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or IL-12 Has Different Effects on Experimental Myasthenia Gravis in C57BL/6 Mice1
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Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, MN 55108; and
Department of Pharmacology, University of Minnesota, School of Medicine, Minneapolis, MN 55455
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12 induce Th1 responses: we investigated whether these cytokines
are necessary for EMG development. We immunized wild-type (WT) C57BL/6
mice and IFN- and IL-12 knockout mutants (IFN--/-,
IL-12-/-) with Torpedo AChR (TAChR). WT
and IFN--/- mice developed EMG with similar frequency,
IL-12-/-mice were resistant to EMG. All strains
synthesized anti-AChR Ab that were not IgM or IgE. WT mice had
anti-AChR IgG1, IgG2b, and IgG2c, IFN--/- mice had
significantly less IgG2c, and IL-12-/- mice less IgG2b
and IgG2c. All mice had IgG bound to muscle synapses, but only WT and
IFN--/- mice had complement; WT mice had both IgG2b
and IgG2c, IFN--/- only IgG2b, and
IL-12-/- neither IgG2b nor IgG2c. CD4+ cells
from all AChR-immunized mice proliferated in response to AChR and
recognized similar epitopes. After stimulation with TAChR,
CD4+ cells from IFN--/- mice secreted less
IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+
cells from IL-12-/- mice secreted less IFN-, but more
IL-4 and IL-10 than WT mice, suggesting that they developed a stronger
Th2 response to TAChR. The EMG resistance of IL-12-/-
mice is likely due to both reduction of anti-TAChR Ab that bind
complement and sensitization of modulatory Th2 cells. The reduced Th1
function of IFN--/- mice does not suffice to reduce
all complement-fixing IgG subclasses, perhaps because as in WT mice a
protective Th2 response is missing. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2, can be cytotoxic, and help the
synthesis of IgG subclasses that bind complement. Th1 cells have been
implicated in the pathogenesis of Ab-mediated autoimmune diseases,
because they help synthesis of Ab able to fix complement, that should
be especially effective in causing tissue damage (1, 2, 3, 4).
IL-12 and IFN- are potent inducers of Th1 cell differentiation
(1, 4). IL-12 facilitates Th1 responses by stimulating the
differentiation of naive Th cells into Th1 cells, and by serving as a
costimulus for maximum IFN- secretion by Ag-activated Th1 cells
(5, 6). Activated Th1 cells secrete IFN-, which
promotes further Th1 cell proliferation and activates macrophages
(1, 4). IFN--deficient mice have reduced Th1 and
macrophage function (7). Mice genetically deficient in
IL-12 have severely reduced Th1 responses and increased secretion of
IL-4 after Ag stimulation, suggesting a polarization of their immune
responses toward Th2 sensitization (8).
Myasthenia gravis
(MG)4 and its animal
model, experimental MG (EMG), are caused by Ab to the muscle
acetylcholine receptor (AChR) (9). Th1 cells are involved
in the pathogenesis of MG and EMG. MG patients had AChR-specific Th1
cells (10, 11, 12, 13). IL-4 knockout mice, that have a reduced
Th2 function but a normal Th1 function, were susceptible to EMG after
immunization with AChR (14, 15). IL-12 knockout mice
(IL-12-/-) immunized with AChR synthesized
anti-AChR Ab, but they seldom developed electromyographical
abnormalities indicative of reduced neuromuscular transmission that
were frequent in AChR-immunized wild-type (WT) mice (16).
Administration of IL-12 at the time of the AChR immunization
facilitated the appearance of abnormality of the neuromuscular
transmission revealed by electromyography (16). Mice
deficient in IFN- and immunized with AChR did not develop
anti-AChR Ab or EMG symptoms (17), whereas mice
genetically deficient in IFN- receptor developed anti-AChR Ab,
although in less amounts than WT mice, and presented EMG, but less
frequently than WT mice (18). Those studies (17, 18) suggest that IFN- affects EMG development by complex
mechanisms that are different from those influenced by IL-12. This is
supported by the finding that transgenic mice that produced IFN- at
the neuromuscular junction did not develop anti-AChR Ab, yet they
had functional disruption of the junction and clinical weakness
reminiscent of MG (19).
Th2 cells help synthesis of Ab that do not bind complement, such as IgA, IgE, and IgG1 in mice (1, 2, 3, 4), and are unlikely to cause severe tissue injury. They modulate immune responses by secreting anti-inflammatory cytokines, like IL-4 and IL-10, that down-regulate the function of APC and Th1 cells (1, 2, 3, 4). Also, Th2 cells may down-regulate immune responses indirectly by stimulating the activity of modulatory CD4+ cells that secrete TGF-ß (sometimes referred to as Th3 cells (4)). The TGF-ß family of cytokines are potent immunomodulators (20) that can polarize CD4+ responses toward a Th2 phenotype (21, 22) and block the effects of IL-12 in the development of Th1 responses (23, 24). Th3 cells are dependent on IL-4 for growth, but they do not produce it; therefore, they may be dependent upon Th2 cells for proliferative signals (4, 25, 26).
Th2 and Th3 cells may have a protective role in EMG. Nasal or s.c. administration to C57BL/6 (B6) mice of synthetic AChR peptides forming CD4+ epitopes activated peptide-specific Th2 cells, caused reduced synthesis of anti-AChR Ab, and prevented EMG (27, 28, 29). IL-4 knockout B6 mice, that have defective Th2 (and presumably Th3) responses, were more susceptible to EMG than WT B6 mice and were not protected from EMG by nasal administration of synthetic AChR CD4+ epitopes (15). An EMG-resistant rat strain had increased up-regulation of TGF-ß as compared with an EMG-susceptible strain (30). Oral administration of purified AChR provided protection from EMG and up-regulated the synthesis of TGF-ß by AChR-specific CD4+ cells (31).
IL-12 is the most important cytokine for Th1 cell differentiation, and
IFN- is an important Th1 effector molecule as well as an autocrine
growth factor for Th1 cells (1, 2, 3, 4). Here, we investigated
the relative importance of these cytokines in the development of EMG.
We immunized WT B6 mice, and their IFN- and IL-12 knockout
(IFN--/-, IL-12-/-)
mutants with purified AChR. We determined the appearance of EMG and of
anti-AChR Ab, their Ig isotypes and IgG subclasses in sera, and the
presence of IgG, IgG subclasses, and complement at the neuromuscular
junction. Also, we assessed the proliferative response of the
CD4+ cells from WT,
IFN--/-, and
IL-12-/- mice to the TAChR, their epitope
repertoire, and the cytokines they secreted after challenge in vitro
with AChR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We bought WT, IFN--/-, and
IL-12-/- B6 mice from The Jackson Laboratory
(Bar Harbor, ME) and bred them at the animal facility of the University
of Minnesota. Because of the unexpected sensitivity to EMG found for
the IFN--/- mice (see below), we verified
the genotype of 10 randomly selected IFN-/- and
5 WT mice by PCR using DNA from 3 to 5 µl of blood from each mouse,
prepared using Chelex 100 resin (Bio-Rad, Hercules, CA)
(32). One microliter of supernatant of the Chelex extract
was used as a template for PCR. Each reaction mixture (total volume, 10
µl) included 1 µl 10x PCR buffer II (Perkin-Elmer, Foster City,
CA), 1 µl of 10x dNTPs (10x dNTPs: 2 mM each dATP, dCTP, dGTP, and
dUTP (Pharmacia, Piscataway, NJ)), 0.8 µl of 25 mM
MgCl2 solution (Perkin-Elmer), 1 µl of 10 µM
stock solutions for each of the primers of the pairs listed below, 2
µl of red sucrose PCR-compatible loading dye (60% sucrose and 1 mM
cresol red), 0.1 µl of 5 U/µl Ampli Taq Gold DNA
polymerase (Perkin-Elmer), and 3.1 µl of sterile double-distilled
H2O. Each sample was covered with 30 µl of
mineral oil. For PCR, the samples were hot started for 10 min at
95°C, followed by a three-step cycling profile using 35 cycles of
30 s at 95°C, 30 s at 53°C, and 45 s at 72°C in a
Perkin-Elmer model 9600 DNA thermal cycler. We used the following pairs
of primers, as recommended by The Jackson Laboratory: for the WT
IFN- allele, IMR126 -5'-AGA AGT AAG TGG AAG GGC CCA GAA G-3' and
IMR127 -5'-AGG GAA ACT GGG AGA GGA GAA ATA T-3' (the resulting product
is 220 bp); and for the disrupted IFN- allele, IMR128 -5'-TCA GCG
CAG GGG CGC CCG GTT CTT T-3' and IMR129 -5'-ATC GAC AAG ACC GGC TTC
CAT CCG A-3' (the resulting product is 375 bp). We used both pairs of
primers for each mouse DNA: each pair was used in a separate tube, so
that only one product could be amplified for each reaction. We assessed
the size of the products by 1.5% agarose (Life Technologies,
Gaithersburg, MD) gel electrophoresis, stained with 0.001% ethidium
bromide (Sigma, St. Louis, MO). When we used DNA from WT mice, we
obtained a PCR product of 220 bp only when we used the primers
corresponding to the unmutated IFN- sequence. When we used DNA from
IFN--/- mice, we obtained a PCR product of
375 bp only when we used the primers corresponding to the mutated
IFN- sequence.
Purification of Torpedo AChR (TAChR)
We purified AChR from the electric tissue of Torpedo
californica fish as alkali-stripped AChR-rich membrane fragments
(TAChR)5
(33). The AChR structure is highly conserved along
evolution: TAChR is quite similar to mammalian muscle AChR and suitable
to induce EMG (9). We measured the protein concentration
by the Lowry assay (34), the TAChR concentration as
-bungarotoxin (-BTX) binding sites (33), and we
assessed the protein composition by SDS-PAGE (35). The
TAChR preparations we used contained 3.8–5.8 nmol of sites/mg protein
and contained only the four TAChR subunits as the main protein bands.
For use in cell cultures, we diluted the TAChR-rich membrane fragments
in RPMI 1640 as needed and sterilized them by UV irradiation. For
immunization and Ab assay, we solubilized the membranes in 1% Triton
X-100 (33), diluted them to 0.5 mg/ml in PBS, and stored
them at -80°C.
Peptides
To determine the epitope repertoire on the TAChR subunit,
which dominates the sensitization of anti-TAChR
CD4+ cells in B6 mice (33), we used
a panel of synthetic (36) peptides, 
20-residue long and
overlapping by 5 residues that spanned the sequence of the TAChR
subunit. We reported their characterization previously
(33). We used solutions of the individual peptides in PBS
sterilized by UV irradiation and stored frozen.
TAChR immunization
We immunized 8- to 10-wk-old female mice by s.c. injections, along the back and at the base of the tail, with solubilized TAChR (20–40 µg in 100 µl PBS) emulsified in an equal volume of CFA. We boosted them twice at 4-wk intervals with the same amount of TAChR in incomplete Freund’s adjuvant and a third time 5–7 days before being euthanized at the end of week 15.
Evaluation of clinical symptoms of EMG
We quantified the EMG symptoms using an inverted hang-forced exercise, sensitized by a minute amount of pancuronium bromide (0.03 mg/kg i.p.), given just before the test (37). The mice hang from a grid: we measured the time it took for the mouse to release its hold and fall three times ("holding time"). The test is parametric and gives a quantitative assessment of the severity of the weakness of the mouse. To verify the myasthenic nature of the weakness, we injected edrophonium chloride (Reversol; Organon, West Orange, NJ) intraperitoneum. Reversol is a cholinesterase inhibitor which immediately increased the strength of mice that have EMG. We tested the mice every 1–3 wk, starting on the day of the first immunization. We performed the test without knowledge of the treatment that the mouse had received.
The average holding time of 285 naive WT mice was 11.4 ± 1.55
min. Naive IL-12-/- and
IFN--/- mice had holding times
indistinguishable from WT mice. We considered myasthenic mice with
holding times of 8.3 min (the holding time of normal mice minus 2 SD)
or less. Paralyzed mice or mice that died of respiratory paralysis are
represented in the figures as having holding time of zero.
Anti-AChR Ab assay
We obtained sera after each clinical testing. We measured the
serum concentration of anti-TAChR Ab by radioimmunoprecipitation
assay (RIPA) using TAChR solubilized in Triton X-100 and labeled by the
binding of 125I--BTX (38). This
assay uses as precipitating Ab a polyclonal serum raised against mouse
Ig that include the 
and 
light chains shared by all Ig;
therefore, the serum precipitates all Ig isotypes and IgG subclasses.
We express the Ab concentration as micromolar precipitated
125I--BTX binding sites.
Assay of anti-TAChR IgM, IgE, IgG, and IgG subclasses
We measured by ELISA the anti-TAChR IgM, IgE, IgG, and the IgG subclasses in mouse sera obtained at 8 and 14 wk after the first TAChR immunization. We coated ELISA plates (Nunc, Karstrup, Denmark) by overnight incubation at 4°C with 100 µl/well of 25 µg/ml TAChR in 10 mM NaHCO3 (pH 9.6). We washed the plates four times with PBS containing 0.05% Tween 20 (PBS-T) and blocked them with 200 µl/well of 2% BSA in PBS-T (PBS-TB) for 1 h at 37°C. After four washings with PBS-T, we added 100 µl/well of three appropriate serum dilutions (in duplicate) in PBS-TB and incubated the plates overnight at 4°C. We used serum from normal untreated mice (NMS) as a negative control. We washed the plates six times with PBS-T, added 100 µl/well of 1 µg/ml biotinylated anti-mouse IgM, IgE, total IgG, or IgG subclass Ab (PharMingen, San Diego, CA) in PBS-TB, and incubated the plates for 1 h at 37°C. Mice carrying the Igh-1b allele, like B6 mice, do not express IgG2a, but rather a related isotype, termed IgG2ab or IgG2c (39, 40). To detect anti-TAChR IgG2c in the mouse sera, we used the mAb biotin anti-mouse IgG2ab (Igh-1b) (PharMingen).
After eight washings with PBS-T, we added 100 µl/well of 1 µg/ml
avidin-peroxidase (Sigma, St. Louis, MO) and incubated the plates for
1 h at 37°C. We washed the plates eight times with PBS-T and
added 100 µl/well of 0.3 mg/ml
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) in 0.01%
H2O2/citric acid buffer
(ABTS peroxidase substrate system; Kirkegaard & Perry Laboratories,
Gaithersburg, MD). After development of the blue-green color at room
temperature, we stopped the enzymatic reaction by adding 100 µl/well
of 1% SDS. We measured the absorbance at 405 nm with an automated
microplate reader ELx800 (Bio-Tek Instruments, Winooski, VT). We
determined the specific absorbance by subtracting the absorbance of
wells incubated with NMS dilutions from that of wells incubated with
the sera from TAChR-immunized mice. The serum concentration of
anti-TAChR Ig isotypes and IgG subclasses was inferred from
standard curves obtained by coating ELISA plates directly with 100
µl/well of purified murine total IgG (Sigma), IgM, and IgE
(PharMingen) or IgG1, Ig2a, and IgG2b subclasses (Sigma) from 100 to
0.097 ng/ml in 10 mM NaHCO3 (pH 9.6) and
detecting their presence with the biotinylated anti-mouse Ig class
or IgG subclass/avidin-peroxidase system, as described above. An ideal
standard curve would have required the use of purified anti-AChR
IgG subclasses binding to the TAChR coating the plate. Therefore, the
values of Ig and IgG subclass concentrations in serum we report here
should not be considered as absolute concentrations. However, since our
goal was to assess the relative amounts of the different anti-AChR
Ig classes and IgG subclasses synthesized in
IFN--/- or IL-12-/-
mice as compared with WT mice, we considered our standard curve a
suitable reference for our evaluations, because it represents an
internal standard that allows comparison of results for the individual
isotypes and subclasses obtained in different experiments. We could not
construct a standard curve for IgG2c because purified IgG2c is not
commercially available. Thus, for IgG2c we simply report the values of
OD, expressed as OD units at a wavelength of 405 nm, obtained for sera
of the three mouse strains assayed simultaneously. We obtained the
results reported here doing duplicate determinations of three serum
dilutions (1:30,000, 1:90,000, and 1:270,000): the OD values in the
linear range were normalized as to represent an arbitrary dilution of
1:90,000 and were averaged.
Detection of IgG, IgM, and IgG subclasses and complement at the muscle synapses by immunofluorescence microscopy
Hind limb muscle tissue of WT,
IFN--/-, and
IL-12-/- mice, frozen in liquid nitrogen and
stored at -70°C, was embedded in OCT Compound Tissue-Tek (Miles
Laboratories, Elkhart, IN) and sectioned it in the transverse direction
into 10-µm sections using a Jung Frigout 2800E Kryostat (Leica,
Nublach, Germany). To detect simultaneously the presence of mouse IgG
and complement at the neuromuscular junction, we incubated the sections
at room temperature for 10 min in PBS and for 1 h with a 1:200
dilution of biotin-conjugated goat anti-mouse IgG polyclonal Ab
(Sigma) in PBS containing 3% BSA. We washed the sections with PBS for
15 min three times and stained them for 1 h at room temperature
with Texas Red-labeled -BTX (Molecular Probes, Eugene, OR),
FITC-labeled goat anti-mouse C3 Ab (Nordic Immunological
Laboratories, Capistrano Beach, CA), and
6-((7-amino-4-methylcoumarin-3hexanoicacetyl)amino) acid, succinimidyl
ester-labeled streptavidin (Molecular Probes) diluted in PBS containing
3% BSA at 1:4000, 1:100, and 1:200 dilutions, respectively. For
detection of mouse IgM and different IgG subclasses, we incubated the
sections for 1 h with 1:20 dilutions in PBS of goat anti-mouse
IgM Ab (Sigma) or mAbs specific for mouse IgG1 (a rat mAb; Sigma),
IgG2b (a rat mAb; Sigma), and IgG2c (PharMingen mAb anti-mouse
IgG2ab
(Igh-1b); a mouse IgG3 mAb). The sections were
washed with PBS for 15 min three times, followed by incubation with a
biotin-conjugated secondary Ab, which was rabbit anti-goat IgG
polyclonal Ab (Sigma) for IgM, goat anti-rat IgG polyclonal Ab
(Sigma) for IgG1 and IgG2b, and a mAb specific for mouse IgG3
(PharMingen) for IgG2c. This was followed by staining with Texas
Red-labeled -BTX (Molecular Probes), FITC-labeled goat
anti-mouse C3 Ab (Nordic Immunological Laboratories), and ALEXA
350-labeled streptavidin (Molecular Probes) as described above. We
washed the sections three times for 15 min with PBS and viewed them in
a fluorescence microscope (Nikon Eclipse E800; Nikon Diaphot, Melville,
NJ). We collected digital images using the program Image Pro Plus
(Media Cybernetics, Silver Spring, MD).
Assay of Ab bound to mouse muscle AChR
We measured the Ab bound to mouse muscle AChR by RIPA
(33) as follows. Carcasses from three to five mice were
skinned, eviscerated, and homogenized individually in two volumes of 50
mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 20 mM PMSF, and 1.2 mM EGTA (pH
7.4, extraction buffer). The homogenates were centrifuged at 30,000 rpm
for 60 min. The pellets were suspended in 1 volume of extraction buffer
and extracted with 2% Triton X-100 for 4 h under shaking. We
centrifuged the extract at 30,000 rpm for 60 min and collected the
supernatant. We added 4 pmol of I125--BTX to
each quadruplicate of 1-ml aliquots of Triton X-100 extract of each
mouse carcass and incubated them for 24 h. Five microliters of NMS
was added to each aliquot. One hundred microliters per sample
anti-mouse IgG Ab were added. The samples were incubated overnight,
centrifuged at 12,000 rpm, washed four times with 10 mM PBS (pH 7.4)
containing 0.1% Triton X-100, and counted in a gamma 5500 counter
(Beckman, Irvine, CA). All procedures were conducted at 4°C.
Proliferation assay
Five to 7 days after the last immunization, we obtained spleen cells (33) from two to three identically treated mice and pooled them. This was necessary to obtain enough cells for testing all of the Ag listed below. We depleted the splenocytes in CD8+ cells using paramagnetic beads and rat anti-mouse CD8 Ab (PharMingen) (33, 37, 38).
For proliferation assays, we suspended the cells in RPMI 1640 (Life
Technologies) supplemented with 10% heat-inactivated FCS (Life
Technologies), 50 µM 2-ME, 1 mM L-glutamine, 10 mM HEPES,
1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin
(1 x 106 cells/ml). We seeded the cells in
triplicate in 96-well flat-bottom plates (200 µl/well). We added one
of the following Ag or stimulants: 10 µg/ml PHA (Sigma), 10 µg/ml
TAChR, and 10 µg/ml of the individual subunit peptides. Controls
were triplicate wells cultivated without any Ag or with a 20-residue
control peptide synthesized by the same method, unrelated to the TAChR
sequence (10 µg/ml). After 4 days, we labeled the cells for 16 h
with [3H]thymidine (1 µCi/well, sp. act., 6.7
Ci/mmol; DuPont, Boston, MA) and harvested them (Titertek; Skatron,
Sterling, VA). We measured the [3H]thymidine
incorporation by liquid scintillation.
Cytokine secretion by CD8+-depleted splenocytes in response to stimulation with TAChR
Five to 7 days after a last boost with TAChR, we prepared pooled
CD8+-depleted splenocytes from three identically
treated mice. We resuspended the cells at 5 x
106 cells/ml and cultured them with and without
10 µg/ml TAChR in 24-well plates. In some experiments, we set up two
independent cultures for each Ag. Cells cultivated without any Ag
served as controls for spontaneous secretion of cytokines. We harvested
the culture supernatants after 24 and 96 h. We measured the
concentrations of IFN-, IL-2, IL-4, and IL-10 by capture ELISA using
duplicate samples. We used anti-IFN-, anti-IL-2,
anti-IL-4, and anti-IL-10 monoclonal and polyclonal Ab
(PharMingen) and recombinant IFN-, IL-2, IL-4, and IL-10
(PharMingen) as standards according to the manufacturer’s
instructions.
Statistical analysis
We determined the significance of the differences of the average responses of two groups using a two-tailed Student’s t test. We considered a difference significant when p < 0.05. To determine the significance of the differences between two curves, such as those describing the time course of the appearance of anti-AChR Ab, we used the program EXCEL-ANOVA. We considered a difference to be significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient mice are susceptible
In the first set of experiments, we immunized five WT, five
IFN--/-, and four
IL-12-/- mice with TAChR. We measured the
strength of the individual mice every 2 wk for 14 wk, starting just
before the first TAChR injection (time 0, Fig. 1
). The horizontal line in Fig. 1
corresponds to the holding time (8.3 min) below which we considered the
mice to have EMG. Two WT mice developed persistent myasthenic weakness
from week 10 onward. The other three mice had holding times indicative
of EMG between 11 and 12 wk and recovered by week 14 (i.e., 6 wk after
the third TAChR immunization). None of the
IL-12-/- mice developed stable EMG weakness,
although two mice had a transient reduction of the holding time to
values slightly below 8.3 min, indicating a modest reduction of the
function of the neuromuscular transmission. All
IFN--/- mice developed EMG. Three had
abnormally reduced holding time after the second TAChR injection when
the WT mice still had normal holding times. From week 9 onward, four
IFN--/- mice had stable EMG and one had a
transient reduction of the holding time to values slightly below 8.3
min, but recovered by week 14.
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-/- mice
to EMG, we immunized a second set of mice that included eight
IFN--/- mice and 19 WT mice. We tested the
mouse strength just before the first TAChR immunization and every 2 wk
for 10 wk. The results of the tests conducted 4, 8, and 10 wk after the
beginning of the immunization are reported in Fig. 2. IFN--/- and
WT mice developed EMG with similar frequency and severity.
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-/-, and IL-12-/- mice
develop comparable amounts of anti-TAChR Ab
We measured the serum anti-TAChR Ab of WT,
IFN--/-, and
IL-12/- mice using the RIPA. Fig. 3 shows the anti-TAChR Ab
concentration in the sera of the mice shown in Fig. 1
that was obtained
4, 8, and 14 wk after the beginning of the TAChR immunization. In
agreement with previous reports (9), we found substantial
variations in the concentration of anti-TAChR Ab from mouse to
mouse that are reflected in the large SDs of the values in Fig. 3. The average concentration of serum anti-TAChR Ab was
similar for all three groups. We obtained similar results when we used
the sera of the IFN--/- and WT mice
illustrated in Fig. 2. After three immunizations with TAChR, WT and
IFN--/- mice had average serum
concentrations of anti-TAChR Ab of 2.8 ± 1.1 and 2.1 ±
0.7 µM: the difference was not statistically significant.
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and IL-12 deficiency on the
synthesis of Th1-driven IgG subclasses
We measured by ELISA the serum concentration of anti-TAChR IgG
subclasses driven by different cytokines. WT mice synthesized primarily
anti-TAChR IgG2b and IgG2c, to a lesser extent IgG1 (Fig. 4), while IgG3 was undetectable (data not
shown); the anti-IgG2a mAb yielded a very small signal that likely
resulted from cross-reactivity with the highly homologous IgG2c (Fig. 4). IFN--/- and
IL-12-/- mice had anti-TAChR IgG1 in
amounts similar to those of the WT mice, whereas their anti-TAChR
IgG2c (and IgG2a) was strongly reduced: in both strains, they were
almost undetectable even 14 wk after beginning the TAChR immunization
(Fig. 4). Absence of IFN- or IL-12 had different effects on
synthesis of anti-TAChR IgG2b. They were significantly reduced in
IL-12-/- mice, whereas in
IFN--/- mice their serum concentration was
comparable to that of WT mice. IFN--/- mice
had a slight reduction in anti-TAChR IgG2b at week 14, which was
not significant as compared with WT mice.
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suggest that the overall serum
concentration of anti-TAChR IgG was higher in WT mice than in the
cytokine mutants. On the other hand, the RIPA experiments indicated
that all strains had similar amounts of serum anti-TAChR Ab. To
identify reasons for this discrepancy, we determined by ELISA the
concentration of anti-TAChR IgG, IgM, and IgE in the mouse sera.
They all had large amounts of anti-TAChR IgG, although
IFN--/- and IL-12-/-
mice had less IgG than WT mice: after three TAChR injections, the
average total IgG in IFN--/- and
IL-12-/- mice was 50 and 40%, respectively, of
the average total IgG in WT mice. None of the strains had measurable
serum anti-TAChR IgM or IgE.
IFN--/- and WT mice immunized with TAChR have
IgG2b and complement at the neuromuscular junction, whereas
IL-12-/- mice do not
We investigated whether the different susceptibility to EMG of these strains was related to the ability of the anti-TAChR Ab to bind to muscle and fix complement.
We measured by RIPA the amounts of mouse muscle AChR that was bound to
Ab in three WT mice, four IFN--/- mice, and
three IL-12-/- mice immunized three times with
TAChR and boosted with a fourth injection 5–7 days before euthanasia.
We found comparable amounts of Ab-complexed AChR in all three groups
(Fig. 5A). Thus, the EMG
resistance of the IL-12-/- mice was not due to
inability of their anti-TAChR Ab to bind mouse muscle
AChR.
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-/-, and three
IL-12-/- mice immunized with TAChR. For each
mouse, we analyzed at least 10 muscle sections in which we could
identify neuromuscular junctions by -BTX binding (red fluorescence).
We identified mouse IgG and the C3 complement component by specific
fluorescent Ab (blue and green fluorescence, respectively). Fig. 5B shows the results of representative sections for each
strain. All mice had IgG bound to the neuromuscular junctions. All WT
and IFN--/- mice also had complement,
whereas we could not detect the presence of complement in any muscle
section from the IL-12-/- mice.
We investigated whether the presence of complement at the neuromuscular
junctions correlated with binding of complement-fixing IgG subclasses.
We stained muscle sections of a mouse from each strain with -BTX and
with mAbs specific for IgG2b and IgG2c. Other sections of the same mice
were stained for total IgG, IgM, and complement. All mice had IgG and
none had IgM bound to the junctions; only WT and IFN- mice had
complement. When stained for the different IgG2 subclasses, the
neuromuscular junctions of the WT mouse stained for both IgG2b and
IgG2c, those of the IFN--/- mouse stained
only for IgG2b, whereas those of the IL-12-/-
mouse did not stain for either IgG2b or IgG2c (Fig. 5C).
CD4+ T cell response to TAChR and TAChR epitopes in WT,
IFN--/-, and IL-12-/- mice
CD4+ cells of mice of B6 background
immunized with TAChR recognize primarily an epitope or overlapping
epitopes within the sequence region 146–169 of the TAChR subunit
(15, 33, 37, 38, 41, 42), and less intensely and
consistently epitopes within the sequences 181–200 and 360–378 of the
TAChR subunit (33) and other minor epitopes
(38). We used CD8+-depleted
splenocytes from WT, IFN--/-, and
IL-12-/- mice immunized three times with TAChR
and boosted with another TAChR injection 5–7 days before being
euthanized to determine their proliferative response to the TAChR and
to overlapping synthetic peptides spanning the sequence of the TAChR
subunit. We conducted two independent experiments that yielded
consistent results. Fig. 6 illustrates
the results of one experiment. All strains recognized the TAChR
vigorously and to comparable extents. They all recognized the peptides
spanning the sequence region 146–169 and peptide 360–378. Other
peptides were recognized inconsistently. In the experiment depicted in
Fig. 6, WT mice also recognized the sequence 30–47,
IFN--/- mice also recognized peptides
106–122 and 181–200, and IL-12-/- mice
also recognized the peptides spanning the sequence 30–60 and
420–438.
|
Stimulation of modulatory Th2 or Th3 cells,
which depend upon IL-4 for their development, is a possible mechanism
of protection from EMG. We investigated whether an increased
anti-TAChR sensitization of Th2 cells correlated with resistance to
EMG by measuring the IL-4 and IL-10 secreted by
CD8+-depleted spleen cells challenged in vitro
with TAChR. We determined also the secretion of IFN- and IL-2 in the
same cultures. We used CD8+-depleted splenocytes
from mice immunized three times with TAChR and boosted with a
fourth injection 5–7 days before the experiments. We cultured the
cells with 10 µg/ml of TAChR and measured the cytokine concentrations
in the culture supernatants. Fig. 7
summarizes the results of one of two consistent experiments. The cells
from IFN--/- mice secreted IL-4 and IL-10 in
amounts comparable to those observed for the cells from the WT mice;
they did not secrete IFN- and secreted moderate, yet significantly
reduced amounts of IL-2 as compared with WT mice. The cells from
IL-12-/- mice secreted much more IL-4 and IL-10
than those from either WT or IFN--/- mice;
they secreted IL-2 in amounts that were slightly, but not
significantly, reduced as compared with WT mice and significantly less
IFN- than the WT mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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have
different roles in the pathogenesis of an autoantibody-mediated
disease, EMG. IL-12, and therefore Th1 cells, have a crucial role,
since IL-12-/- mice had minimal or no EMG
symptoms: this is in excellent agreement with the conclusions of a
previous study (16). On the other hand, in B6 mice IFN-
is not necessary for EMG development, since
IFN--/- mice developed anti-TAChR Ab
(Fig. 3) and EMG symptoms (Figs. 1 and 2) with the same frequency and
intensity as WT mice.
Two studies (17, 18) investigated the effects of
disrupting the genes for IFN- and its receptor on EMG using mice of
mixed genetic background ((129/SvEv x
C57BL/6)F2). The 129/SvEv and the C57BL/6 strains
both have the H-2b allele, and the I-A allele
influences EMG susceptibility; however, genetic factors unrelated to
the MHC haplotype also influence susceptibility to EMG
(43, 44, 45). The different genetic background of the mice
used in those studies (17, 18) and in the present study
complicates the comparison of their results.
One study (17) found that (129/SvEv x
C57BL/6)F2 mice genetically deficient in IFN-
did not synthesize anti-TAChR Ab (including Th2-driven IgG
subclasses) or develop EMG after TAChR immunization. Absence of
IFN- reduces the effectiveness of Ag presentation.
IFN--/- mice have reduced macrophage
function and expression of MHC class II molecules (7). An
insufficient immunization, perhaps due to TAChR degradation by
proteases that copurify with the TAChR (46, 47), might
explain the lack of anti-TAChR response in that study
(17).
The second study (18) found that (129/SvEv x
C57BL/6)F2 mice genetically deficient in IFN-
receptor developed less anti-AChR Ab than the nonmutated mice and
less frequent and severe EMG. This indicated that IFN- signaling was
a disposable, albeit facilitating, factor for EMG development. Thus,
EMG can develop in the absence of IFN- signaling if the TAChR
immunization suffices to elicit a good anti-AChR Ab response.
The more important facilitating effect of IFN- on EMG in
(129/SvEv x C57BL/6)F2 (17, 18) than in B6 mice may be because it stimulates macrophage
function and expression of class II molecules (1, 2, 3, 4).
Also, IFN- may trigger pathogenic mechanisms other than those
mediated by anti-TAChR Ab: transgenic expression of IFN- in
mouse muscle resulted in cellular infiltrates, deposition of IgG at the
neuromuscular junction, and disruption of neuromuscular transmission
without synthesis of anti-AChR Ab (19). IFN- has
complex and sometimes contrasting effects in other autoimmune responses
(see below). This and the previous studies (17, 18)
indicate that also in EMG the effects of IFN- are complex and may be
influenced by other genetic factors.
The serum concentration of anti-AChR Ab does not correlate with
symptom severity in EMG or MG, indicating that not every subpopulation
of anti-AChR Ab causes the disease (9). In agreement
with those findings, the mouse strains we used, irrespective of their
susceptibility to EMG, had levels of serum anti-TAChR Ab that were
not statistically different. Since none of the strains developed
anti-TAChR IgM or IgE and the cytokine mutants had less
anti-TAChR IgG, the similar levels of anti-TAChR Ab found in
the RIPA experiments in all strains might be due to the presence of
anti-TAChR IgA in the Th1 cytokine-deficient mice, because of their
propensity to develop Th2-induced Ab responses (7, 8). In
our mice the ability of anti-TAChR Ab to induce EMG was not due
just to cross-reactivity with muscle AChR, since all
IL-12-/- mice had Ab bound to their
neuromuscular junctions (Fig. 5A), yet they did not have EMG
weakness. The ability of anti-AChR Ab to fix complement likely
contributes to their pathogenic potential. The diminished ability to
synthesize complement-binding anti-TAChR IgG subclasses in
EMG-resistant IL-12-/- mice (Fig. 4) and the
absence of complement and of complement-binding IgG2b and IgG2c at
their muscle synapses (Fig. 5, B and C) support
an important role of complement activation in EMG development.
WT mice made large amounts of anti-TAChR IgG2b and 2c (Fig. 4),
which bind complement effectively. IFN--/-
mice had less anti-TAChR IgG2c, whereas the
IL-12-/- had less of both anti-TAChR IgG2b
and IgG2c (Fig. 4). Also, IL-12-/- mice was the
only strain that did not have IgG2b (and complement) at the muscle
synapses. These data suggest that anti-AChR IgG2b have a prominent
role in triggering the complement-mediated destruction of the
neuromuscular junction. The effective synthesis of anti-TAChR IgG2b and
their binding to mouse muscle AChR likely explain the consistent
presence of complement at the neuromuscular junctions of
IFN--/- mice (Fig. 5) and their frequent EMG
symptoms (Figs. 1 and 2). The finding that
IFN--/- mice had a reduced synthesis of
anti-TAChR IgG2c (Fig. 4), yet were susceptible to EMG (Figs. 1 and 2) down plays the importance of the anti-AChR IgG2c in EMG.
The serum concentration of anti-TAChR IgG1 was comparable in
all strains, suggesting that Th2-driven IgG subclasses have limited
importance in EMG development.
Two IL-12-/- mice after three TAChR
immunizations had reduced holding times indicative of EMG (Fig. 1).
Also Moiola et al. (16) found that a few
IL-12-/- mice immunized with TAChR had
defective neuromuscular transmission. This might be due to the small
amounts of anti-TAChR IgG2b in these mice, perhaps driven by the
action of IL-2 or other cytokines (48).
Other mechanisms might cause the reduced synthesis of pathogenic
anti-AChR Ab and the EMG resistance of
IL-12-/- mice. A protective mechanism might be
the increased sensitization of anti-TAChR Th2 cells. This is
suggested by the increased secretion of IL-4 and IL-10 after challenge
with TAChR by splenocytes from IL-12-/- mice as
compared with WT and IFN--/- mice (Fig. 7).
Also Moiola et al. (16) found that
IL-12-/- B6 mice had increased TAChR-specific
IL-4 producing cells. Anti-TAChR Th2 cells might have a direct
protective action by down-regulating the activity of Th1 cells and APC
and by exerting a local anti-inflammatory activity. They may also
exert indirect protective effects mediated by IL-4 through the
activation of regulatory cells that secrete TGF-ß (4, 49).
All three mice strains we tested had a good sensitization of
CD4+ cells to the TAChR. Their spleen
CD4+ cells recognized most strongly the same
sequence regions of the TAChR subunit (residues 146–169 and
residues 360–378, Fig. 6). We observed small differences in the
CD4+ repertoire of WT- and cytokine-deficient
mice, perhaps due to individual variations in the
CD4+ response to minor TAChR epitopes
(9). One exception may be peptide 420–438, that elicited
a strong response of CD4+ splenocytes from
IL-12-/- mice: CD4+ cells
from WT- or IL-4-deficient B6 mice never recognized this sequence
(Refs. 15, 33, 37, 38, 41, 42 and Fig. 6), that might
sensitize Th2 cells.
Development of several T cell-mediated experimental autoimmune diseases requires IL-12 (50, 51, 52, 53, 54, 55). IL-12 has been implicated also in the pathogenesis of multiple sclerosis (56, 57). In those diseases, Th1 effector cells cause tissue injury by local secretion of proinflammatory cytokines (50, 51, 52, 53, 54, 55, 56, 57, 58, 59). Development of Ab-mediated autoimmune diseases does not always require IL-12 (60, 61, 62).
IFN- has complex and contrasting effects in the development of T
cell-mediated autoimmune diseases. Most EAE-inducing T cell clones
isolated from diseased mice secreted IFN- (62) and Ab
that neutralized IFN--inducing factor prevented EAE
(59). Treatment with IFN- exacerbated multiple
sclerosis (63). Yet, mice with a disrupted IFN- gene
were susceptible to EAE (64), and IFN- may even play a
role in down-regulating EAE (65). IL-12, but not IFN-,
appeared to be crucial for development of mouse Th1 cell-mediated
colitis (66, 67). IFN- transgenic rats that expressed
IFN- in the eye had increased severity and accelerated onset of
autoimmune uveitis (68), yet IFN--deficient mice could
develop this disease (69). Transgenic expression of
IFN- in pancreatic ß cells caused loss of islet tolerance
(58), and treatment of nonobese diabetic mice with
anti-IFN- Ab prevented diabetes (70, 71); yet,
genetic absence of IFN- in nonobese diabetic mice delayed but did
not prevent diabetes (72).
The present results underline the importance of IL-12 and complement-induced destruction of the neuromuscular junction in the development of EMG. Complement-fixing Ab are likely important in MG as well, since MG patients always have complement at the neuromuscular junction (9) and anti-AChR Th1 cells (12, 13). The present results suggest that IL-12 might be a target for therapeutic approaches in MG.
|
Footnotes |
|---|
2 Previously published under the name Bianca M. Conti-Tronconi.
3 Address correspondence and reprint requests to Dr. Bianca M. Conti-Fine, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108.
4 Abbreviations used in this paper: MG, myasthenia gravis; -BTX, -bungarotoxin; AChR, muscle acetylcholine receptor; EMG, experimental MG; NMS, normal mouse serum; TAChR, Torpedo AChR; RIPA, radioimmunoprecipitation assay; WT, wild type; EAE, experimental autoimmune encephalomyelitis.
5 We use TAChR to indicate the AChR purified from Torpedo electric organ, whereas AChR indicates AChR irrespective of its tissue source and animal species. In this paper, the abbreviation AChR frequently refers to mouse muscle AChR.
Received for publication July 29, 1999. Accepted for publication March 2, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit of human muscle acetylcholine receptor in myasthenia gravis. Neurology 48:1643. (IFN-) is necessary for the genesis of acetylcholine receptor-induced clinical experimental autoimmune myasthenia gravis in mice. J. Exp. Med. 186:385. receptor deficiency are less susceptible to experimental myasthenia gravis. J. Immunol. 162:3775. in neuromuscular junction. J. Exp. Med. 181:547. have opposing effects, while TGF-ß positively regulates its own production. J. Immunol. 160:5719.-expressing Th1-like cells and up-regulation of TGF-ß mRNA in mononuclear cells. Ann. NY Acad. Sci. 778:273.[Medline]
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), IFN-
),
and IL-12-/- (
) mice. The mice had been
immunized with TAChR at weeks 0, 4, and 8 and had received a further
boost 5–7 days before being sacrificed. The columns represent the
average ± SD of duplicate ELISA determinations in the
supernatant of cells cultivated in the presence of 10 µg/ml TAChR.
The spontaneous secretion of the different cytokines by cells
cultivated in the absence of any stimulus was subtracted. The
asterisks indicate a significant difference in the secretion of
cytokines by the cells of the knockout mutants as compared with the
secretion by the cells from WT mice (*, p < 0.03;
**, p < 0.02; ***, p <
0.01, ****, p < 0.002). See text for
experimental details.