
immunization. We conducted one experiment at 2 wk and two independent
experiments at 4 wk. At 2 wk, consistent with the much smaller
proliferative responses to the TAChR than at later times, we did not
detect TAChR-specific, IFN--secreting cells. In contrast, at 4 wk we
observed substantial numbers of TAChR-specific, IFN--secreting cells
in both strains. In both experiments WT mice had slightly, yet
significantly, higher numbers of TAChR-specific Th1 cells than
IL-4-/- mice. Fig. 11 reports the results of one of the
two consistent experiments.
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by
CD4+ splenocytes of WT and
IL-4-/- mice 4 wk after TAChR immunization. In
two independent experiments the supernatant of
CD4+ splenocytes from both strains, cultured
48 h with 2.5 µg/ml of TAChR, contained similar concentrations
of IFN-, comparable to those induced by the presence of Con A (Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study suggests that in EMG regulatory mechanisms that
involve IL-4 are responsible for contribute to preventing the
development of chronic Ab-mediated autoimmune responses to the AChR
even in the presence of an acute self-reactive Ab response. The absence
of IL-4 allowed the development of a chronic self-reactive Ab and T
cell response to autologous AChR after immunization with the xenogenic
TAChR. IL-4-/- mice developed EMG weakness that
lasted for the entire observation period, at times when WT mice were
recovering. They had serum anti-mouse AChR Ab much more frequently
than WT mice, which persisted for the duration of our observation, even
at times when Ab to the xenogenic TAChR had dropped to undetectable
levels. TAChR-immunized IL-4-/- mice had
CD4+ T cells that responded well to mouse AChR
sequences shortly after TAChR immunization. The same immunizing
protocol in WT mice resulted in a short-lived myasthenic syndrome
mediated by cross-reactive Ab, induced by TAChR immunization, but
without detectable sensitization of CD4+ T cells
to mouse AChR sequences during the first 16 wk, when EMG symptoms were
the most strong. Interestingly, we detected responses of
CD4+ splenocytes from WT mice to mouse AChR
sequence from wk 24 onward, when their frequency of EMG was minimal
(Figs. 1 and 10); this suggests that in B6 mice with intact
IL-4-dependent modulatory mechanisms, the presentation of self epitopes
at the inflamed neuromuscular junction led to sensitization of
self-reactive protective T CD4+ cells specific
for autologous AChR. The present results suggest that IL-4, perhaps
through the action of TGF- made by IL-4-dependent regulatory Th3
cells (18), has an important role as a gatekeeper in
immune responses to exogenous Ag potentially cross-reactive with
self Ag.
The increased susceptibility of IL-4-/- mice to EMG compared with that of WT mice could be due to an increased function of pathogenic Th1 cells, or to enhanced costimulation during the activation of anti-AChR CD4+ T cells. However, the present results suggest that neither of these hypotheses explains the enhanced incidence and sustained time course of EMG in IL-4-/- mice; both strains had similar numbers of splenocytes expressing the costimulatory molecules CD40 and CD80. Also, although IL-4-/- mice had numerous TAChR-specific Th1 cells in the spleen, WT mice had comparable and even more elevated numbers. These results are consistent with the hypothesis that the lesser susceptibility to EMG and the lack of progression of the symptoms to a chronic phase in WT mice are related to regulatory circuits that involve IL-4.
The present results agree well with those of several previous studies
on the anti-AChR response in WT mice (1, 2, 3). Similar
to the conclusions of those studies, we found that EMG symptoms
appeared several weeks (8–12 wk) after the beginning of TAChR
immunization, and that a single TAChR injection did not induce EMG
effectively in WT mice; the two groups of WT mice we studied had
maximum EMG frequencies of 50 and 22%, respectively. Even after more
intense TAChR immunization procedures than the one we used, WT mice
developed EMG with frequencies that varied in different studies, but
were never >80% and were as low as 25% in some studies
(1, 2, 3). Also in agreement with previous studies (5, 13), we found that the CD4+ T cells of WT
mice recognized most strongly and consistently the sequence region
146–169 of the TAChR subunit, and during the first 16 wk after
TAChR immunization they did not cross-react with mouse AChR sequences.
The frequency and severity of the EMG symptoms were higher in
IL-4-/- mice than in WT mice even during the
first few months after TAChR immunization (Figs. 1 and 2), consistent
with an important role of IL-4 in the modulatory mechanisms that
prevent EMG development. A previous study suggested that IL-4 did not
have a pathogenic role in EMG (19), and another study
found that IL-4-/- mice were more susceptible
to EMG than WT mice (8).
Several WT mice had EMG, which was severe in a few of them (Fig. 1),
whereas only one WT mouse had anti-mouse AChR Ab in its serum.
These seemingly conflicting observations can be reconciled by
considering that only a very small fraction of the anti-TAChR Ab
cross-reacts with mouse muscle AChR (1). Because mouse
muscles are very rich in AChR (20), they probably entrap
and remove from the bloodstream the high affinity cross-reactive Ab
that cause the myasthenic symptoms. The present observations agree with
the reported lack of correlation between the severity of myasthenic
symptoms in both MG and EMG and the concentration of serum
anti-AChR Ab (1, 9).
The WT mice, although less susceptible to EMG than the
IL-4-/- mice, developed symptoms 8 wk after
TAChR immunization, when 10% of them had severe EMG (Fig. 1). Yet,
even 16 wk after the immunization, they had no detectable response to
mouse-specific AChR sequences, even though the EMG-affected WT mice
must have high affinity Ab cross-reactive with the mouse muscle AChR.
This phenomenon, which has been observed in other previous studies
(1, 2), suggests that EMG-inducing, high affinity
anti-AChR Ab are generated with the help of
CD4+ T cells specific for xenogenic TAChR
sequences.
The repertoire of TAChR and mouse AChR peptide sequences
recognized by CD4+ T cells of
IL-4-/- and WT mice after TAChR
immunization and the intensity of those responses are consistent
with a modulatory role of IL-4 in both the xenogenic response to the
TAChR and the development of a self-reactive anti-AChR
CD4+ T cell response. CD4+
T cells from IL-4-/- mice recognized the TAChR
more strongly (Fig. 5) and recognized a richer repertoire of TAChR
subunit sequences than CD4+ cells from WT mice
(Fig. 8). Also, they recognized mouse AChR sequences in the early
phases of the anti-AChR immune response, when the
CD4+ T cells of WT mice did not (Fig. 10). In the
late phases of the anti-AChR response (88 wk), the
CD4+ T cell epitope repertoire of
IL-4-/- mice was much more limited than at
earlier times (Fig. 10). This is reminiscent of the behavior over time
of anti-AChR CD4+ T cells from MG patients,
which respond strongly to human AChR Ags during the first several years
of symptoms, but respond poorly in patients that had MG for many years
(21). The finding that CD4+ T cells of WT mice
did not recognize mouse sequences during the first months after TAChR
immunization, when they were developing EMG, whereas they did so when
they were recovering from EMG raises the possibility that the absence
of chronic EMG in WT mice with intact IL-4-dependent modulatory
circuits is related to sensitization of self-reactive, protective
CD4+ T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Monica Milani, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 6/155 Jackson Hall, 321 Church Street, Minneapolis, Minnesota 55455. E-mail address: mmilani{at}cbs.umn.edu
3 Previously known as Bianca M. Conti-Tronconi.
4 Abbreviations used in this paper: MG, myasthenia gravis; AChR, acetylcholine receptor; B6 mice, C57BL6 mice; BTX, -bungarotoxin; EMG, experimental MG; SI, stimulation index; TAChR, Torpedo AChR; WT, wild type.
Received for publication March 18, 2002. Accepted for publication October 23, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits of Torpedo californica and murine acetylcholine receptors. Eur. J. Immunol. 21:2303.[Medline]
or interleukin-12 has different effects on experimental myasthenia gravis in C57BL/6 mice. J. Immunol. 164:5236. subunit in myasthenia gravis: a study with synthetic peptides. J. Immunol. 144:1276.[Abstract]
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W. Wang, M. Milani, N. Ostlie, D. Okita, R. K. Agarwal, R. Caspi, and B. M. Conti-Fine C57BL/6 Mice Genetically Deficient in IL-12/IL-23 and IFN-{gamma} Are Susceptible to Experimental Autoimmune Myasthenia Gravis, Suggesting a Pathogenic Role of Non-Th1 Cells J. Immunol., June 1, 2007; 178(11): 7072 - 7080. [Abstract] [Full Text] [PDF]
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N. E. Standifer, S. Stacy, E. Kraig, and A. J. Infante Discrete T Cell Populations with Specificity for a Neo-Self-Antigen Bear Distinct Imprints of Tolerance J. Immunol., March 15, 2007; 178(6): 3544 - 3550. [Abstract] [Full Text] [PDF]
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R. Liu, A. La Cava, X.-F. Bai, Y. Jee, M. Price, D. I. Campagnolo, P. Christadoss, T. L. Vollmer, L. Van Kaer, and F.-D. Shi Cooperation of Invariant NKT Cells and CD4+CD25+ T Regulatory Cells in the Prevention of Autoimmune Myasthenia J. Immunol., December 15, 2005; 175(12): 7898 - 7904. [Abstract] [Full Text] [PDF]
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W. Wang, N. S. Ostlie, B. M. Conti-Fine, and M. Milani The Susceptibility to Experimental Myasthenia Gravis of STAT6-/- and STAT4-/- BALB/c Mice Suggests a Pathogenic Role of Th1 Cells J. Immunol., January 1, 2004; 172(1): 97 - 103. [Abstract] [Full Text] [PDF]
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