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*
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; and
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(Th1) and
IL-4 (Th2) cytokines in the initiation and progression of this disease
are not fully understood. Recently, we have demonstrated that IFN-
is necessary for the initiation of tAChR-induced EAMG in mice. However,
the role of IL-4 in the progression of clinical EAMG remained
undetermined. In this study we have addressed the contribution of IL-4
in the disease progression in IL-4-/- C57BL/6j mice whose
IL-4 gene has been disrupted. Following immunization with
Torpedo (t) AChR, the IL-4-/- mice readily
developed signs of muscle weakness and succumbed to clinical EAMG with
kinetics similar to the susceptibility of IL-4+/+ mice. The
tAChR-primed lymph node cells from IL-4-/- mice
vigorously proliferated to tAChR and to its dominant
146–162 sequence associated with disease pathogenesis.
However, these T cells secreted higher levels of IFN- and IL-2,
suggesting the development of a Th1 default pathway in these mice.
Nevertheless, the IL-4 mutation had no effect on the recruitment of
CD4+ Vß6+ T cells specific to the dominant
tAChR 146–162 sequence in vivo. Immune sera from
IL-4-/- mice showed a dramatic increase in mouse
AChR-specific IgG2a levels followed by a concomitant decrease in IgG1
levels, but these mice did not exhibit an accelerated disease. In
conclusion, we have demonstrated for the first time that IL-4 is not
required either for the generation of a pathogenic anti-AChR
humoral immune response or for progression of clinical EAMG in
mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Production of the pathogenic AAbs by B lymphocytes is dependent upon
CD4+ Th lymphocytes (9, 10) and effective T-B cell
interactions. The role of cytokines in the initiation and progression
of AChR-induced EAMG in mice is not fully understood. Because EAMG is
an Ab-mediated disease (11, 12, 13, 14, 15, 16), it has been suggested that Th2 (IL-4),
but not Th1 (IFN-), cytokines regulate the unfolding of MG. In
contrast, we have recently demonstrated that the Th1 cytokine IFN-
is necessary for 1) the initiation of AChR-induced MG and 2) mounting
an effective pathogenic anti-M-AChR response in vivo (17). However,
the role of the Th2 cytokine IL-4 in the disease pathogenesis
(progression) was not examined. More recently, it was further suggested
that IL-4 might be involved in the activation of autoreactive B cells
and the induction of MG (18). Therefore, in the current study we tested
the requirement for IL-4 in the progression of AChR-induced EAMG in
mice. For this purpose we used IL-4-/- mice, in which
IL-4 gene activity was disrupted, and IL-4+/+ mice, in
which the IL-4 gene was intact.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-4 wild-type (IL-4+/+) and mutant (IL-4-/-) mice were purchased (The Jackson Laboratory, Bar Harbor, ME). The IL-4-/- mice (19) were derived after 10 backcrosses to the C57BL/6j strain. As controls, we used C57BL/6j mice. Mice were 8 to 10 wk old when used in the experiments in compliance with institutional guidelines.
Culture medium
RPMI 10 consisted of RPMI 1640 supplemented with 10% heat-inactivated FBS, 20 mM HEPES, 3 x 10-5 M 2-ME, 2 x 10-3 M L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. RPMI 20 is similar to RPMI 10, except it contains 20% heat-inactivated FBS.
Antigens
The tAChR was purified from Triton X-100 extracts of electric
organ membranes from Torpedo californica by affinity
chromatography on a conjugate of -bungaro toxin coupled to agarose
(20). M-AChR was obtained from crude carcass extracts of C57BL/6 mice.
The tAChR 111–126 (DYTGKIMWTPPAIFKS), tAChR
146–162 (LGIWTYDGTKVSISPES), and tAChR
182–198 (RGWKHWVYYTCCPDTPY) sequences were synthesized
at >70% purity at the core facility of the Scripps Research Institute
(La Jolla, CA). OVA was purchased (Sigma, St. Louis, MO).
Lymphocyte proliferation assay
Lymphocyte proliferation assays were performed as described
previously (17). Age-matched IL-4+/+ and
IL-4-/- mice were immunized at the base of the tail with
20 µg of tAChR in 100 µl of CFA emulsion. After 7 days 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 x 105/well in 96-well, flat-bottom microtiter
plates (Becton Dickinson, Rutherford, NJ) with and without graded doses
of tAChR or its 17 mer 146–162 sequence. OVA (control
Ag) was used at 20 µg/ml in RPMI 10. Cultures were incubated for 3
days at 37°C in humidified 5% CO2-enriched air and were
pulsed with 1 µCi of [3H]TdR/well during the last
18 h of incubation. [3H]TdR uptake was measured in a
Beckman beta scintillation counter (Beckman, Palo Alto, CA). The
results were expressed as a stimulation index, i.e., mean cpm with
Ag/mean cpm without Ag.
Cytokine ELISA
Single cell suspensions of draining LNC from tAChR-primed mice
were cultured at 106/ml in RPMI 10 and 2.5 µg/ml tAChR in
24-well, flat-bottom 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- (PharMingen, San Diego, CA). Concentrations of
IFN- were determined using a standard curve based on known
quantities of mouse rIFN- (Genzyme, Cambridge, MA). The standard
curve was linear in the range of 10 to 1000 pg/ml. Concentrations of
IL-2 and IL-4 were determined by bioassay by measuring proliferation of
the NK-3 cell line, which responds to both cytokines. The assay is
rendered specific for IL-2 when IL-4 activity is blocked by the
addition of anti-IL-4 mAb (11B11) at 1 µg/ml and specific for
IL-4 when IL-2 activity is blocked by the addition of anti-IL-2R
mAb (7D4 and PC.61) at a 1/2000 dilution and JES6 at 1 µg/ml. NK-3
cells were diluted to 1 x 105 cells/well of a
96-well, flat-bottom culture plate, and proliferation was determined by
the addition of 1 µCi of [3H]TdR/well for the
last 18 h of a 2-day culture. For IL-2 and IL-4, the standard
curves were linear in the range of 5 to 1000 pg/ml.
Generation of tAChR-specific T cell hybridomas
To analyze the Vß profile of AChR-specific T cells more
precisely, we generated T cell hybridomas instead of analyzing the Vß
profile of T cells in AChR-boosted LNC (4 days in vitro) from
AChR-primed mice (7 days in vivo). T cell hybridomas were generated
following the protocol described previously (21). IL-4+/+
(n = 3) and IL-4-/- (n =
3) mice were immunized s.c. at the base of the tail with 20 µg of
tAChR in 100 µl of PBS/CFA (Mycobacterium tuberculosis
H37RA; Difco, Detroit, MI) emulsion. Seven days later, the draining
inguinal and para-aortic lymph nodes were aseptically collected and
pooled. Single cell suspensions of lymph nodes were prepared in RPMI 10
medium. Cells (4 x 106/ml) were cultured in the
presence of 10 µg/ml tAChR at 37°C in a 5% CO2/95%
air, humidified incubator for 4 days. Later, the viable lymphoblasts
were separated on Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) and
fused with BW5147-/ß- cells, using prewarmed 50%
(w/v) polyethylene glycol 1500 in 75 mM HEPES (Boehringer Mannheim,
Mannheim, Germany) at a 1:2 ratio of lymphocytes (15 x
106) to tumor cells (30 x 106). After the
fusion step, gradual addition of prewarmed serum-free RPMI 1640 and
suspension of the cells in 31.5 ml of prewarmed RPMI 20 (containing
20% FBS) were performed. Growth of the hybridoma was usually observed
5 days postfusion. The cultures were gradually transferred from
hypoxanthine-aminopterin-thymidine-containing (Sigma) to
hypoxanthine-aminopterin-containing (Sigma) medium and finally
to RPMI 10 before they were screened for specificity in IL-2 release
assays.
Specificity testing of T cell hybridomas and IL-2 release assays
The specificity of T cell hybridomas was screened following a
modified protocol described previously (21). T cell hybridomas (5
x 104) and irradiated (1500 rad) syngeneic splenocytes
(106) were cultured together with or without indicated Ags
in 200 µl of RPMI 10/well. The tAChR (5 µg/ml), tAChR
111–126 (10 µg/ml), tAChR 146–162 (10
µg/ml), and tAChR 182–193 (10 µg/ml) sequences were
used. Then, 100 µl of supernatant was harvested from each well,
transferred into new 96-well, flat-bottom plates, and kept frozen at
-20°C until use. Upon subsequent thawing, 104 CTLL were
added per well. Eighteen hours later, [3H]thymidine (1
µCi/well; ICN Radiochemicals, Irvine, CA) was added. The cells were
harvested 6 h later using a semiautomated cell harvester (Skatron,
Sterling, VA), and incorporated thymidine was counted in a liquid beta
scintillation counter. The results were expressed as the mean of
duplicate or triplicate wells. The SD between wells was <20% of their
mean values.
FACS analysis
The FACS analysis was performed as described previously with some modifications (21). Expression of CD4 and TCR Vß was assessed by direct labeling of T cell hybridomas. Briefly, 2 to 3 x 105 cells were washed once with PBS containing 0.02% sodium azide and 0.5% BSA (PAB) and were incubated with 2% normal mouse serum in PAB for 15 min to block potential Fc receptors. Without further washing, the cells were incubated with 1 µl of anti-CD4-phycoerythrin and 1 µl of anti-Vß-FITC mAb (PharMingen, San Diego, CA) in 100 µl of PAB for 30 min on ice. The cells were washed twice at 4°C and 1200 rpm for 5 min and fixed in 0.5 ml of 1% paraformaldehyde. FACS analysis was performed using a FACStar Plus analyzer (Becton Dickinson, Mountain View, CA).
ELISA for IgG isotype determination
Anti-M-AChR responses were measured as described previously (22). The 96-well, flat-bottom polystyrene plates (Corning Glass Works) 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 1/2000 for IgG1 and IgG2b, 1/100 for IgG2a and IgG3) were added and incubated at 37°C for 90 min. After four washes, horseradish peroxidase-conjugated goat anti-mouse IgG isotypes (1/2000; Caltag, San Francisco, CA) were added and incubated at 37°C for 90 min. After washing the plates, 0.3 mg/ml 2.2'-azino-di-3-ethyl-benzthiazolinsulfonate (Boehringer Mannheim) substrate solution was added and allowed to develop color at room temperature in the dark. Serially diluted anti-AChR and normal mouse serum were used as positive and negative controls, respectively. Plates were read at OD410 nm, and results were expressed as OD values.
Statistical analysis
Statistical analyses were performed using Student’s t test, two-way analysis of variance, and the log-rank test, using StatView software (Abacus Concepts, Berkeley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the role of IL-4 in the development of tAChR-induced
EAMG of susceptible C57BL/6j (H-2b) mice, we immunized
IL-4+/+ (n = 13) and IL-4-/-
(n = 13) mice with 20 µg of AChR in CFA on day 0 and
again on day 30 (first boost) and day 75 (second boost; third
immunization). After the first boost (second immunization), the mice
were monitored daily for clinical symptoms (muscle weakness) of EAMG.
The final results appear in Table I
. At
40 days after the first immunization, 7 of 13 (54%)
IL-4+/+ mice and 6 of 13 (46%) IL-4-/- mice
developed muscle weakness. At 45 days after first immunization, 9 of 13
(69%) IL-4+/+ mice and 10 of 13 (77%)
IL-4-/- mice developed muscle weakness. The incidence of
disease did not differ between the two groups of mice
(p > 0.05). The kinetics of the disease
progression were similar between the groups (not shown). These results
directly demonstrate that IL-4 does not affect the genesis of EAMG and
is totally dispensable in tAChR-induced EAMG.
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CD4+ T cells reactive to tAChR and its 17 mer
immunodominant 146–162 sequence are pivotal in
facilitating B cells to generate pathogenic anti-AChR Abs (23, 24, 25, 26).
The role of Th2 cytokine IL-4 in the induction of T cell responses to
tAChR is not known. Therefore, to directly examine the immune effects
of IL-4 on T cell responses to tAChR and its 146–162
peptide, we employed IL-4+/+ and IL-4-/-
C57BL/6 mice in the experiments. We immunized IL-4+/+
(n = 4) and IL-4-/- (n =
4) mice with 20 µg of AChR in CFA. Seven days later, proliferation of
the draining inguinal and para-aortic LNC from individual mice was
assayed. As illustrated in Figure 1A, the tAChR-primed LNC from
IL-4+/+ as well as from IL-4-/- mice
proliferated equally well in a dose-dependent manner against the tAChR.
There was no difference in the proliferative responses between groups
to graded doses of tAChR tested (p =
0.5366). Similarly, a significant proliferative response to the
immunodominant tAChR 146–162 sequence was observed with
tAChR-primed LNC from IL-4+/+ and IL-4-/-
mice (Fig. 1B). There was no difference in the proliferative
responses between groups to graded doses of the tAChR
146–162 sequence tested (p =
0.4539). These findings indicate that disruption of the IL-4 gene does
not affect tAChR-specific T cell responses in vivo.
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cytokines by Th1
lymphocytes) upon challenge of mice with parasitic infections (27, 28).
Therefore, we examined whether the IL-4 gene disruption would similarly
result in a Th1 default pathway in tAChR-primed mice. To test this
possibility, we estimated the Th1 and Th2 cytokine profiles in
tAChR-primed and boosted LNC culture supernatants. The results appear
in Figure 2, A–C.
The LNC from IL-4-/- mice (n = 2),
following 7 to 8 days of tAChR immunization, secreted higher levels of
IFN- (77.9 ng/ml; p < 0.05) and IL-2 (2.9 ng/ml;
p < 0.05) and no detectable IL-4. In contrast, LNC
from IL-4+/+ mice (n = 2) secreted lower
levels of IFN- (32.3 pg/ml) and IL-2 (1.2 ng/ml) and no detectable
IL-4. However, these LNC from AChR-immunized IL-4+/+ mice,
upon further in vitro stimulation with plate-bound anti-CD3 mAb for
24 h, produced significant levels of IL-4 (73 pg/ml). No
detectable IL-4 was detected in the LNC culture supernatants of
IL-4-/- mice. These results demonstrate and confirm that
the IL-4 gene disruption results in the development of a Th1 default
pathway in vivo.
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146–162 sequence
The tAChR priming of C57BL/6 mice elicits T cells that
predominantly recognize tAChR 146–162 sequence
(29, 30, 31). CD4+ Vß6+ T cells recognizing the
146–162 sequence are preferentially expanded in
tAChR-primed mice (32, 33). In this study we examined whether IL-4 gene
disruption would affect the recruitment of specific CD4+
Vß6+ T cells in tAChR-primed IL-4-/- mice
by immunizing IL-4-/- and IL-4+/+ mice with
tAChR. The draining LNC from these tAChR-primed mice were boosted in
vitro with tAChR for 3 to 4 days, and the viable lymphoblasts were
immortalized by fusing them with BW5147
-/ß- cells. The resulting hybridomas
were randomly expanded and tested for specificity in IL-2 release
assays and for TCR Vß expression by flow cytometry. The results of
the FACS analysis and IL-2 release assays are summarized in Figure 3. A representative FACS profile was
depicted in Figure 4.
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-/ß- cells, 474 (62%) of the 768
seeded wells showed hybridoma growth; of these, 37 hybridomas were
tested for specificity against the three tAChR peptides (see
Materials and Methods). Of these 37 hybridomas, 24
hybridomas were found to be specific. Twenty-two of these 24 hybridomas
(92%) responded to AChR 146–162 sequence. This finding
further confirms the immunodominance of this sequence in tAChR (29, 30). Similarly, after fusion of tAChR-primed LNC from
IL-4-/- mice with BW5147
-/ß- cells, 520 (68%) of the 768 seeded
wells showed hybridoma growth, and of these, eight hybridomas were
expanded and tested for specificity. Of these, four hybridomas were
specific, and all (100%) responded to tAChR 146–162
sequence. The hybridomas that responded to the immunodominant tAChR
146–162 sequence were phenotyped by flow cytometry for
TCR Vß expression (Fig. 3
). As expected, in IL-4+/+ mice,
six of the seven (86%) AChR 146–162 sequence-specific
CD4+ T cells expressed Vß6+ TCR. Note that
TCR Vß expression for four hybridomas was shown in Figure 3.
Interestingly, three of the four (75%) AChR 146–162
sequence-specific CD4+ T cell hybridomas in
IL-4-/- mice also expressed Vß6+ TCR. These
results indicate that disruption of the IL-4 gene had no effect on the
recruitment of CD4+ Vß6+ T cells specific for
the tAChR 146–162 sequence. IL-4 gene disruption does not affect the serum anti-AChR Ab response but affects the anti-AChR IgG1 response
The primary pathology of EAMG, the end-plate AChR loss, stems from
the deleterious effects of pathogenic AAbs to the AChR (13, 34).
Therefore, to learn whether susceptibility of IL-4-/-
mice to EAMG correlates with effective AAb response, we used RIA to
compare the levels of serum anti-M-AChR Ab to those in
IL-4+/+ mice on day 14 of second and third AChR
immunizations. The results appear in Figure 5. The values were expressed as
bungarotoxin binding sites (nM) precipitated per liter of serum.
The humoral immune response in individual mice varied dramatically.
However, the results indicate that the susceptibility of mutant (-/-)
mice to EAMG correlated with efficient production of anti-AChR Abs.
These IgG titers in IL-4-/- mice did not differ from
those observed in the sera of IL-4+/+ mice
(p = 0.293 for day 14 sera of second AChR
immunization; p = 0.796 for day 14 sera of third dose
AChR immunization). There was also no statistical significance in the
IgG titers between the time points within each group (for
IL-4-/- mice, p = 0.242; for
IL-4+/+ mice, p = 0.589).
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and IL-2)
cytokines by AChR-specific T cells in vitro (Fig. 2, A–C)
in IL-4-/- mice. Because IL-4 has been associated with
the IgG1 response (35), we examined AChR-immunized mice for levels of
IgG1 and IgG2a with an IgG isotype-specific ELISA. The results are
depicted in Figure 6. Comparison of
immune sera from IL-4-/- mice with that from
IL-4+/+ mice revealed a dramatic reduction in the IgG1
response (p = 0.001 for day 14 sera of second
dose AChR immunization; p = 0.002 for day 14 sera of
third dose (second boost) AChR immunization) followed by a concomitant
increase in the IgG2a response (p = 0.003 for
day 14 sera of second dose AChR immunization; p = 0.003
for day 14 sera of third dose AChR immunization). There was no
significant difference in the IgG2b response (p
= 0.251 for day 14 sera of second dose AChR immunization;
p = 0.121 for day 14 sera of third dose AChR
immunization) and the IgG3 response (p = 0.592
for day 14 sera of second dose AChR immunization; p =
0.211 for day 14 sera of third dose AChR immunization) in sera from
both groups of mice. There was no statistical significance in the
difference between IgG2b levels in the serum samples from
tAChR-immunized IL-4-/- and IL-4+/+ mice
(p = 0.251 for day 14 sera of second dose AChR
immunization; p = 0.121 for day 14 sera of third dose
AChR immunization; data not shown). Therefore, AChR priming of
IL-4-/- mice apparently failed to elicit significant
amounts of IgG1 Abs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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146–162 sequence was
unaffected in tAChR-primed IL-4-/- C57BL/6 mice.
We have shown previously that IFN--driven Th1 responses are
necessary for the genesis of tAChR-induced EAMG (17). The current
results show that IL-4-driven Th2 responses are not required for the
progression of EAMG. These results further affirm an immunoregulatory
role for IFN- in EAMG. Additionally, 
-IFN- transgenic mice
expressing the IFN- transgene in the neuromuscular junction
exhibited an autoimmune humoral response to an unidentified 87-kDa
protein and an MG-like syndrome (36). Furthermore, tAChR-immunized B
cell-deficient C57BL/6 mice failed to develop EAMG, and their
AChR-specific T cells produced lowered levels of IL-2 and IFN- (37).
Therefore, IFN-, not IL-4, plays a dominant role in Ab-dependent (B
cell dominant) EAMG via the breakdown of peripheral tolerance toward
self AChR. Evidence for the role of IFN- in humoral autoimmunity has
recently been emerging in other animal models of B cell-dominant
autoimmune diseases such as systemic lupus erythematosis (38, 39, 40, 41) and
mercury-induced autoimmunity (42). Paradoxically, several animal models
of cell-mediated autoimmunity, such as diabetes (43), encephalomyelitis
(44), uveitis (45), arthritis (46), and thyroiditis (47), are found to
be IFN- independent, since these diseases readily develop in
IFN-- or IFN- receptor-deficient mice. Thus, humorally mediated
autoimmune diseases have been found to be IFN- dependent regardless
of whether cytokines and IgG isotypes in diseased animals are of Th1,
Th2, or both types.
Elsewhere, it was shown that disruption of the IL-4 gene did not influence the Th1 cytokine-dependent MBP-induced EAE in H-2u (48) and thyroglobulin-induced granulomatous thyroiditis in H-2k (49) haplotype mice. Additionally, IL-4-/- C57BL/6 (H-2b) mice did not develop MBP-induced EAE (50), suggesting that IL-4 is not necessary for conferring resistance to EAE. We have shown in this study that IL-4 gene disruption did not influence AChR-induced EAMG. Taken together, these results demonstrate that IL-4 gene disruption in mice of susceptible background does not influence the cell- or Ab-mediated autoimmune diseases.
Examination of the IL-4 gene disruption in mice found to be susceptible to a variety of infectious agents provided interesting findings. IL-4-/- BALB/c mice were susceptible (51) and resistant (28) to Leishmania major infection. The reasons for these disparate outcomes are currently unknown. Interestingly, the susceptibility of BALB/c mice to infection with L. major was shown to be dependent upon the loss of the ability to generate an IL-12-induced Th1 response rather than from an IL-4-induced Th2 response (52). On the other hand, IL-4-/- mice were resistant to experimental onchocercal keratitis, indicating that IL-4 confers on mice susceptibility to this corneal disease (53).
In agreement with the role of IL-4 in IgG isotype switching (35) and
previous studies showing that IL-4-/- C57BL/6 mice upon
parasitic infections produced lowered levels of IgG1 and increased
levels of IgG2a Abs (54, 55, 56), we also observed an enhancement in IgG2a
titers followed by a concomitant decrement in IgG1 titers in
tAChR-immunized IL-4-/- mice (Th1 predominance). This was
in agreement with our current findings that AChR-specific T cells from
IL-4-/- mice secreted higher levels of IFN- and IL-2
(Th1 cytokines). As a result, we expected an increase in the incidence
and severity of disease in IL-4-/- mice relative to those
in IL-4+/+ mice. Apparently, both these parameters were
similar in IL-4-/- and IL-4+/+ mice.
Furthermore, these IL-4-/- mice did not exhibit an
accelerated form of the disease.
Additionally, we did not find a correlation between IgG2a titers and
the presence and/or the severity of disease (data not shown). These
findings suggest that 1) a threshold level of IFN- is sufficient to
activate the immune system to respond to self Ags and elicit antihost
immunity, leading to the onset of clinical disease, and beyond that
concentration the enhanced levels of IFN- appear to be innocuous;
and 2) IFN- is the orchestrator in facilitating appropriate T-B cell
interactions leading to the generation of pathogenic anti-M-AChR
Abs. In support of the role of IFN- in EAMG,
IFN--/- mice failed to develop clinical EAMG (17), and
a decrease in IFN- levels correlated with suppression of EAMG in
tAChR -peptide-tolerized mice (25, 57) and tAChR-tolerized rats
(58). Moreover, susceptibility and resistance of rat strains to
tAChR-induced EAMG correlated with the number of IFN--producing T
cells (59). Although IL-4 is dispensable in the disease process, the
disease in wild-type mice may be a consequence of both IgG1 and IgG2a
anti-AChR AAbs, while only IgG2a Abs are at play in IL-4-deficient
mice. Adoptive induction of EAMG through serum transfer experiments
using purified IgG2a (from IL-4-deficient mice) will resolve this
issue.
It can be argued that the induction of EAMG in IL-4-/-
mice may be due in part to a redundancy in genes conferring Th2
function. We cannot rule out this possibility, but it is probably
unlikely, since earlier studies have established that
IL-4-/- mice have little or no Th2 cell activity, as
evidenced by the lack of expression of other Th2 cytokines in T cells
and very low IgG1 serum levels (19, 27, 60). Furthermore, in vitro
studies have provided compelling evidence that IL-4 is essential for
the generation of Th2 cells (61, 62). We did not find a difference in
the levels of other Th2 (IL-5 and IL-10) cytokines in the culture
supernatants of tAChR-boosted (in vitro) LNC of tAChR-primed
IL-4-/- and IL-4-/- mice (data not shown).
Nevertheless, our findings of an ability to induce disease in
IL-4-/- mice (current study) and a failure to induce
disease in IFN--/- mice (17) strongly argue against a
Th2 (IL-4) cytokine dependence in EAMG.
Our present and previous (17) studies have highlighted the importance
of IFN- in the pathogenesis of EAMG in mice. However, the
application of these findings to the rat EAMG model remains to be seen.
This consideration stems from the suggestion, addressed by kinetic
analysis of cytokine mRNA synthesis by mononuclear cells during the
course of EAMG in rats, that both IFN-- and IL-4-producing cells
might be involved in the genesis of this autoimmune syndrome (63, 64).
Additionally, mercury-induced autoimmunity in mice is IFN- dependent
(42), whereas in rats it is IL-4 dependent (65, 66). Therefore,
species-specific factors may influence the Ab-dependent autoimmune
diseases.
Immunotherapeutic strategies aimed at reducing the number of
IFN--secreting AChR-specific T cells would be beneficial at
subjugating the antihost pathogenic immunity. Therefore, identification
of the immunodominant peptides within the self AChR that are closely
associated with disease pathogenesis is extremely necessary. In that
direction, several groups have mapped the T cell epitopes on xenogenic
(foreign) tAChR (29, 30, 31). From these studies it was found that the
tAChR 146–162 sequence, bearing 71% amino acid
sequence similarity with its homologue on M-AChR, is immunodominant in
C57BL/6 mice. However, the tAChR 146–162
sequence is not pathogenic in mice upon direct immunization, but its
specific CD4+ T cells participate in the pathogenesis of
EAMG by providing T cell help to M-AChR-specific B cells, leading to
the generation of a pathogenic anti-M-AChR Ab response in vivo
(23, 24, 25, 26). Synthetic peptides of AChR with myasthenogenic potential (67)
or with the potential to provide excellent T cell help to
M-AChR-specific B cells (26) have been used to prevent MG in
experimental animals by decreasing the number of IFN--secreting
AChR-specific T cells in vivo (25). Additionally, tAChR-primed T cells
from EAMG-resistant, B cell-deficient C57BL/6 mice secreted lower
levels of IFN- (37).
Until now, the concept of the Th1/Th2 paradigm has provided a useful
and simple model for defining the roles of Th subsets (68). However,
delineation of the role of specific cytokines may be critical for
understanding particular immune responses in relation to autoimmunity.
The utility of this approach has been shown in several animal models of
autoimmune diseases, such as myasthenia (17, 36), lupus (39), diabetes
(43, 69, 70), and mercury-induced autoimmunity (42). This approach is
more likely to be relevant than simply categorizing immune responses
into classical Th1 and Th2 types. In conclusion, our results directly
demonstrated that the Th2 cytokine IL-4 is not necessary for the
progression of EAMG and reaffirmed that IFN- shapes the outcome of
the EAMG pathogenesis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nora Sarvetnick, Department of Immunology, Mail Code IMM23, The Scripps Research Institute, 10555 North Torrey Pines Rd., La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: MG, myasthenia gravis; AAb, autoantibodies; AChR, acetylcholine receptor; tAChR, Torpedo AChR; EAMG, experimental autoimmune myasthenia gravis; M-AChR, mouse AChR; LNC, lymph node cells.
Received for publication April 8, 1998. Accepted for publication May 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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