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Receptor Deficiency Are Less Susceptible to Experimental Autoimmune Myasthenia Gravis1
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Division of Neurology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden;
Biomedical Primate Research Center, Rijswijk, The Netherlands; and
Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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can either adversely or beneficially affect certain
experimental autoimmune diseases. To study the role of IFN- in the
autoantibody-mediated experimental autoimmune myasthenia gravis (EAMG),
an animal model of myasthenia gravis in humans, IFN-R-deficient
(IFN-R-/-) mutant C57BL/6 mice and congenic wild-type
mice were immunized with Torpedo acetylcholine receptor
(AChR) plus CFA. IFN-R-/- mice exhibited significantly
lower incidence and severity of muscle weakness, lower anti-AChR
IgG Ab levels, and lower Ab affinity to AChR compared with wild-type
mice. Passive transfer of serum from IFN-R-/- mice
induced less muscular weakness compared with serum from wild-type mice.
In contrast, numbers of lymph node cells secreting IFN- and of those
expressing IFN- mRNA were strongly augmented in the
IFN-R-/- mice, reflecting a failure of negative
feedback circuits. Cytokine studies by in situ hybridization revealed
lower levels of lymphoid cells expressing AChR-reactive IL-1ß and
TNF-
mRNA in AChR + CFA-immunized IFN-R-/- mice
compared with wild-type mice. No differences were found for
AChR-reactive cells expressing IL-4, IL-10, or TGF-ß mRNA. These
results indicate that IFN- promotes systemic humoral responses in
EAMG by up-regulating the production and the affinity of anti-AChR
autoantibodies, thereby contributing to susceptibility to EAMG in
C57BL/6-type mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(4) and IL-2 and TNF- (5), as well as the Th2 cytokines IL-4
(3, 6, 7), IL-6 (8), and IL-10 (9), might be involved in the
development of both MG and EAMG, while TGF-ß (10) and IFN- (11)
may have a suppressive effect in both diseases.
The particular importance of IFN- for autoimmunity is the apparent
ability of this cytokine to critically contribute to the conversion of
anergic autoreactive T cells into active effector cells, as illustrated
by experiments with organ-specific overexpression of IFN- (12).
IFN- has been considered to play a pivotal disease-promoting role in
cell- and Ab-mediated autoimmunity. However, IFN- is also one of the
factors inhibiting the disease in a mouse strain resistant to
experimental allergic encephalomyelitis, indicating that IFN- also
exerts immunosuppressive activity (13).
Recently, transgenic mice that lack the IFN-R
(IFN-R-/-) have become available, offering an
excellent in vivo model to study the immune-regulatory functions of
endogenous IFN-. These mice develop a normal immune system, possess
IFN--independent macrophage and NK cell activity, and constitutively
express MHC class I and II Ags (14). To further study the systemic
immune response mediated by IFN- in EAMG, IFN-R-/-
mice and wild-type littermates were inoculated with Torpedo
AChR emulsified in CFA. They were evaluated regarding clinical course,
AChR-induced T and B cell responses, and cytokine profiles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mice that were used in this study have been described (14).
Briefly, to disrupt IFN-R, embryonic stem cells were transfected
with a replacement vector containing 11 kb of the murine IFN-R gene
with the neomycin resistance gene at exon V. Chimeric founder males
were mated with C57BL/6 females to yield heterozygous F1
offspring. These mice were interbred to yield wild-type and
IFN-R-/- (129/Sv/Ev x C57BL/6)F2
offspring. These mice were obtained from Dr. M. Aguet (Genentech, San
Francisco, CA) and maintained and bred at the animal housing facilities
of the Microbiology and Tumor Biology Center, Karolinska
Institute. All mice used were females 8–12 wk of age and weighed
20–30 g.
Ag preparation
Torpedo AChR was purified from the electric organs of
T. californica (Pacific Biomarine, Venice, CA) by affinity
chromatography on an -cobrotoxin-agarose resin (Sigma, St. Louis,
MO) as described previously (15). The isolated product was pure
as judged by SDS-PAGE. Muscle AChR extract from normal C57BL mouse
(M-AChR) was prepared (15). Myelin basic protein (MBP) used as control
Ag was purified from normal mouse brains.
Induction of EAMG
Mice were immunized with 20 µg of Torpedo AChR emulsified in CFA in a total volume of 100 µl injected into five intradermal sites along the back, the hind footpads, and the base of the tail, and they were boosted on days 30 and 60 after the primary immunization (postinjection; p.i.). The mice were observed every other day in a blinded fashion for signs of clinical muscle weakness. The disease symptoms were graded as follows (16): 0, no definite muscle weakness; 1+, normal strength at rest but weak with chin on the floor and inability to raise the head after exercise consisting of 20 consecutive paw grips; 2+, same as grade 1+ and weakness at rest; and 3+, moribund, dehydrated, and paralyzed. Clinical EAMG was confirmed by i.p. administration of neostigmine bromide and atropine sulfate (16). Mice were sacrificed 100 days p.i.
RIA for measurements of serum anti-AChR Abs
Blood specimens were collected from the tail vein just before
immunization and then at weekly intervals. Serum Ab concentrations to
AChR were measured by RIA (15). Briefly, 1 nM M-AChR was incubated with
2 nM 125I-labeled -bungarotoxin (Amersham, Arlington
Heights, IL). To 1 ml of labeled M-AChR, 1 µl of serum was added,
followed by rabbit anti-mouse Ig (Dakopatts, Glostrup, Denmark).
The samples were centrifuged, washed, and counted in a gamma counter.
The AChR precipitated minus the background value permits calculation of
the titer in mol of toxin binding sites bound per liter of serum.
Detection of relative affinity of serum anti-AChR IgG Abs
The relative affinity of anti-AChR IgG Abs was determined by ELISA using thiocyanate elution (17). Briefly, microtiter plates (Costar, Cambridge, MA) were coated with Torpedo AChR (5 µg/ml). Uncoated sites were blocked with 10% FCS (Life Technologies, Paisley, U.K.). Diluted serum with a constant concentration of anti-AChR Abs (1 pM, as determined by RIA) was added and incubated. Then, appropriate molarities of potassium thiocyanate (KSCN) were added in duplicate and incubated, followed by biotinylated goat anti-rat IgG (Harlan, Grawley Down, U.K.) and alkaline phosphatase-avidin (Vector Laboratories, Burlingame, CA). The color was developed with p-nitrophenyl phosphate and expressed as OD at 405 nm. The relative affinity was expressed as affinity index equal to the molarity of KSCN, resulting in 50% of the absorbance obtained in the absence of KSCN.
Ab isotype assay
Isotypes for mouse anti-AChR Abs were detected as described (18). Briefly, microtiter plates (Costar) were coated with 100 µl per well of Torpedo AChR at 4°C overnight. Uncoated sites were blocked with 10% FCS (Life Technologies). Diluted serum with a predetermined amount of anti-AChR Abs was added and incubated for 2 h at room temperature. Then, plates were incubated for 2 h with rabbit anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (Dakopatts), followed by biotinylated swine anti-rabbit Ig (Dakopatts) and alkaline phosphatase-conjugated avidin-biotin complex (Vector). The color was developed with p-nitrophenyl phosphate and expressed as OD at 405 nm.
Passive serum transfer
Muscle strength of recipient mice (16 female wild-type mice) was
predetermined using an automated grip strength meter (Columbus
Instruments, Columbus, OH) as described (19) after 6.0 µg of
D-tubocurarine to establish their individual baseline.
Briefly, mice were placed on a platform, allowed to grasp a rectangular
ring, and then steadily pulled away (
1 inch/s) until the grip was
broken. Grip strength was determined with a computerized electronic
pull strain gauge that was fitted directly to the grasping ring. The
peak values of 10 measurements were automatically recorded at the end
of 3 min of the exercise. The mice were randomly divided into two
groups. Pooled sera from IFN-R-/- mice or from
wild-type mice, obtained 4–10 wk p.i. with AChR + CFA, were injected
i.p. into the recipient mice at a dose of 0.3 ml/20 g body weight (20, 21). Each of the sera was assayed for anti-M-AChR Abs, and the
titers were made uniform at 1.5 nM before injection. Forty-eight hours
after passive transfer, the mice were again given 6.0 µg of
D-tubocurarine/20 g body weight and remeasured blindly by
two observers on the grip strength meter to detect muscle weakness.
Mononuclear cell (MNC) suspensions
Eight mice in each group were randomly sacrificed, and suspensions of MNC from the popliteal and inguinal lymph nodes (PILN) were prepared. Cells were suspended in DMEM (Life Technologies) supplemented with 1% (v/v) MEM (Life Technologies), 2 mM glutamine (Flow Laboratories, Irvine, U.K.), 50 IU/ml penicillin, and 50 µg/ml streptomycin (Life Technologies) and with 10% (v/v) FCS (Life Technologies). The cells were washed three times and then rediluted to a cell concentration of 2 x 106/ml.
Enumeration of anti-AChR IgG Ab-secreting cells
A solid-phase enzyme-linked immunospot assay was used, with some modifications (7). Briefly, wells of microtiter plates with nitrocellulose bottoms were coated with 100 µl of AChR or MBP (10 µg/ml in PBS). Aliquots of 100-µl cell suspensions containing 2 x 105 MNC were added in triplicate to individual wells. After incubation for 24 h, the wells were emptied, followed by addition of rabbit anti-mouse IgG (Sigma), biotinylated swine anti-rabbit IgG (Dakopatts), and avidin-biotin-peroxidase complex (Dakopatts). After peroxidase staining, the red-brown immunospots that corresponded to cells having secreted anti-AChR IgG were counted and standardized to numbers per 105 MNC.
AChR-reactive IFN--secreting cells
Nitrocellulose-bottomed microtiter plates were coated with 100
µl of rat IFN- capture Ab (DB1; Innogenetics, Genth, Belgium) at
15 µg/ml. Aliquots of 200 µl of suspension containing 4 x
105 MNC were added to individual wells in triplicate,
followed by Ag (AChR or MBP) or the mitogen Con A (Sigma) in 10-µl
aliquots to a final concentration of 10 µg/ml AChR or MBP or 5
µg/ml Con A. These Ag or mitogen concentrations resulted in optimal
stimulatory effects in preliminary experiments (4, 7). After 48 h
of culture, the wells were emptied. Secreted and bound IFN- was
visualized by sequential application of polyclonal rabbit anti-rat
IFN- (Immogenetics), biotinylated anti-rabbit IgG, and
avidin-biotin-peroxidase complex (both from Dakopatts). After
peroxidase staining, the red-brown immunospots, which corresponded to
the cells that had secreted IFN-, were enumerated in a dissection
microscope. To calculate the numbers of T cells responding to a
particular Ag or mitogen, numbers of spots in culture without Ag added
were subtracted from the values obtained after Ag or mitogen exposure.
The data were expressed as number of spots per 105
MNC.
Lymphocyte proliferation responses
Triplicate aliquots (200 µl) of MNC suspensions were applied into 96-well round-bottomed microtiter plates (Nunc, Copenhagen, Denmark) at a cell density of 2 x 106/ml. Aliquots (10 µl) of either AChR, MBP, or Con A were added into appropriate wells at a final concentration of 10 µg/ml (AChR or MBP) or 5 µg/ml (Con A). After 60 h of incubation, the cells were pulsed for 12 h with 10-µl aliquots containing 1 µCi of [3H]methylthymidine (specific activity 42 Ci/mmol; Amersham). Cells were harvested onto glass fiber filters, and thymidine incorporation was measured. The results were expressed as stimulation index, which was calculated by dividing the cpm from culture in the presence of Ag or mitogen by the cpm from culture without Ag.
Detection of cytokine mRNA expression by in situ hybridization (ISH)
ISH was performed as described (6). Briefly, 200-µl aliquots
of suspensions from PILN containing 4 x 105 MNC were
plated into round-bottomed microtiter plates (Nunc) in triplicate.
Aliquots (10 µl) of either AChR, MBP, or Con A were added into
appropriate wells at a final concentration of 10 µg/ml (AChR or MBP)
or 5 µg/ml (Con A). After culture for 24 h, the cells were
washed, counted, and dried onto restricted areas of electrically
charged glass slides (ProbeOn slides; Fisher Scientific, Pittsburgh,
PA). Synthetic oligonucleotide probes (Scandinavian Gene Synthesis AB,
Köping, Sweden) were labeled using 35S-labeled
deoxyadenosine-5'--(thio)-triphosphate with terminal
deoxynucleotidyl transferase (Amersham). To increase the sensitivity of
the method, a mixture of four different oligonucleotide probes was
employed for each cytokine. The oligonucleotide sequences were obtained
from GenBank using the MacVector software (IBI). Probes used in this
study are listed to in Table I
(22, 23, 24, 25, 26) (W.
Feeser and B. D. Frimark, unpublished data). The oligonucleotide
probes were 48 bases long and checked for the absence of palindromes
or long sequences of homology within the species against available
GenBank data. Cells were hybridized with 106 cpm of labeled
probe per 100 µl of hybridization mixture. After emulsion
autoradiography, development, and fixation, the coded slides were
examined by dark-field microscopy for positive cells containing >15
grains per cell in a starlike distribution. The intracellular
distribution of the grains was always checked by light microscopy.
Labeled cells were expressed as numbers per 105 MNC.
Numbers of cells expressing cytokine mRNA after culture without Ag
added, which are considered as cells spontaneously expressing cytokine
mRNA, were subtracted from the values obtained after Ag exposure. In
many positive cells, the grains were so heavily accumulated within and
around the cells that it was not possible to count every single grain.
In cells judged negative, the number of grains was mostly zero to two
per cell, and the grains were scattered randomly over the cells and not
distributed in a starlike fashion. There were, therefore, no
difficulties in differentiating between positive and negative cells.
Variation between duplicates was <10%. The number of cells used in
ISH was not equal to the number that was ultimately detected on the
slide. Cell losses were 10–50% (mean 30%) from cell application to
cell counting. The preferential loss of certain cell types is not ruled
out. To compensate for cell losses, the total number of cells on the
slides was regularly counted. With the help of a microscope grid used
as a measuring unit, the radius (r) of the round area
(A) covered by cells was determined. The area A
was calculated by the formula A =
r2 x 
. Cells were usually counted in two
grids at the periphery and one grid at the center of the surface
covered by cells. The mean value of the number of cells per grid was
determined, and the total number of cells in area A was
calculated by the formula mentioned. In case of uneven distribution,
cells in additional grids were counted. There were no positive cells in
a control probe consisting of the sense nucleotide sequence for rat
IFN- exon 4, which was used in parallel with the cytokine probes on
the cells from each rat.
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Differences between groups were tested with the two-tailed
Student’s t test. Differences between groups with respect
to the incidence were analyzed with a 
2 test. The level
of significance was set at p = 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clinical signs were evaluated from two parallel experiments. In
experiment 1, 7 of 15 wild-type mice developed muscle weakness before
(3 mice) and after (4 mice) the first boosting with Torpedo
AChR + CFA. Among them, 2 mice exhibited mild and moderate muscle
weakness. Three mice deteriorated progressively, and 2 of them died,
whereas 1 was humanely sacrificed. In contrast,
IFN-R-/- mice exhibited less severe disease than the
wild-type mice. After first boosting on day 31, 3 of 13
IFN-R-/- mice exhibited mild or moderate weakness, and
1 of them recovered at day 80 p.i. Clinical myasthenia was further
confirmed by injection of the anticholinesterase drug neostigmine that
improved muscle strength. This improvement started 15 min after
administration and lasted for several hours. There was no difference
regarding the time of onset of clinical disease between
IFN-R-/- and wild-type mice.
In experiment 2, the same tendencies of clinical onset and disease
progression were observed as in experiment 1. After immunization with
Torpedo AChR + CFA, 7 of 11 wild-type mice developed
clinical EAMG, and 1 of them was humanely killed when upon reaching
disease severity grade 3. In the IFN-R-/- group, 1 of
8 mice developed mild or moderate clinical disease. Muscle weakness was
confirmed by clinical improvement after administration of neostigmine
(Table II).
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The levels of serum anti-M-AChR Abs were detected from both
experiments. These Abs increased gradually over the observation period
in both groups after immunization with Torpedo AChR + CFA.
IFN-R-/- mice had significantly lower serum
anti-AChR Ab levels at 4, 6, and 10 wk p.i. compared with wild-type
mice (p < 0.01, 0.05, and 0.001, respectively)
(Fig. 1). No differences were found at other time points
under study.
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R-/- mice. Significant differences between the two
groups were noticed at weeks 6, 8, and 10 p.i. (Fig. 2).
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To study the influence of IFN-R deficiency on the Ab isotypes,
we used ELISA to determine anti-AChR IgG1, IgG2a, IgG2b, and IgG3
Abs, which may reflect the preferential production of IL-4 and IFN-,
respectively. Serum samples were examined 4 and 10 wk p.i., when the
anti-AChR IgG Ab levels were significantly different (Fig. 1
).
Although IFN-R-/- mice and wild-type mice had similar
levels of anti-AChR IgG Abs of the IgG1 subclass (Fig. 3), a down-regulation of the IgG2a, IgG2b, and IgG3
subclasses of anti-AChR Ab titers was observed in
IFN-R-/- mice vs wild-type mice at 4 and 10 wk p.i.
(p < 0.05 and p < 0.01,
respectively) (Fig. 3).
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As shown before (20, 27), we also had difficulties in detecting
clinically measurable muscle weakness after passive transfer of immune
sera to MG-susceptible mice. Therefore, we adopted an automated grip
strength meter to quantitate muscle weakness. Fig. 4
shows the muscle strength of individual mice examined before and
48 h after passive transfer of immune sera. Muscle weakness was
dramatically induced in mice receiving sera from AChR-immunized
wild-type mice, while mice receiving sera from AChR-immunized
IFN-R-/- mice showed only minute muscle weakness
(p < 0.01). The decreased muscle weakness
registered in IFN-R-/- compared with wild-type mice
upon immunization with AChR + CFA was thus associated with
reduced ability of imune sera from the IFN-R-/-
mice to transfer muscle weakness.
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The MNC from PILN of wild-type mice, which had higher incidence
and more severe muscle weakness upon immunization with AChR + CFA,
contained higher numbers (18.9 ± 3.9/105 MNC) of
anti-AChR IgG Ab-secreting cells compared with
IFN-R-/- mice (12.5 ± 2.2/105 MNC)
(p < 0.05). Only low numbers of such cells
secreting IgG Abs to the control Ag MBP in lymphoid organs were
observed. There were no differences for such cells between the two
groups.
AChR-reactive IFN--secreting cells and AChR-induced lymphocyte
proliferation
As shown in Table III, the lymph nodes from
IFN-R-/- EAMG mice contained much higher numbers of
AChR-reactive IFN- secreting T cells compared with wild-type mice.
When cells were evaluated in control experiments after culture without
Ag or mitogen, representing cells spontaneously secreting IFN-,
higher numbers were found in IFN-R-/- mice. After
culture of MNC from lymph nodes in the presence of the mitogen Con A,
higher numbers of IFN--secreting cells were also found in the
IFN-R-/- mice (44.1 ± 19.5/105 MNC)
than in the wild-type mice (16.2 ± 18/105 MNC)
(p < 0.05). No difference between the two
groups was seen after culture of cells in the presence of the control
Ag MBP.
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R-/- mice and wild-type mice
upon culture without Ag or mitogen, nor after culture in the presence
of AChR (2.6 ± 0.6 vs 2.7 ± 1.3), the control Ag MBP
(1.0 ± 0.2 vs 1.4 ± 0.8), or Con A (4.1 ± 0.9 vs
5.2 ± 1.7), respectively, which suggests that there were no
modulatory effects on IL-2 production. AChR-reactive cytokine mRNA-expressing cells
To identify the influence of IFN-R deficiency on the cytokine
profiles after inducing EAMG, we determined the numbers of cells
expressing mRNA for the Th1-related cytokines IFN- and TNF-, the
Th2 cytokines IL-4 and IL-10, the immunosuppressive cytokine TGF-ß,
and an additional proinflammatory cytokine (IL-1ß) in MNC from lymph
nodes. IFN-R-/- mice revealed strongly elevated
numbers of AChR-reactive IFN- mRNA-expressing cells when compared
with wild-type mice. The numbers of TNF- and IL-1ß mRNA-expressing
cells in the IFN-R-/- mice were lower than in the
wild-type mice. There were no differences between the two groups for
AChR-reactive IL-4, IL-10, or TGF-ß mRNA-expressing cells (Fig. 5).
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mRNA-expressing cells were dramatically elevated in the
IFN-R-/- mice (27.3 ± 15/105 MNC)
compared with wild-type mice (4.2 ± 1.5/105 MNC) in
the control experiments with cultures of MNC without Ag. Higher numbers
were also observed in the IFN-R-/- mice after culture
in the presence of Con A but not in the presence of the irrelevant Ag
MBP (Table IV). For IL-4, Con A induced a
small but significantly higher number of mRNA-expressing cells in the
IFN-R-/- mice (11.6 ± 2.3/105 MNC)
compared with the wild-type mice (9.1 ± 1.0/105 MNC)
(p = 0.041). There were no differences for the
other cytokines under study between the two groups (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R become less
susceptible to EAMG. These mice express decreased systemic humoral
responses to AChR, reflected by lower IgG and subclass levels, as well
as decreased affinity of anti-AChR Abs in serum and lower numbers
of anti-AChR IgG Ab-secreting cells in lymphoid organs. In
addition, decreased AChR-reactive TNF- and IL-1ß mRNA-expressing
cells were found in lymphoid organs of these mice, which may also
contribute to the low anti-AChR production and susceptibility of
IFN-R-/- mice for EAMG. These findings indicate that
IFN- plays a critical role in the induction of EAMG.
The production of IFN- is mostly of benefit for the host in the
defense against microorganisms. However, many of the activities of
IFN- may throw the normal immune response "off balance" and
cause profound changes that may lead to autoimmunity (28). Abnormal T
cell responses to AChR and the four subunits of AChR are characteristic
for both EAMG and human MG (4, 29, 30). Elevated levels of circulating
cells secreting IFN- or expressing IFN- mRNA in response to AChR
could reflect escalated AChR-specific Th1 responses (6). After
tolerance induction in Lewis rats by oral or nasal administration of
AChR, the numbers of AChR-reactive IFN--secreting cells are reduced
in parallel with decreased muscle weakness (31). These observations may
implicate an active role of IFN- in the immunopathogenesis of EAMG.
It has also been shown that IFN- is required during the initiation
of certain organ-specific autoimmune diseases (32, 33). Although ISH
has the disadvantage that detected cytokine mRNA-expressing cell levels
may not necessarily parallel secretion levels, the procedure
nonetheless avoids many of the drawbacks inherent to other approaches
and, with its high sensitivity and specificity, represents a useful
approach to evaluate cytokine profiles. This is exemplified in the
present study, in which both the number of cells producing
AChR-reactive IFN- and the number of cells expressing IFN- mRNA
in MNC preparations were strongly elevated in the IFN-R-deficient
mice. This elevation may well reflect a failure of a negative
feedback loop due to a blockade of signal transduction pathways
normally activated via IFN-R.
It is clear that an Ab-mediated abnormality at the NMJ is the final
common pathway and a prominent feature of MG, even for seronegative MG
patients (2). Any of the Ab-mediated disturbances might be influenced
by various quantitative and qualitative characteristics of the
anti-AChR Abs produced, including their amount, binding affinity,
fine specificity for AChR epitopes, and isotype (34). IFN- functions
as a direct B cell-maturing cytokine, driving non-Ig-secreting normal B
cells to active Ig secretion and isotype switches to IgG2a and IgG3
(35, 36). After expressing IFN- within the NMJ by a transgenic
technique, EAMG-resistant BALB/c mice exhibited clinical weakness and
functional disruption of NMJ, accompanied by infiltration of MNC
(mainly macrophages) and autoantibody deposition at motor end plates,
implying that IFN- in the milieu of the muscle tissue induces
humoral autoimmunity without circulating anti-AChR Abs (37).
Furthermore, IFN- is an important cytokine to induce B cell memory
and affinity maturation, and these responses can be completely blocked
by anti-IFN- mAb but not by anti-IL-4 (38). It is reasonable
to suspect that blocking the signal of IFN- in receptor-deficient
mice for this cytokine will lead to a change in the quantity and
quality of Abs. This study also showed that the blockade of IFN-
function results in decreased Ag-specific Ab affinity and production,
as has been shown before (39). The subclasses of AChR-specific IgG2b
and IgG3 were reduced in IFN-R-/- mice, with the
prominent reduction of IgG2a. These results imply the effects of
IFN- on the B cell memory, isotype switch, and affinity maturation.
On the other hand, although anti-AChR Abs are involved in the
pathogenesis of MG and EAMG, the serum Ab levels often do not correlate
with clinical severity of disease (2). In the present study, levels of
anti-AChR Abs in IFN-R-/- mice were not always
significantly lower than in wild-type mice. However, the passive
transfer of sera from immunized IFN-R-/- mice induced
much less muscle weakness compared with passive transfer of sera
obtained from immunized wild-type mice containing the same
concentration of anti-AChR Abs, implying that other factors, such as
affinity of Abs present in the IFN-R-/- mice, may
contribute to the low incidence of EAMG in these mice.
Within the cytokine network, IFN- promotes the differentiation of
precursors into Th1 cells, which produce IFN-, IL-2, and lymphotoxin
(40). Other effects of IFN-, including induction of MHC Ag
expression on APCs (41), macrophage activation (42), and up-regulation
of other cytokines such as TNF- and IL-1 (43), are probably also
important in the induction of EAMG. It has also been noticed that the
combination of IFN- and TNF- is highly effective in inducing the
production of chemokines (44), which may play an important role in
attract phagocytes in the early phase of EAMG (45). The thymus with
hyperplasia in MG patients revealed increased production or mRNA
expression of IL-1ß (46, 47). MG patients and EAMG animals have
elevated numbers of AChR-reactive TNF- and lymphotoxin
mRNA-expressing MNC in peripheral blood and lymphoid organs (10, 48).
Severe MG is associated with augmented spontaneous production of
TNF- in cultures compared with mild disease (47). TNF- is known
to enhance B cell functions by up-regulating B cell proliferation and
differentiation (49). It can also be anticipated that TNF- acts in
the context of cytokines within the cytokine network and influences
immune responses by, e.g., stimulating IL-6 production. It is
reasonable to assume that blocking of the signal for IFN- as
achieved in the present study leads to decreased levels of
proinflammatory cytokines, might be involved in the low levels of
anti-AChR Abs, and might explain in part the low susceptibility to
EAMG in IFN-R-/- mice.
The involvement of Th2 cytokines in MG and EAMG is expected on the
basis of the strong B cell responses in these diseases. IL-4 is
necessary for Ig synthesis and class switches (50) and provides help
for the production of anti-AChR Abs in EAMG and MG (3). IL-10 has
similar effects on Ab production, and we have recently shown that
injection of rIL-10 into EAMG rats enhances the disease in parallel
with up-regulated B cell responses (9). In the present study, lack of
IFN-R does not substantially influence the expression of IL-4 or
IL-10 mRNA, nor does it modulate the expression of TGF-ß. These data
indicate that the stimulatory effect of IFN- on EAMG is not mediated
via modulation of the expression of these particular cytokines. These
Th2-type cytokines might be responsible for the clinical weakness,
despite occurring at decreased frequency and being less severe, and for
the anti-AChR Ab production in the IFN-R-/- mice.
Balasa et al. (51) have recently reported that
IFN--/- mice immunized with AChR + CFA did not
develop signs of muscle weakness. This resistance to EAMG was
associated with reduced levels of whole Abs, including the
AChR-specific IgG1, IgG2a, IgG2b, and IgG3 subclasses. Although the
authors show a similar effect and draw some of the same
conclusions as we do in the present study, disruption of
IFN- seems more potent than disruption of IFN-R-/-
in blocking the induction of EAMG. The mechanism behind this difference
is not clear. Cognate T cell-B cell interactions involving
AChR-specific T cells may require a higher level of costimulation to
achieve the threshold necessary for contact-dependent signals to cause
stimulation and differentiation of B cells and subsequent Ab production
(51). IFN- regulates the expression of costimulatory molecules and
is thus essential to mount the humoral responses to AChR (51). Our data
imply that IFN- plays an important role, but is not essential, to
establish B cell responses to AChR. In general,
IFN-R-/- mice show no interference with the normal
development of lymphocyte subsets and MHC Ag expression (14), and it
seems that IFN-R-/- mice had immune outcome similar to
that of IFN--/- mice (14, 52). Whether and how
IFN-R or IFN- itself exerts different influence(s) on a
particular autoantigen, e.g., AChR, is currently under investigation.
Taken together, our results indicate that IFN- is critically
involved in the systemic humoral responses in EAMG by up-regulating the
production and affinity of autoantibodies against AChR. Decreased
levels of proinflammatory cytokines may also contribute to the low
production of anti-AChR Abs and, therefore, to the low
susceptibility to EAMG in IFN-R-/- mice.
|
Acknowledgments |
|---|
R-/-
mice and wild-type mice, and Dr. A. M. Rostami and Dr.
S. M. Phillips for helpful discussion of the
experiments. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Guang-Xian Zhang, Division of Neurology, 464 Stemmler Hall, 36th and Hamilton Walk, University of Pennsylvania, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: EAMG, experimental autoimmune myasthenia gravis; AChR, acetylcholine receptor; M-AChR, mouse acetylcholine receptor; MG, myasthenia gravis; NMJ, neuromuscular junction; MBP, myelin basic protein; KSCN, potassium thiocyanate; MNC, mononuclear cell; PILN, popliteal and inguinal lymph node; ISH, in situ hybridization; p.i., postinjection.
Received for publication May 29, 1998. Accepted for publication December 17, 1998.
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