By
From * The Department of Immunology, The Scripps Research Institute, La Jolla, California 92037; The Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston,
Texas 77555-1019; § Trudeau Institute, Saranac Lake, New York 12983
Experimental autoimmune myasthenia gravis (EAMG) is an animal model of human myasthenia gravis (MG). In mice, EAMG is induced by immunization with Torpedo californica acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the role of cytokines in the pathogenesis of EAMG is not clear. Because EAMG is an antibody-mediated
disease, it is of the prevailing notion that Th2 but not Th1 cytokines play a role in the pathogenesis of this disease. To test the hypothesis that the Th1 cytokine, interferon (IFN)-, plays a
role in the development of EAMG, we immunized IFN-
knockout (IFN-gko) (
/
) mice
and wild-type (WT) (+/+) mice of H-2b haplotype with AChR in CFA. We observed that
AChR-primed lymph node cells from IFN-gko mice proliferated normally to AChR and to its
dominant pathogenic
146-162 sequence when compared with these cells from the WT mice.
However, the IFN-gko mice had no signs of muscle weakness and remained resistant to clinical
EAMG at a time when the WT mice exhibited severe muscle weakness and some died. The
resistance of IFN-gko mice was associated with greatly reduced levels of circulating anti-AChR antibody levels compared with those in the WT mice. Comparatively, immune sera from IFN-gko mice showed a dramatic reduction in mouse AChR-specific IgG1 and IgG2a
antibodies. However, keyhole limpet hemocyanin (KLH)-priming of IFN-gko mice readily
elicited both T cell and antibody responses, suggesting that IFN-
regulates the humoral immune response distinctly to self (AChR) versus foreign (KLH) antigens. We conclude that
IFN-
is required for the generation of a pathogenic anti-AChR humoral immune response
and for conferring susceptibility of mice to clinical EAMG.
Myasthenia gravis (MG)1 is a T cell-dependent antibody-mediated disease whose hallmark is an autoimmune
neuromuscular disorder (1). The cause is a loss of functional
acetylcholine receptors (AChR) at the postsynaptic membrane,
mediated by autoantibodies (AAbs) and complement (2). Experimental autoimmune MG (EAMG) is a well-established animal model for exploring the pathogenesis of this disease in
humans. In EAMG, the autoimmune destruction of AChR
produces a defect in neuromuscular transmission causing the
characteristic muscle weakness and fatigue of the disease. EAMG can be induced in mice of the H-2b haplotype by
repeated immunizations with AChR emulsified in CFA (3).
The role of cytokines in the immunopathogenesis of
AChR-induced EAMG is not clear. Because EAMG is an
antibody-mediated disease, it has been thought Th2 cytokines play a major role in the pathogenesis of this disease.
The prevailing notion in autoimmunity is that Th1 cytokines (IFN- Mice.
IFN-gko mice of the H-2b haplotype (5) were provided by Dr. D. Dalton (Trudeau Institute, NY). Heterozygous
IFN-gko (+/ Isolation of Genomic DNA and PCR.
IFN-gko ( Antigens.
AChR was purified from Triton X-100 extracts of
electric organ membranes from Torpedo californica by affinity chromatography on a conjugate of Lymphocyte Proliferation Assay.
Mice were immunized at the
base of the tail with 20 µg of AChR or 100 µg of KLH in 100 µl
of CFA emulsion. After 5 d of immunization, the mice were
killed, and their draining para aortic and inguinal lymph node
cells (LNC) were cultured in 0.2 ml of RPMI-10 at 4 × 105/well
in 96-well, flat-bottomed microtiter plates (Becton Dickinson, Franklin Lakes, NJ) with and without AChR (10 µg/ml) or
Cytokine ELISA.
Single cell suspensions of draining LNC of
AChR-primed mice were cultured at 106/ml in RPMI-10 and
2.5 µg/ml AChR in 24-well, flat-bottomed plates (Corning Glass
Works, Corning, NY) at 37°C in 5% CO2 and 95% humidity. The supernatants were collected after 48 h of in vitro boosting. An ELISA kit was used for detection of IFN- Induction and Assessment of EAMG.
Groups of mice (n = 13 to 15) were immunized subcutaneously in both hind footpads and
at two shoulder regions with 20 µg of Torpedo californica AChR in
CFA (Mycobacterium tuberculosis, H37Ra) (Difco Laboratories, Detroit, MI). Each site received ~5 µg of AChR in 50 µl of emulsion. Mice were boosted on days 30 and 75 with 20 µg of AChR
in CFA at selected sites in the shoulders and also in thigh regions
(four sites). In a blind study, these mice were assessed daily for the
characteristic symptom of EAMG: muscle weakness graded from
0 to 3 as described earlier (3). Muscle weakness attributable to MG
was further confirmed by administering intraperitoneally the anticholinesterase, neostigmine bromide, combined with atrophine sulfate, and assessing for temporary improvement in muscle strength (4).
Radioimmunoassay for Anti-mouse-AChR (M-AChR) Abs.
Serum anti-M-AChR Ab levels were determined by using an established protocol (7). M-AChR (1 × 10 ELISA for IgG Isotype Determination.
Anti-M-AChR responses
were measured as described earlier (9). The 96-well flat-bottomed
polysterene plates (Corning Glass Works, Corning, NY) coated
with M-AChR (0.5 µg/ml) in 0.1 M carbonate-bicarbonate buffer (pH 9.6) were incubated overnight at 4°C. The wells were blocked with 2% BSA in PBS at room temperature for 30 min.
Serum samples (diluted 1:4,000 for IgG1, and IgG2b; 1:200 for
IgG2a) were added and incubated at 37°C for 90 min. After four
washes, horseradish peroxidase (HRPO)-conjugated goat anti-
mouse IgG isotypes (1:2,000) (CALTAG Labs., San Francisco,
CA) were added and incubated at 37°C for 90 min. After washing
the plates, 0.3 mg/ml 2.2 Measurement of the Ab Response to KLH.
Mice (n = 6) were
primed with 100 µg KLH in CFA on day 0 and boosted on days
30 and 75 as in the AChR immunization protocol. In brief, we
coated the 96-well flat-bottomed plates (Corning Glass Works,
Corning, NY) with 5 µg/ml KLH in PBS overnight at 4°C.
Later, the wells were blocked for 2 h at 37°C with PBS containing 1% BSA, 10% heat-inactivated fetal bovine serum, and 0.05%
Tween-20. Immune sera (diluted 1:800,000 for IgG1; 1:6,400 for
IgG2a) were added and incubated for 2 h at room temperature.
For IgG isotype measurement, HRPO-labeled Ab to murine IgG
isotypes was used at 1:2,000 dilution in plates incubated for 2 h at
room temperature. After three washes, color was developed with
the substrate, o-phenylene-diamine dihydrochloride (Sigma Chemical Co.), and OD read at 492 nm. Serially diluted anti-KLH sera
and NMS were used as positive and negative controls, respectively. The results were expressed as OD values.
To test the hypothesis that IFN- Table 1.
Ablation of Endogenous IFN-) are associated with cell-mediated rather than
antibody-mediated diseases. However, in previous studies from our laboratory, the ectopic expression of proinflammatory Th1 cytokine IFN-
in the neuromuscular junction elicited a humoral IgG response to an unidentified antigen within the motor end plate, yielding a MG-like
syndrome in mice (4). Therefore, we tested here the requirement of IFN-
in the development of AChR-induced EAMG in mice. For this purpose, we used IFN-
knock-out (IFN-gko) mice in which IFN-
gene activity was disrupted and wild-type (WT) mice whose IFN-
gene was
intact.
) (129/SvEv × C57BL/6)F1 mice were intercrossed in our animal facility to generate homozygous (
/
) gko
(129/SvEv × C57BL/6) F2 mice. WT (129/SvJ × C57BL/6)F2
mice (+/+) were used as positive control mice and were purchased from The Jackson Laboratory (Bar Harbor, ME). In addition, C57BL/6 mice were used as additional controls (The Jackson Laboratory). Both 129/SvJ and 129/SvEV are derived from
the same parental strain. The difference between the two substrains is that the SvEv was crossed once with C3H, then the F1
were backcrossed again 14 times back to Sv parental strain but
SvEv substrain is very much (99.99%) similar to Svj (6). Mice
were 8- to 10-wk-old when used in the experiments in compliance with institutional guidelines.
/
) mice were
screened by PCR of tail DNA. An aliquot of the genomic DNA
was amplified in a PCR using primers binding to the neo gene
(Neo490V, 5
-CGGTTCTTTTTGTCAAGAC-3
; NB3, 5
-ATCCTCGCCGTCGGGC ATGC-3
) (391-bp product) and exon 2 and 3 of the IFN gene (A1, 5
-AAGTGGCATAGATGTGGAAG-3
; B1, 5
-GAATGCATCCTTTTTCGCCT-3
) (223-bp product). PCR conditions were as follows: one step at 94°C for 1 min, 30 s; then 33 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The final step was at 72°C for 5 min.
-bungarotoxin coupled to agarose (7).
AChR-
146-162 peptide LGIWTYDGTKVSISPES (8) was synthesized at >70% purity. KLH (Cal Biochem, San Diego, CA), OVA
(Sigma Chemical Co., St. Louis, MO), purified protein derivative
(PPD; Statens Seruminstitute, Copenhagen, Denmark), and Con A
(Pharmacia Biotech, Piscataway, NJ) were purchased as designated.
146-162 peptide (20 µg/ml). KLH and OVA were used at 50 µg/ml and 20 µg/ml, respectively. RPMI-10 consisted of RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum,
20 mM Hepes, 3 × 10
5 M 2-ME, 2 × 10
3 M L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cultures were incubated for 4 d at 37°C in humidified 5% CO2-enriched air and
were pulsed with 1 µCi [3H]TdR/well during the last 18 h of incubation. [3H]TdR uptake was measured in a Beckman
scintillation counter. The results were expressed as a stimulation index,
i.e., (mean cpm with antigen) / (mean cpm without antigen).
(PharMingen, San Diego, CA). Concentrations of IFN-
were determined using a
standard curve based on known quantities of mouse recombinant
IFN-
(Genzyme, Cambridge, MA).
9) was incubated at
4°C in Triton buffer with [125I]
-bungarotoxin (2 × 10
9 M) for
4 h. To 1 ml of labeled M-AChR, 1 µl sera from experimental mice was added. Normal mouse serum (NMS) was used as a control. After overnight reaction at 4°C, rabbit anti-mouse Ig (100 µl) was added. After 4-h incubation at room temperature, the tubes were centrifuged, and the pellet was washed with 1 ml Triton
buffer, centrifuged again, and counted in a
counter. The difference in AChR counts precipitated in the experimental versus the
control samples was used to calculate the Ab response in nanomoles of toxin binding sites per liter of serum.
-Azino-di 3-athyl benzthiazolinsulfonat
(Boehringer Mannheim GmbH, Mannheim, Germany) substrate
solution was added and allowed to develop color at room temperature in the dark. Serially diluted anti-AChR and NMS were
used as positive and negative controls, respectively. Plates were
read at OD410 nm and results were expressed as OD values.
The IFN- Gene Disruption Prevents the AChR-induced
Clinical EAMG.
plays a
role in the development of AChR-induced EAMG of susceptible H-2b mice, we immunized C57BL/6 (+/+) (n = 15), WT (+/+) (n = 13), and IFN-gko (
/
) (n = 14)
mice with 20 µg of AChR in CFA on day 0 and again on
days 30 (first boost) and 75 (second boost). After the first
boost, the mice were monitored daily for clinical symptoms (muscle weakness) of EAMG. The final results appear in
Table 1. At 39 d after the first immunization, 7 of 15 C57BL/6 (+/+) mice and 8 of 13 WT (+/+) mice developed muscle weakness, but none of the 14 IFN-gko
(
/
) mice exhibited such weakness. At 42 d after first
immunization, one C57BL/6 (+/+) mouse died from severe clinical EAMG; then 11 and 14 d later, two more
died. Although none of the WT (+/+) mice died as a result of severe disease, two were killed for humanitarian reasons because of their severe muscle weakness 11 d after the
second boost. However, the IFN-gko (
/
) mice remained resistant to clinical EAMG (P values: IFN-gko versus WT = <0.001; P value: IFN-gko versus C57BL/6 = <0.001). These results indicate that IFN-
influences the
genesis of EAMG.
Prevents the Development of AChR-induced EAMG in Mice
In vivo
No. of mice
per group
Muscle weakness (grade)
No. of mice
died due to
severe disease
0
1
2
3
Disease incidence
IFN-gko (
/
)
14
14
0/15 (0%)
Wild type (+/+)
13
1
7
3
2
2
12/13 (92.3%)
C57BL/6 (+/+)
15
2
6
4
1
2
13/15 (86.7%)
Clinical manisfestation of EAMG was graded as follows: grade 0 indicated no muscle weakness even after exercise consisting of 20-30 consecutive
paw grips on cage top steel grids. Grade 1 was defined as normal at rest, but weak with chin on the floor and inability to raise head after exercise.
Grade 2 was defined as grade 1 weakness at rest. Grade 3 was moribund, dehydrated, or paralyzed. Clinical EAMG was further confirmed by assessing for temporary improvement after administering a combination neostigmine bromide and atrophine sulfate (4).
CD4+ T cells reactive to AChR and
its dominant 146-162 peptide are pivotal in the ability of
B cells to generate pathogenic anti-AChR Abs (3, 10).
To address whether the resistance of IFN-gko (
/
) mice
results from their inability to evoke T cell responses to
AChR and its
146-162 peptide, we immunized WT (+/+)
and IFN-gko (
/
) mice with 20 µg of AChR in CFA. 5 d later, proliferation of their draining LNC was assayed. As
illustrated in Fig. 1 A, the AChR-primed LNC from
C57BL/6 and WT (+/+) mice proliferated strongly in
response to AChR, as expected. LNC from IFN-gko
(
/
) mice also proliferated significantly against the AChR.
Significant proliferative responses (SI = 25.5 ± 1.8) to
146-162 peptide of AChR were also observed with
AChR-primed LNC from IFN-gko mice (data not shown).
A similar magnitude of proliferative responses were observed
with LNC from IFN-gko and control group mice after 14 d
of AChR priming (data not shown). Interestingly, when
the mice were immunized with KLH, the intensity of the
proliferative response of draining LNC from IFN-gko (
/
)
and WT (+/+) mice appear similar (Fig. 1 B). Further examination of IFN-
levels in the culture supernatants collected 48 h after an in vitro boost with AChR revealed the
presence of IFN-
in cultured LNC of the C57BL/6 and
WT (+/+) mice but not of the IFN-gko (
/
) mice
(data not shown).
The IFN-
The primary pathology of EAMG,
the end plate AChR loss, stems from the deleterious effect
of pathogenic AAbs to the AChR (2). Therefore, to learn
whether a reduced anti-AChR Ab response underlies the
resistance of IFN-gko (/
) mice to EAMG, we used
RIA to compare their levels of serum anti-M-AChR Ab to
those in C57BL/6 (+/+), and WT (+/+) mice on day 89 after immunization with AChR in CFA. The results appear
in Fig. 2. The results indicate that the resistance of the mutants (
/
) to EAMG correlated with their dramatic decrease in the level of AAbs to AChR compared with significantly higher levels in the C57BL/6 (+/+) and WT (+/+)
mice. The individual AChR-immunized C57BL/6 (+/+)
and WT (+/+) mice responded heterogeneously. A wide
heterogeneity in anti-AChR response has earlier been documented in inbred C57BL/6 mice (13). However, we did
not find a correlation between the anti-AChR antibody response magnitude of individual mice (C57BL/6 and WT)
and severity of EAMG. Our findings further support the
earlier findings that no correlation exists between the magnitude of the anti-AChR antibody response and severity of
EAMG (8, 13).
The IFN-
Because IFN- has been associated with the IgG2a response (16), we examined
AChR-immunized mice for levels of IgG2a and IgG1 with
an IgG isotype-specific ELISA. Comparison of immune sera from IFN-gko (
/
) mice and WT (+/+) mice revealed dramatic reductions in both IgG1 and IgG2a Ab
levels in the IFN-gko (
/
) mice (Fig. 3, A-B). Similarly,
the IgG2b and IgG3 Ab levels of the latters were significantly reduced (data not shown). Therefore, AChR priming of IFN-gko (
/
) mice apparently failed to elicit significant amounts of Ab in any IgG subclass.
Finally, to assess whether the observed IgG subclass distribution was unique to AChR stimulation, we used KLH
as the immunogen. Sera from mice immunized with KLH
on the same schedule used for AChR priming were subsequently analyzed for IgG isotypes. As the results in Fig.
3, C-D, show, immune sera from IFN--containing WT
(+/+) mice, when compared with such sera from IFN-gko (
/
) mice, had decreased levels of IgG2a and increased levels of IgG1, confirming the association between
IFN-
and the IgG2a response (17).
Our report documents that IFN- is essential for the development of EAMG in mice. Herein, we demonstrate that
the lack of endogenous IFN-
dramatically affected the humoral autoimmune response to AChR and prevented clinical disease in H-2b mice. However, T cell responses to
AChR were found relatively normal. Unexpectedly, our
findings also demonstrate a dichotomy of the requirement
for IFN-
in the induction of humoral immune response
to self (AChR) and foreign (KLH) antigens.
The EAMG-resistant IFN-gko mice failed to mount significant M-AChR-specific Ab responses (Fig. 2). The importance of this lapse may be that their AChR-specific Ab levels were too low (a) to provoke antigenic modulation at the motor end plate (18, 19); (b) to competitively block AChR function (20); and/or (c) to destroy postsynaptic membranes through complement fixation (21, 22), thereby preventing immune destruction.
The IFN-gko (/
) and the control WT (+/+) mice
used in our study were derived from intercrossing of (129/
SvEv × C57BL/6)F1 mice and (129/SvJ × C57BL/6)F1
mice, respectively. Therefore, the genetic origin (129/Sv
substrain) of the embryonic stem cells used provides a
source of genetic heterogeneity in mice, although the SvEv
and SvJ substrains are very (99.99%) much similar (see Materials and Methods). However, this genetic heterogeneity
may have little influence, if any, on the dramatic effect
that we were measuring. This interpretation stems from
three pieces of observations: (a) the complete resistance
(100%) of IFN-gko mice to clinical EMAG and a higher
incidence (92.3%) of disease in WT mice (Table 1); (b) a
dramatic reduction in anti-M-AChR response in all of the
IFN-gko mice (Fig. 2); and (c) a similar heterogeneous humoral immune response among individual WT (+/+) and
C57BL/6 mice (Fig. 2). Thus, the simplest and most direct
interpretation of our findings is that the phenotype (resistance to EAMG) reflects an effect of IFN-
. Therefore, the
resistance of IFN-gko mice to EAMG is attributed to the
absence of IFN-
.
The dichotomy observed in the humoral immune response to AChR versus KLH-immunized IFN-gko (/
)
mice is unexpected. Because a significant AChR-specific T
cell proliferative response is observed in WT (+/+) and
IFN-gko (
/
) mice, the loss of M-AChR-specific Ab
responses in IFN-gko (
/
) mice probably results from
defective T cell-B cell cognate interactions. What role could
IFN-
play in that process? It is known that T cells responding to self-antigen(s) are generally of low affinity
compared with those responding to foreign antigens (23,
24). Additionally, the expression of AChR in the thymus
(25) may enhance negative selection of higher affinity autoreactive T cells, resulting in a particularly low affinity
AChR T cell repertoire. During cognate T cell-B cell interactions, these AChR-specific low affinity T cells may require a higher level of costimulation to achieve the threshold for contact-dependent signals to cause stimulation and
differentiation of B cells and subsequent Ab production.
Therefore, we speculate that mounting an effective humoral immune response to this (AChR) self-antigen is
IFN-
dependent, because the cytokine regulates the expression of costimulatory molecules (26, 27). On the other
hand, the KLH-specific high affinity T cells do not require
IFN-
-dependent costimulation by B cells to effectively participate in humoral response. The hypothetical model is
shown in Fig. 4. The data reported in our manuscript are
suggestive of differential requirements for IFN-
in the induction of humoral immune response to self (AChR) and
foreign (KLH) antigens; however, the study of other self-antigens would substantiate this notion. The findings of humoral immune responses observed in KLH-immunized
IFN-gko (
/
) mice are further supported by previous
findings in which IFN-gko (
/
) mice produce antibodies to a wide variety of pathogens; influenza (17), Leishmania major (28), and Herpes simplex virus (29).
In support of our hypothesis, it has long been known
that cognate T cell-B cell interactions involve several reciprocal receptor-counterreceptor interactions. One of such
interactions is CD28-CTLA-4/B7 signaling. A role for
CD28/B7 signaling in humoral immune response has been documented; human CTLA-4Ig therapy (30) and anti-B7-2
mAb treatment (31) of mice profoundly blocked antibody
responses to a nominal antigen priming. This notion is further supported by the report that transgenic expression of
soluble murine CTLA4-H 1 in mice profoundly blocked
antibody response to a protein antigen without affecting
the T cell responses (32). It is likely that IFN-
by virtue of
its ability to upregulate the basal levels of B7-2 on B cells
(33) may influence the CD28/B7 costimulation during cognate T cell-B cell interactions, which in turn affect the
humoral immune response for a self-antigen (M-AChR).
The fact that anti-self-humoral responses require additional cytokine inducible signals seems evolutionarily appropriate. In the case of the AChR, even extremely low levels of autoantibodies are highly detrimental to the functioning of the neuromuscular junction. Our findings suggest that under nonstimulatory circumstances the process of cognate help by low affinity anti-AChR T cells is not favored.
Our results demonstrate that the lack of IFN- converts
an otherwise EAMG-susceptible mouse strain to a disease-resistant one; that is, in H-2b mice, IFN-
confers susceptibility to EAMG (Table 1). The freedom from EAMG in
IFN-gko mice was somewhat surprising for two reasons: first, EAMG is an Ab-mediated disease (36), and second, the proinflammatory Th1 cytokine IFN-
is strongly
associated with cell-mediated autoimmune diseases (39-
41). However, elsewhere it was shown that IFN-
is not
required for the cell-mediated autoimmune diseases, autoimmune encephalomyelitis and diabetes (42, 43), or confers resistance to them (44). Thus, it appears that IFN-
plays a more essential role in antibody-mediated than in
cell-mediated autoimmune diseases.
We have shown in this study that the endogenous absence of IFN- in the periphery prevented the development of EAMG in mice to AChR challenge. Work from
our laboratory has shown earlier that the localized expression of IFN-
transgene in the neuromuscular junction
elicited a humoral autoimmune response to an unidentified 87-kD antigen and provoked a MG-like syndrome in mice
of the H-2d haplotype (4). These findings, as demonstrated
by diverse approaches in mice of disparate haplotypes, implicate IFN-
as a pivotal player in the pathogenesis of humoral autoimmunity in the neuromuscular junction.
In conclusion, the results of this study reveal a differential requirement for IFN- (a) in humoral immune response to self (AChR) versus foreign (KLH) antigens, and
(b) the cytokine plays an important immunomodulatory
role in the pathogenesis of EAMG. Therefore, IFN-
antagonists may prove beneficial in the treatment of Ab-mediated autoimmune diseases such as MG.
Address correspondence to Dr. Nora Sarvetnick, Ph.D., Department of Immunology, The Scripss Research Institute, 10555 North Torrey Pines Road, La Jolla, CA 92037. Phone: 619-784-9066; FAX: 619-784-9383; E-mail: noras{at}scripps.edu
Received for publication 23 April 1997 and in revised form 4 June 1997.
B. Balasa (DVM., Ph.D) has been supported successively by postdoctoral fellowships from the Myasthenia Gravis Foundation of America, Inc., and the Juvenile Diabetes Foundation International. C. Deng is a Myasthenia Gravis Foundation Osserman postdoctoral fellow. This work was supported by the Diabetes Interdisciplinary Research Program grant from Juvenile Diabetes Foundation International (N. Sarvetnick), and the Muscular Dystrophy Association (P. Christadoss). This is manuscript No. 10815-IMM.We thank P. Minnick for editorial corrections on this manuscript.
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