HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ostlie, N. S. Articles by Conti-Fine, B. M. Search for Related Content PubMed PubMed Citation Articles by Ostlie, N. S. Articles by Conti-Fine, B. M.

Transgenic Expression of IL-10 in T Cells Facilitates Development of Experimental Myasthenia Gravis¹

Norma S. Ostlie^*, Peter I. Karachunski^*, Wei Wang^*, Cristina Monfardini^*, Mitchell Kronenberg and Bianca M. Conti-Fine^2,3,*,

^* Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, MN 55108; Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; and Department of Pharmacology, School of Medicine, Universitiy of Minnesota, Minneapolis, MN 55455

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ab to the acetylcholine receptor (AChR) cause experimental myasthenia gravis (EMG). Th1 cytokines facilitate EMG, whereas Th2 cytokines might be protective. IL-10 inhibits Th1 responses but facilitates B cell proliferation and Ig production. We examined the role of IL-10 in EMG by using wild-type (WT) C57BL/6 mice and transgenic (TG) C57BL/6 mice that express IL-10 under control of the IL-2 promoter. We immunized the mice with doses of AChR that cause EMG in WT mice or with low doses ineffective at causing EMG in WT mice. After low-dose AChR immunization, WT mice did not develop EMG and had very little anti-AChR serum Ab, which were mainly IgG1, whereas TG mice developed EMG and had higher levels of anti-AChR serum Ab, which were mainly IgG2, in addition to IgG1. At the higher doses, TG mice developed EMG earlier and more frequently than WT mice and had more serum anti-AChR Ab. Both strains had similar relative serum concentrations of anti-AChR IgG subclasses and IgG and complement at the muscle synapses. CD8⁺-depleted splenocytes from all AChR-immunized mice proliferated in the presence of AChR and recognized a similar epitope repertoire. CD8⁺-depleted splenocytes from AChR-immunized TG mice stimulated in vitro with AChR secreted significantly more IL-10, but less of the prototypic Th1 cytokine IFN-, than those from WT mice. They secreted comparable amounts of IL-4 and slightly but not significantly reduced amounts of IL-2. This suggests that TG mice had reduced activation of anti-Torpedo AChR Th1 cells, but increased anti-AChR Ab synthesis, that likely resulted from IL-10-mediated stimulation of anti-AChR B cells. Thus, EMG development is not strictly dependent on Th1 cell activity.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD4⁺ T cells comprise Th1 and Th2 cells, which differ in their function and the cytokines they secrete (1, 2, 3). Th1 cells have been implicated in the pathogenesis of both T cell-mediated and Ab-mediated autoimmune diseases because they secrete proinflammatory cytokines, can be cytotoxic, and help synthesis of complement-binding IgG subclasses that should be effective in causing tissue damage (1, 2, 3). Th2 cells are not cytotoxic and might protect from some autoimmune responses, because they down-regulate the activity of both Th1 cells and APC by secreting antiinflammatory cytokines such as IL-4, IL-5, and IL-10 (1, 2, 3). However, Th2 cytokines have a variety of effects on the immune functions that may result in contrasting consequences on normal and autoimmune responses.

Ab against the muscle acetylcholine receptor (MAChR)⁴ cause myasthenia gravis (MG) and its animal model, experimental MG (EMG) (4). Th1 cells and/or Th1 cytokines are involved in the pathogenesis of MG (5, 6, 7) and EMG (8, 9, 10, 11, 12), whereas Th2 or TGF--secreting cells (sometimes referred to as Th3 cells) may have a protective role (13, 14, 15, 16, 17, 18, 19, 20).

IL-10 is an important effector cytokine of Th2 cells and other cell types, including Ly-1 B cells, keratinocytes, activated macrophages, and mast cells. IL-10 has a broad range of activities that include anti-inflammatory activity, costimulation of thymocytes, stimulation of mast cell proliferation, and stimulation of B cells differentiation (1, 2, 21).

To understand the role of IL-10 in EMG, we have examined whether transgenic (TG) expression of IL-10 influences susceptibility to EMG. We have used wild-type (WT) C57BL/6 (B6) mice, and IL-10-TG mutants of B6 background in which the T cell-specific human IL-2 promoter drives the expression of mouse IL-10 transgene (22). The T cells that synthesize TG IL-10 include activated, IL-2-secreting Th1 cells, which produce IL-10 only transiently in response to T cell activation (22). Thus, activated Th1 cells in these mice may down-regulate their own activity, including secretion of TG IL-10. Because of the transient expression of the transgene, the immune system of these mice is not significantly different from the control littermates: serum IgG levels and numbers and phenotype of T and B cells are normal (22). Moreover, although IL-10 is a potent down regulator of Th1 cells (1, 2, 3), IFN- synthesis is reduced but not eliminated in these TG mice (22). We have immunized IL-10 TG and WT mice with acetylcholine receptor (AChR) and examined the appearance of EMG and the characteristics of their anti-AChR Ab and T cell responses.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

WT breeders were obtained from The Jackson Laboratory (Bar Harbor, ME). TG breeders had been obtained from eggs of (B6 x C3H)F₁ females mated to B6 males. Their establishment and characterization were described previously (22). Because the genetic background of mice influences their susceptibility to EMG (4) and the mice used to establish the TG mice were of mixed background, littermates with or without the IL-10 transgene could not be used to investigate the effects of TG expression of IL-10 on susceptibility to EMG. The susceptibility to EMG of B6 mice is well characterized (4). To obtain TG mice suitable for these studies, the IL-10 TG founders were back-crossed with B6 mice six times. Therefore, the genetic background of the resulting TG mice we used was almost entirely B6, and we could compare their susceptibility to EMG with that of WT mice.

Previous studies have characterized the properties of the immune system and immune responses of the TG mice we used (22, 23). They have normal T and B lymphocyte development. Their Th2 activity, as judged by the secretion of IL-4, is similar to the control littermates (22). They secrete more IL-10 than the control littermates but less IFN-, indicating that TG IL-10 secretion by the Th1 cells results in a self-limitation of the activity of Th1 cells (22). We bred both strains at the animal facility of the University of Minnesota.

Purification of Torpedo AChR (TAChR)

We purified TAChR from electric tissue of Torpedo californica as alkali-stripped TAChR-rich membrane fragments (24). The AChR structure is very conserved along evolution: TAChR is highly homologous to mammalian MAChR and suitable to induce EMG (4). We measured the protein concentration by the Lowry assay (25) and the TAChR concentration as -bungarotoxin (BTX)-binding sites (24). The TAChR preparations we used contained 3.8–5.8 nmol of sites/mg protein. SDS-PAGE analysis (24) showed that the TAChR preparations contained only the four TAChR subunits as the main protein bands. For 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 (24), diluted them to 0.5 mg/ml in PBS, and stored them at -80°C.

Peptides

We determined the epitope repertoire on the AChR subunit, which dominates the sensitization of anti-TAChR CD4⁺ cells in B6 mice (24, 26), by using a panel of synthetic peptides 20 residue long and overlapping by 5 residues that spanned the sequence of the TAChR subunit. We reported their characterization previously (24). We used solutions of the individual peptides in PBS sterilized by UV irradiation and stored frozen.

Immunization

We immunized 8- to 10-wk-old female mice by s.c. injections along the back and at the base of the tail of solubilized TAChR (2 or 30 µg in 100 µl of PBS) emulsified with 100 µl of CFA. We boosted them twice at 4-wk intervals with the same amount of TAChR in incomplete CFA, and a third time 5–7 days before sacrifice. We have shown previously that this immunization procedure, when using 20–40 µg of AChR, induces EMG (e.g., Refs. 12, 15, 17, 18, 24, 26, 27). We used female mice because after they reach maturity and until 6 mo of age, their weight is constant, both in the WT and the TG strain, whereas the weight of males increases with age, thus making it impossible to measure their strength with the holding test we used, described below.

Evaluation of clinical symptoms of EMG

EMG symptoms in mice may not be obvious, and are difficult to quantify by inspection (4). We quantified the EMG weakness with a forced exercise, sensitized by a small amount of pancuronium bromide (0.03 mg/kg i.p.) just before the test (27). The mice hang from a grid: we measured the time it took them to release the hold and fall three times (holding time). To verify the myasthenic nature of the weakness, we injected i.p. edrophonium chloride (Reversol; Organon, West Orange, NJ). Reversol is a cholinesterase inhibitor and it immediately increased the strength of mice that have EMG. We described this test in detail previously (12, 15, 17, 18, 27). The test is parametric and gives a quantitative and reliable assessment of the severity of the muscle weakness, as verified by the finding that repeated tests of the same mouse yield comparable holding times (e.g., Ref. 12 ; also see left panels of Fig. 1, A and B). The average holding time of 285 naive WT mice was 11.4 ± 1.55 min (12). Naive TG mice had holding times indistinguishable from WT mice (also, see Ref. 18). We considered myasthenic the mice with holding times of 8.3 min (holding time of normal mice - 2 SD) or less. Among EMG mice, we differentiated the severity of the myasthenic weakness as follows: mild EMG, holding times between 8.3 and 6.75 min (holding time of normal mice - 3 SD); moderate EMG, holding times between 6.75 and 5.2 min (holding time of normal mice - 4 SD); severe EMG, holding times of 5.2 min or less. Mice that were paralyzed or that died of EMG are represented in the figures as having holding time of zero.

View larger version (63K): [in this window] [in a new window]   FIGURE 1. TG mice are more susceptible to EMG than WT mice. Muscle strength and appearance of EMG in WT and TG mice, as indicated inside the panels, after immunization with 2 µg of TAChR (A) or 30 µg of TAChR (B) at the times indicated with arrows (0, 4, and 8 wk). The muscle strength was measured by the pancuronium-sensitized hanging test at different times after beginning of the TAChR immunization, as indicated. The tests at 0, 4, and 8 wk were conducted just before the TAChR injections. In the left panels, we report the muscle strength of the individual WT and TG mice, expressed as holding times, at different times after beginning of the TAChR immunization. The shaded area indicates the holding times indicative of EMG (8.3 min or less). The dashed lines indicate holding times below which we considered the animals to have moderate or severe EMG (6.75 and 5.2 min, respectively). The horizontal bars indicate a statistically significant difference in the holding times of the TG group as compared with the WT group. The right panels report the percentage of mice with clinical EMG at the different times. The presence of mild, moderate, or severe EMG is indicated with increasingly dark shading. See Materials and Methods for experimental details.

Reduction of the MAChR content correlates with EMG severity (4). However, it can be measured only after sacrificing the mouse, whereas the holding test permits to follow the time course of the disease. In another study,⁵ we verified that a reduction in holding time correlated with reduced MAChR content of the muscle, measured at the time of the clinical observation: for mice with EMG there was a good statistical correlation between holding time and MAChR content.

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 ¹²⁵I-BTX (23, 25). We express the Ab concentration as µM precipitated ¹²⁵I-BTX binding sites. We measured the concentration of Ab that cross-reacted with mouse MAChR by a modification of the anti-TAChR Ab assay, with Triton X-100 extract of muscle from naive B6 mice as the source of solubilized MAChR, at a concentration of 1 nM/ml of BTX binding sites. We labeled the MAChR by overnight incubation with 4 nM ¹²⁵I-BTX. We set up precipitation curves by using increasing amounts of serum (0.2–5 µl) in 96-well Immulon-4 Removawell plates (Dynex Technologies, Chantilly, VA), precoated by overnight incubation at 4°C with PBS containing 0.01% sodium azide, 0.05% Tween 20, and 3% BSA (all from Sigma, St. Louis, MO). We used 0.2 pmol of MAChR per sample, and we usually set up triplicate or quadruplicate samples for each of the serum amounts. After overnight incubation, we precipitated the MAChR-Ab complexes by incubation with 20 µl of Zysorbin (Zymed Laboratories)/well for 2 h. All incubations were at 4°C. We washed the plates four times with wash buffer (24) and counted the individual wells in a 5500 gamma counter (Beckman, Fullerton, CA). We express the Ab concentration as nanomolars precipitated ¹²⁵I-BTX binding sites.

We used the two-factor ANOVA test with the MacANOVA program to determine the statistical significance between the serum concentration over time of anti-TAChR and anti-MAChR Ab in TG mice and WT mice that had received the same TAChR immunization treatment. We used the following model: response = treatment + time + E(mouse) + treatment x time, where response was the Ab concentration, treatment was the presence (or absence) of the IL-10 transgene, time was the time of the individual test (measured as weeks after the beginning of the immunization), and E(mouse) was the degrees of freedom (a function of the number of the mice in the groups analyzed). We considered a difference to be significant when p 0.05.

Assay of serum anti-TAChR IgG subclasses

We measured by ELISA the anti-TAChR IgG subclasses in sera obtained 4 wk after the second and third TAChR immunizations. 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 sera from normal, untreated mice as negative control. We washed the plates six times with PBS-T, added 100 µl/well of 1 µg/ml biotinylated anti-mouse IgG subclass Ab (BD 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 the IgG2a gene, but rather a related isotype, termed IgG2ab or IgG2c (28, 29). To detect anti-TAChR IgG2c in the mouse sera we used the mAb biotin anti-mouse IgG2ab (Igh-1b; BD PharMingen). After eight washings with PBS-T, we added 100 µl/well of 1 µg/ml avidin-peroxidase (Sigma) 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 ABTS 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 samples incubated with normal, untreated mice from that of samples incubated with the sera from TAChR-immunized mice. The serum concentration of anti-TAChR IgG subclasses was inferred from standard curves obtained by coating ELISA plates directly with 100 µl/well of purified mouse IgG subclasses (Sigma; from 0.097 to 100 ng/ml in 10 mM NaHCO₃, pH 9.6) and detecting their presence with the biotinilated anti-mouse IgG subclass/avidin-peroxidase system, described above. We could not construct a standard curve by using purified IgG2c, which is not commercially available; for IgG2c we used the standard curve obtained for IgG2a, which is highly homologous to IgG2c and likely to yield similar readings.

The standard curves we used are not ideal: ideal curves would have required the use of purified anti-TAChR IgG subclasses to bind to the TAChR coating the plate. Thus, the values of serum concentrations of the IgG subclasses that we obtained are not absolute. However, because our goal was to assess the percentage of the different anti-TAChR IgG subclasses in TG mice as compared with WT mice, we considered our standard curves satisfactory references, as internal standards that allowed comparison of results for the individual subclasses of different experiments.

We determined the serum concentration of IgG subclasses in naive WT and TG mice by using an ELISA similar to that described above and coating the plate with dilutions of the sera to be tested.

Detection of IgG, IgG subclasses, and complement at muscle synapses by immunofluorescence microscopy

We used hind limb muscle samples of TAChR-immunized or naive WT and TG mice frozen in liquid nitrogen and stored at -80°C. We used tissue from three different mice for each group (WT or TG mice, immunized with 30 or 2 µg of TAChR). We embedded the frozen tissues in OCT Compound Tissue-TEK (Miles Laboratories, Elkhart, IN) and sectioned it in the transverse direction into 10-µm sections with a Jung Frigout 2800E Kryostat (Leica, Nublach, Germany). We analyzed at least 10 sections from each mouse.

To detect simultaneously the presence of mouse IgG and complement at the neuromuscular junction, we incubated the sections in PBS for 10 min, 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 with Texas Red-labeled BTX (Molecular Probes, Eugene, OR), FITC-labeled goat anti-mouse C3 Ab (Nordic Immunological Laboratories, Capistrano Beach, CA), and AMCA-S-labeled streptavidin (Molecular Probes) diluted, respectively, at 1:4000, 1:100, and 1:200 in PBS containing 3% BSA. For detection of different IgG subclasses, we incubated the sections for 1 h with 1:20 dilutions in PBS of mAbs specific for mouse IgG2b (Sigma; a rat mAb) or IgG2c (BD PharMingen; mAb anti-mouse IgG2ab (Igh-1b) a mouse IgG3 mAb). We washed the sections three times for 15 min with PBS followed by incubation with a biotin-conjugated secondary Ab, which was goat anti-rat IgG polyclonal Ab (Sigma) for IgG2b and a mAb specific for mouse IgG3 (BD PharMingen) for IgG2c. This was followed by staining with Texas Red-labeled BTX (Molecular Probes), 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 microscopy (Eclipse E800; Nikon, Tokyo, Japan). All procedures were at room temperature. We collected digital images with the program Image Pro Plus (Media Cybernetics, Silver Spring, MD).

Lymphocyte proliferation assay

Five to 7 days after the last TAChR immunization, we obtained splenocytes (24) from two to three identically treated mice. Because we administered the immunizations at multiple spots along the mouse back and conducted the proliferation experiments 5–7 days after the last boost, the spleen is a good source of TAChR-specific CD4⁺ cells (e.g., Refs. 12, 24, 26). For the experiments described here, the splenocytes are a better choice than lymph node cells because they can be obtained in large numbers, and this allows both the depletion in CD8⁺ cells (necessary to assess the cytokine secretion by CD4⁺ cells), and the recovery of enough cells to test a large number of Ags (necessary to determine the epitope repertoire on the TAChR subunit). We pooled the splenocytes and depleted them in CD8⁺ cells by using paramagnetic beads and rat anti-mouse CD8⁺ Ab (BD PharMingen). We suspended the CD8⁺-depleted splenocytes in RPMI 1640 (Life Technologies, Grand Island, NY) 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), and seeded them 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); 5 and 10 µg/ml TAChR; or 10 µg/ml of the individual subunit peptides. Controls were triplicate wells cultured 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 per well; sp. act. 6.7 Ci/mmol; DuPont, Boston, MA), harvested them (Titertek, Skatron, Sterling, VA), and measured the [³H]thymidine incorporation by liquid scintillation. We determined the significance of the difference in the average incorporation of [³H]thymidine in cultures exposed to a given Ag and their basal incorporation in the absence of any stimulus by using a two-tailed Students’ t test. We considered a difference to be significant when p < 0.05.

Cytokine secretion by CD8⁺-depleted spleen cells in response to stimulation with TAChR

Five to 7 days after the last TAChR boost, we prepared CD8⁺-depleted splenocytes from two to 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 duplicate cultures. CD8⁺-depleted splenocytes cultivated without any Ag served as controls for spontaneous secretion of cytokines. We harvested the culture supernatants after 24 and 72 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 (BD PharMingen), and recombinant IFN-, IL-2, IL-4, and IL-10 (BD PharMingen) as standards, and followed the manufacturer’s instructions. We determined the significance of the difference in the average cytokine secretion of cultures exposed to the TAChR and their average basal secretion in the absence of any stimulus by using a two-tailed Students’ t test. We considered a difference to be significant when p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TG mice are very susceptible to EMG

In a first experiment, we immunized 10 WT and 5 TG mice three times with 2 µg of TAChR and used the hanging test to measure their strength every 2–4 wk for 14 wk, starting just before the first TAChR injection (time 0) (Fig. 1A). The shaded area in Fig. 1A (left) represents the holding times (8.3 min or less) that are indicative of EMG. None of the WT mice developed EMG weakness. Some TG mice had reduced holding time at week 4, and most or all of them after week 10: at this time the difference in the average holding time of TG and WT mice was significant (bar in left panel of Fig. 1A). The dashed lines in the left panels of Fig. 1A represent the holding time below which we consider the mice to have moderate and severe EMG (6.75 and 5.2 min, respectively). In the right panels of Fig. 1A, we represent the results of this experiment as the percentage of mice with clinical EMG at the different times. We have indicated the presence of mild, moderate, and severe disease with increasingly dark shaded areas.

In two other experiments, we immunized TG and WT mice three times with 30 µg of TAChR, a dose that induces EMG in WT B6 mice (4). In the first of those experiments, we used 5 TG 5 five WT mice, and in the second we used 10 TG and 6 WT mice. We used the hanging test to measure their strength every 2–4 wk for 14 wk, starting just before the first TAChR injection (time 0). The two experiments yielded consistent results, which are reported together in Fig. 1B. Similar to Fig. 1A, we report in the left panels the holding times of the individual mice observed in the different tests. They illustrate well the consistency and reliability of the holding test we used. In the right panels, we represent the results of the same experiments as the percentage of mice with clinical EMG at the different times. Among the mice with EMG, we have differentiated, as in Fig. 1A, the presence of mild, moderate, or severe disease. TG mice developed more frequent and more severe EMG than WT mice. About one-third of the WT mice had EMG, usually mild or moderate, during the first 10 wk. At week 12, all WT mice had EMG, which was mild in many of them, but >50% of them had recovered by the end of the observation period. Of the TG mice, 75% had EMG during the first 9 wk, which was moderate or severe in 50% of the animals. By week 11, all TG mice had EMG, which was usually moderate or severe and persisted for the whole duration of the observation period. The difference in the holding times of TG and WT mice was significant for the whole duration of the observation period (p < 0.05; horizontal bar in the left panel of Fig. 1B).

Serum anti-AChR Ab concentrations in TG and WT mice

We measured by RIPA the anti-TAChR Ab concentration in sera of WT and TG mice immunized with low or high doses of TAChR.

Fig. 2A reports the serum anti-TAChR Ab concentrations of individual mice immunized with 2 µg of TAChR, and their averages. The sera were obtained 4, 8, and 14 wk after beginning the TAChR immunization. In agreement with previous reports (4), we found mouse-to-mouse variations of the serum anti-TAChR Ab. At each tested time, the average Ab concentration in TG mice was higher than in WT mice. The curves reflecting the time course of the appearance of serum anti-TAChR Ab in the two strains were significantly different (p < 0.001).

View larger version (33K): [in this window] [in a new window]   FIGURE 2. TG mice had more serum anti-TAChR Ab than WT mice. Anti-TAChR Ab concentration, measured by RIPA, in the sera of WT mice () and TG mice (•), immunized with injections of 2 µg of TAChR (A) or 30 µg of TAChR (B) at weeks 0, 4, and 8. We obtained the sera 4, 8, and 14 wk after beginning the TAChR immunization as indicated below the plots. In both A and B, the top plots report the Ab concentrations in the sera of individual mice and the bottom plots the average concentrations ± SD in the two groups. At all times TG mice had higher concentrations of anti-TAChR Ab than the WT mice, both after low-dose (p < 0.001) and high-dose (p < 0.0015) immunization. See Materials and Methods for experimental details.

We measured by RIPA the concentration of Ab that cross-reacted with MAChR in the sera of WT and TG mice immunized with low or high doses of TAChR. Assay of the anti-MAChR Ab, the concentration of which in EMG induced by TAChR immunization is much lower than that of the anti-TAChR Ab (4), required rather large amounts of serum (up to 30–40 µl/curve). For a few mice, we did not have enough serum, at least for some of the time points. As controls we used sera of WT mice sham-immunized with adjuvant alone in the same amounts and with the same injection schedule as the TAChR-immunizations.

We measured the serum anti-MAChR Ab concentration of individual WT and TG mice immunized with 2 µg of TAChR. The sera were obtained 4, 8, and 14 wk after the beginning of the immunization. The WT mice never had anti-MAChR Ab at 4 and 14 wk, and only one of them had a small amount of Ab (2.04 nM) at week 8. The IL-10 mice did not have measurable anti-MAChR Ab at weeks 4 and 14, but three of five of them had anti-MAChR Ab (12.13, 8.7, and 16.03 nM, respectively) at week 8.

We measured the anti-MAChR Ab in the sera of WT and TG mice immunized with 30 µg of TAChR in the two experiments described above. For both experiments we collected sera at 2–4 wk, at 8 wk, and at 14 wk. For the second experiments, we collected sera also at 6 and 11 wk. For a few mice, we did not have enough serum for the week 14 samples. Fig. 3 summarizes the results of both those experiments.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Serum anti-MAChR Ab in TG and WT mice. The mice were immunized with 30 µg of TAChR at weeks 0, 4, and 8. The symbols represent the anti-MAChR Ab concentration, measured by RIPA, in the sera of WT mice () and TG mice () at different times after beginning of the immunization, as indicated below the plots. Notice that at 6 wk and 11 wk we had sera for only some of the mice. The black symbols represent the average anti-MAChR Ab in the two groups at the different times. See Materials and Methods for experimental details.

Anti-TAChR IgG subclasses in WT and TG mice

We measured the percentage of anti-TAChR IgG subclasses in sera obtained 4, 8, and 14 wk after beginning the TAChR immunization in the mice immunized with low doses of TAChR and in the mice used in the first experiment in which we used high-dose TAChR immunization. In WT mice, the anti-TAChR Ab have been reported to be IgG2b and IgG1, with undetectable IgG3 (15, 17). The presence of IgG2c had not been investigated previously.

Fig. 4A reports the averages of the relative concentrations of anti-TAChR IgG1, IgG2b, and IgG2c in sera of individual mice immunized with the low TAChR dose. We do not show the values for the anti-AChR IgG3 because they were undetectable. WT mice synthesized primarily IgG1 at weeks 4 and 8 and comparable amounts of IgG1 and IgG2b by week 14. At all time points, the IgG2c were a minor component of the anti-TAChR Ab. In contrast, TG mice synthesized primarily anti-TAChR IgG2b at all time points tested. They also synthesized substantial amounts of anti-TAChR IgG2c (30% of the anti-TAChR Ab at weeks 8 and 14), whereas the synthesis of anti-TAChR IgG 1 was minimal (<10% at weeks 8 and 14). The relative serum concentration of anti-TAChR IgG1 was significantly lower in the TG mice than in the WT mice at all the time points. The relative concentrations of anti-TAChR IgG2b and IgG2c were significantly higher in the TG mouse sera at all the time points, with the only exception of IgG2c at week 4.

View larger version (41K): [in this window] [in a new window]   FIGURE 4. Percentage of anti-TAChR IgG subclasses in the sera of WT and IL-10 TG mice. The mice had received injections of 2 µg of TAChR (A) or 30 µg of TAChR (B) at weeks 0, 4, and 8. The sera were obtained 4, 8, and 14 wk after beginning the TAChR immunization, as indicated below the plots. The columns represent averages ± SD of the relative amounts of the different anti-TAChR IgG subclasses (relative to the total IgG concentration) obtained by ELISA in sera from individual mice (*, p < 0.05; **, p < 0.005; ***, p < 0.0005). , WT mice; , TG mice. See Materials and Methods for experimental details.

We measured amounts of serum IgG subclasses in naive WT and TG to see whether the two strains have a different profiles in IgG subclass production: no difference was detected (not shown).

TG and WT mice immunized with TAChR have IgG and complement at the neuromuscular junctions

We used immunofluorescence to determine the presence of mouse IgG and complement at the neuromuscular junctions of WT and TG mice immunized with either 2 or 30 µg of TAChR. We used three mice for each group. As negative controls for unspecific binding of the fluorescent probes to muscle, we used muscle sections from three naive WT mice. For each mouse we analyzed at least 10 muscle sections in which we could identify neuromuscular junctions by the binding of fluorescent BTX (red fluorescence). We identified the presence of mouse IgG, IgG2b, IgG2c, and the C3 complement component by binding of specific fluorescent Ab (blue and green fluorescence, respectively). Fig. 5 reports representative muscle sections from WT and TG mice immunized with 30 µg of TAChR. All mice of both strains had IgG and complement bound to the neuromuscular junctions, and they had both IgG2b and IgG2c. Also WT and TG mice immunized with 2 µg of TAChR had detectable IgG and complement at the neuromuscular junctions (data not shown). We could not detect IgG, IgG2b, IgG2c or complement at the neuromuscular junctions of naive mice (not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Mouse IgG and complement at the neuromuscular junction of WT and TG mice. Muscle sections from mice immunized three times with 30 µg of TAChR (at weeks 0, 4, and 8) and that had received a further boost 5–7 days before being sacrificed. A, Staining for mouse IgG (blue fluorescence) and the C3 component of complement (green fluorescence) as indicated below the panels. B, Staining for mouse IgG2b or IgG2c (blue fluorescence) as indicated below the panels. We localized the neuromuscular synapses by using immunofluorescence staining with BTX (red fluorescence). Magnification, x1000. See Materials and Methods for experimental details.

After TAChR immunization, CD4⁺ cells of mice with B6 background recognize primarily epitopes within the sequence region 146–169 of the TAChR subunit (24, 26, 30, 31, 32). CD4⁺ cells that recognize the immunodominant sequence 146–169 are strongly pathogenic, because lack of recognition of the sequence by CD4⁺ cells correlates with resistance to EMG induction (30, 31, 32). They recognize also epitopes within residues 181–200 and 360–378 of the TAChR subunit (24) and other minor epitopes (26, 31). We wished to examine whether the increased susceptibility to EMG of the TG mice correlated with enhanced recognition of the TAChR by their CD4⁺ cells, or with recognition of a different CD4⁺ epitope repertoire. We used CD8⁺-depleted splenocytes from WT and TG mice immunized with 30 or 2 µg of TAChR, to determine their proliferative response to the TAChR and to overlapping synthetic peptides spanning the TAChR subunit sequence.

We conducted one experiment with WT mice and two experiments with TG mice immunized with 30 µg of TAChR that yielded consistent results (Fig. 6). Both strains recognized TAChR vigorously. The CD8⁺-depleted splenocytes from TG mice recognized the TAChR less strongly than those from the WT mice. Given the limited number of experiments, we cannot decide whether this was attributable to a reduced Th1 response (22) or to the described variations in the proliferative response of individual mice to the TAChR (15, 17, 18, 24, 26, 27). CD8⁺-depleted splenocytes from WT and TG mice immunized with 30 µg of TAChR recognized a similar repertoire of subunit peptides. They always recognized most strongly the peptides spanning the immunodominant sequence region 146–169. Other peptides were recognized less consistently and/or less intensely. WT mice recognized also peptides 30–47 and 360–378. TG mice also recognized, at least in one experiment, peptides 1–20, 30–47, 63–80, and 276–295.

View larger version (37K): [in this window] [in a new window]   FIGURE 6. Proliferative responses to the TAChR and to TAChR subunit sequences of CD8+-depleted splenocytes from WT and TG mice. Responses of CD8+-depleted splenocytes from WT and TG mice immunized with 30 µg of TAChR, as indicated inside the plots, to the TAChR and to individual overlapping synthetic peptides spanning the TAChR subunit sequence: the residues of the subunit sequence included in the individual peptides are indicated at the bottom of the panels. The mice had been immunized three times with TAChR, at weeks 0, 4, and 8, and had received a further boost 5–7 days before being sacrificed (at week 15). We conducted one experiment with splenocytes from WT mice, and two experiments with splenocytes from TG mice. The columns represent average cpm ± SD of triplicate cultures (*, p < 0.05; **, p < 0.005; ***, p < 0.0005). The baseline values obtained in the absence of any stimulus, or in the presence of a synthetic 20-residue peptide unrelated to the TAChR sequence, have been subtracted, and they were: 8602 ± 503 cpm for the WT (top) and 4511 ± 654 and 5519 ± 898 cpm for the TG mice (middle and bottom, respectively). See Materials and Methods for experimental details.

Thus, these experiments did not detect any substantial change in the pattern of the recognition of TAChR subunit epitopes in the TG mice.

Cytokine secretion by anti-TAChR CD4⁺ cells

We investigated the production of cytokines in response to TAChR stimulation in vitro by CD8⁺-depleted splenocytes of WT and TG mice immunized with 30 µg of TAChR. We cultured CD8⁺-depleted splenocytes with TAChR or without any stimulus and measured IFN-, IL-2, IL-4, and IL-10 in the culture supernatants by ELISA (Fig. 7). In agreement with the characteristics of the immune response of the TG mice (22), their CD8⁺-depleted splenocytes secreted more IL-10 and less IFN- than those from WT mice, whereas their synthesis of IL-2 was slightly, but not significantly, decreased as compared with the WT mice, and that of IL-4 was the same in both strains. Thus, TG IL-10 secretion led to a decrease of the prototypic Th1 cytokine, IFN-, but it neither caused an increase in Th2 activity nor it disrupted significantly the synthesis of IL-2 by Th1 cells.

View larger version (35K): [in this window] [in a new window]   FIGURE 7. Cytokine secretion by CD8+-depleted splenocytes from WT and IL-10 TG mice immunized with TAChR after challenge in vitro with TAChR. IFN- and IL-2 (top), IL-4 and IL-10 (bottom) in the supernatant of cultures of CD8+-depleted splenocytes from WT mice () and TG mice () immunized with TAChR. The mice had been immunized three times with 30 µg of 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 with supernatant of cells cultured in the presence of 10 µg/ml TAChR. The spontaneous secretion of the different cytokines by cells cultivated without any stimulus was subtracted from these data. The star indicates a significant (p < 0.02) difference in the secretion of cytokines by the cells from the TG mice, as compared with the secretion by cells from WT mice (n.s., not significant). See Materials and Methods for experimental details.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The TG expression of IL-10 did not have a significant effect on the serum concentration of anti-MAChR Ab, although after immunization with 30 µg of TAChR, anti-MAChR Ab appeared in the sera of TG mice earlier than in WT mice, and the few mice with high concentrations of serum anti-MAChR Ab were in the TG group (Fig. 3). Also, after immunization with low doses of TAChR, TG mice had serum anti-MAChR Ab more frequently than WT mice. The immune cross-reactivity between the TAChR and MAChR is small, and anti-MAChR Ab may accumulate in the serum only after the MAChR in the mouse muscles has been saturated. Mice have a large MAChR content, and Ab binding to muscle AChR accelerates its degradation and resynthesis (4), and therefore absorption of more anti-MAChR Ab. Thus, the absence or the presence of low amounts of anti-MAChR Ab in the serum may not correlate with absent or mild EMG symptoms. A lack of correlation between the concentration of anti-MAChR Ab and severity of EMG in mice has been described (33). Also in MG, there is poor or no correlation between the concentration of anti-AChR Ab in the serum and the symptom severity (4). Paradoxically, although Ab cross reactive with MAChR are the direct cause of EMG, measurement of the total concentration of the anti-TAChR Ab in our mice provided a better indicator of the effect of TG IL-10 expression on the anti-AChR Ab response.

Complement activation at the neuromuscular junction is a likely pathogenic mechanism in EMG. For example, IL-12-deficient mice do not develop EMG after TAChR immunization and complement cannot be detected at their endplates (12). However, despite the increased synthesis of anti-TAChR IgG subclasses that bind complement in TG mice immunized with low doses of TAChR, we could not detect an increased deposition of complement at the neuromuscular junctions of TG mice, as compared with WT mice: all mice immunized with TAChR had C3 at their endplates. The immunofluorescence method we used to detect complement is strictly qualitative and may not detect small yet clinically significant changes in the amount of complement at the neuromuscular junction.

IL-10 facilitates the differentiation and proliferation of B cells and production of Ig. It is a switch factor for a variety of Ig classes and subclasses (34, 35, 36, 37, 38) and possibly for all IgG isotypes (39). At the higher TAChR doses, the percentages of all anti-TAChR IgG subclasses were similar in WT and TG mice (Fig. 4B), indicating that TG mice had increased synthesis of all anti-TAChR IgG subclasses, including those induced by Th1 cytokines. This suggests that the increased synthesis of anti-TAChR Ab in TG mice was caused by the action of IL-10 on B cells, not by the reduced anti-TAChR Th1 response (Fig. 7). This is verified by the finding that immunization of the TG mice with suboptimal doses of TAChR resulted in preferential synthesis of Th1-induced IgG2b and IgG2c (Fig. 4A). This is likely explained by the focal synthesis of the TG IL-10 by Th1 cells simultaneous with IL-2 synthesis. In WT mice immunized with low TAChR doses, the small amounts of anti-TAChR Ab were primarily Th2-induced IgG1 after the first two TAChR injections, and even after the third injection (week 14) the IgG1 were almost half of the anti-TAChR IgG (Fig. 4A). This is consistent with a preferential activation of Th2 cells when their TCR are ligated at low density by the MHC class II/epitope complexes (3, 21).

The action of IL-10 on B cells may be synergistic with IL-2, with or without additional costimulatory signals (40, 41, 42, 43). The TG expression of IL-10 in our TG mice might have been especially effective in stimulating IgG synthesis by anti-TAChR B cells, because it occurred simultaneously with secretion of IL-2, and at the same location. The TG mice do not have overall increased levels of serum IgG or of individual IgG subclasses (Ref. 22 and this study), yet they had significantly higher concentrations of anti-TAChR Ab than WT mice (Fig. 2). This argues for a specific stimulatory effect of the TG IL-10 on the B cells that synthesize anti-TAChR Ab, resulting from the cognate interaction between TAChR-specific CD4⁺ Th and B cells.

The TG expression of IL-10 occurs simultaneously with that of IL-2, which might also contribute to EMG development and to the proliferation and differentiation of anti-TAChR B cells. However, IL-2 is unlikely to have had a role in the enhancement of EMG development in the TG mice, because the IL-2 synthesis induced by challenge with TAChR in cultures of CD8⁺-depleted splenocytes of TAChR-immunized mice was modestly, albeit not significantly, decreased in the TG mice as compared with the WT mice. This is consistent with the decreased synthesis of IFN- (Fig. 7), described previously in TG mice (22), which suggests that secretion of TG IL-10 by the Th1 cells down-regulates the activity of the Th1 cells that express the transgene. An important role of IL-10 in EMG development is supported by studies of IL-10 gene knockout mice, which appeared to be resistant to EMG induction (P. Christadoss, unpublished observations).

IL-10 down-regulates Th1 responses by reducing the expression of costimulatory molecules and cytokines by APC (44, 45, 46, 47) and by inhibiting the transcription of the IL-12 genes during the primary Ag stimulus (48). IL-10 also affects CD4⁺ T cells and resting T cells by inhibiting IL-2 production and T cell growth (49, 50). It induces a long-term Ag-specific anergic state in human CD4⁺ cells when present during Ag challenge (51, 52) and might be responsible for the increased T cell death mediated by Fas/Fas ligand that occurs in human systemic lupus (53). Also, the shift toward Th2 responses caused by TGF- may be attributable to inhibition of Th1 cells mediated by IL-10 (54). The decreased secretion of IFN- by CD8⁺-depleted splenocytes from our TG mice, after stimulation in vitro with TAChR (Fig. 7), suggests that they had a reduced Th1 response to the TAChR. This might have occurred because the activated Th1 cells down-regulated their own activity because of the TG IL-10 expression induced by the stimulation of IL-2 synthesis. Some Th1-mediated responses, like anti-tumor activity and colitis, were reduced in the TG mice we used (22).

IL-10, especially in combination with IL-2, facilitates CD8⁺ CTL responses (55, 56, 57, 58, 59). The role of CD8⁺ cells in EMG is not clear. Mice deficient in MHC class I molecules and CD8⁺ cells developed EMG with high frequency after immunization with AChR (60), suggesting that CD8⁺ cells do not have a pathogenic role in EMG. However, other studies suggested that CD8⁺ cells are necessary for EMG development. In Lewis rats, Ab-mediated depletion of CD8⁺ cells suppressed EMG development and reduced the synthesis of anti-AChR Ab (61). Also, mutant B6 mice that lacked CD8⁺ cells did not develop EMG and had reduced anti-AChR Ab responses (62). If CD8⁺ cells have a facilitating role in rodent EMG, a stimulation of CD8⁺ cells by the TG IL-10 may have a role in the increased susceptibility of the TG mice to EMG.

Nasal, s.c., or oral administration of TAChR sequences to B6 mice caused activation of specific Th2 cells and protected from EMG (e.g., 15, 16, 17, 18, 19, 20). How can these results be reconciled with those we report here? Activated Th2 cells secrete IL-4, which might help prevent EMG directly because of its antiinflammatory properties and its down-regulating action on Th1 cells (1, 2, 3). Also, IL-4 may be a growth factor for modulatory CD4⁺ cells that secrete TGF- (also referred to as Th3 cells, Refs. 62, 63, 64 ; however, see Ref. 65). The TGF- family of cytokines are potent immunomodulators (66) that polarize CD4⁺ responses toward a Th2 phenotype (66, 67) and block the effects of IL-12 in the development of Th1 responses (66, 68, 69). Because Th3 cells do not produce IL-4, they may be dependent on Th2 cells for proliferative signals (3, 63). Thus, Th2 cells may down-regulate immune responses indirectly, through the action of IL-4-induced Th3 cells.

In WT mice, activation of anti-TAChR Th2 cells might prevent EMG because the protective action of IL-4 overshadows the stimulation of B cells by IL-10. IL-10 itself may down-regulate Th1 cells and APC, and contribute to protect from EMG after s.c. or nasal "tolerization" procedures. TG mice did not have an increased production of IL-4 (Fig. 7). Moreover, their transient secretion of large amounts of IL-10 by activated anti-TAChR Th1 cells that also secreted IL-2 enhanced the anti-TAChR B cell response. In TG mice, the anti-TAChR Th1 cells were down-regulated by their own TG IL-10 secretion, because they produced less IFN- than WT mice. However, this effect did not suffice to curb the overstimulation of the anti-TAChR Ab response.

Because of its ability to down-regulate Th1 cells and the synthesis of a variety of cytokines, IL-10 is considered as a possible therapy of undesirable immune responses (70, 71). However, the effects of IL-10 on T cell-mediated experimental autoimmune responses are complex and conflicting. For example, in nonobese diabetic mice, TG pancreatic expression of IL-10 accelerated autoimmune diabetes, and treatment with anti-IL-10 Ab prevented insulitis (72, 73, 74). Yet treatment with IL-10 or an adoptive transfer of islet specific T clones transfected with IL-10 cDNA prevented diabetes (75, 76, 77). The regulated production of IL-10 in T cells by the transgene we used here had a weak inhibitory effect in some mouse models of diabetes (23). Also in experimental autoimmune encephalomyelitis (EAE) the effects of IL-10 are contrasting. IL-10 TG mice were resistant to EAE, and genetically IL-10-deficient mice were more susceptible than WT mice (78). Yet administration of IL-10 did not affect EAE development (79). IL-10 has an important pathogenic role in an Ab-mediated autoimmune disease, systemic lupus erythematosus (SLE). In SLE, the Ig production is IL-10 dependent, and an increased production of IL-10 by B cells and monocytes may be a critical pathogenic mechanism (80, 81). The finding that MG patients have increased blood levels of AChR-specific cells that secrete IL-10 is consistent with a pathogenic role of this cytokine in MG (82).

These results, and the pathogenic role of IL-10 in SLE (80, 81), raise concerns about the suitability of IL-10 to curb Ab-mediated autoimmune diseases, and suggest that IL-10 might be a target, rather than a tool, in the suppression of undesirable Ab responses.

	Footnotes

 
¹ This work was supported by the National Institute of Neurological Disorders and Stroke Grant NS23919 (to B.M.C.-F.).

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: MAChR, muscle acetylcholine receptor; AChR, acetylcholine receptor; MG, myasthenia gravis; EMG, experimental MG; B6, C57BL/6 mice; BTX, bungarotoxin; RIPA, radioimmunoprecipitation assay; TAChR, Torpedo AChR; TG, transgenic; WT, wild type; EAE, experimental autoimmune encephalomyelitis; SLE, systemic lupus erythematosus.

⁵ C. Monfardini, P. I. Karachunski, N. Ostlie, W. Wang, M. Milani, D. K. Okita, J. Lindstrom, and B. M. Conti-Fine. Adoptive protection from experimental myasthenia gravis with T cells from mice treated nasally with acetylcholine receptor epitopes. Submitted for publication.

Received for publication March 6, 2000. Accepted for publication February 2, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Abbas, A. K., K. M. Murphy, A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383:787.[Medline]
2. Romagnani, S.. 1997. The Th1/Th2 paradigm. Immunol. Today 18:263.[Medline]
3. O’Garra, A.. 1998. Cytokines induce the development of functionally heterogenous T helper cell subsets. Immunity 8:275.[Medline]
4. Conti-Fine, B. M., M. Bellone, J. F. Howard, and M. P. Protti. 1997. Myasthenia Gravis. The Immunobiology of Autoimmune Disease. Landes RG, Chapman & Hall, Springer, Austin, TX, pp. 230.
5. Moiola, L., M. P. Protti, J. F. Howard, B. M. Conti-Tronconi. 1994. Myasthenia gravis: residues of the and subunits of muscle acetylcholine receptor involved in formation of immunodominant CD4⁺ epitopes. J. Immunol. 152:4686.[Abstract]
6. Wang, Z.-Y., D. Okita, J. F. Howard, B. M. Conti-Fine. 1997. Th1 epitope repertoire on the subunit of human muscle acetylcholine receptor in myasthenia gravis. Neurology 48:1643.[Abstract]
7. Wang, Z.-Y., J. F. Howard Okita, B. M. Conti-Fine. 1998. T-cell recognition of muscle acetylcholine receptor subunits in generalized and ocular myasthenia gravis. Neurology 50:1045.[Abstract/Free Full Text]
8. Balasa, B., C. Deng, J. Lee, L. M. Bradley, D. K. Dalton, P. Christadoss, N. Sarvetnick. 1997. Interferon (IFN-) is necessary for the genesis of acetylcholine receptor-induced clinical experimental autoimmune myasthenia gravis in mice. J. Exp. Med. 186:385.[Abstract/Free Full Text]
9. Moiola, L., F. Galbiati, G. Martino, S. Amadio, E. Brambilla, G. Comi, A. Vincent, L. M. E. Grimaldi, L. Adorini. 1998. IL-12 is involved in the induction of experimental autoimmune myasthenia gravis, an Ab-mediated diseases. Eur. J. Immunol. 28:2487.[Medline]
10. Zhang, G. X., B. G. Xiao, X. F. Bai, P. H. van der Meide, A. Orn, H. Link. 1999. Mice with IFN- receptor deficiency are less susceptible to experimental myasthenia gravis. J. Immunol. 162:3775.[Abstract/Free Full Text]
11. Gu, D., L. Wogensen, N. A. Calcutt, C. Xia, S. Zhu, J. P. Merlie, H. S. Fox, J. Lindstrom, H. C. Powell, N. Sarvetnick. 1995. Myasthenia gravis-like syndrome induced expression of interferon- in neuromuscular junction. J. Exp. Med. 181:547.[Abstract/Free Full Text]
12. Karachunski, P. I., N. S. Ostlie, C. Monfardini, B. M. Conti-Fine. 2000. Absence of interferon- or interleukin-12 has different effects on experimental myasthenia gravis in C57BL/6 mice. J. Immunol. 164:5236.[Abstract/Free Full Text]
13. Zhang, G. X., C. G. Ma, B. G. Xiao, P. H. van de Meide, H. Link. 1996. Autoreactive T cell responses and cytokine patterns reflect resistance to experimental autoimmune myasthenia gravis in Wistar Furth rats. Eur. J. Immunol. 26:2552.[Medline]
14. Ma, C. G., G. X. Zhang, B. G. Xiao, Z. Y. Wang, J. Link, T. Olson, H. Link. 1996. Mucosal tolerance to experimental autoimmune myasthenia gravis is associated with down-regulation of AChR-specific IFN--expressing Th1-like cells and up-regulation of TGF- mRNA in mononuclear cells. Ann. NY Acad. Sci. 778:273.[Abstract]
15. Karachunski, P. I., N. Ostlie, D. Okita, B. M. Conti-Fine. 1997. Prevention of experimental myasthenia gravis by nasal administration of synthetic acetylcholine receptor T epitope sequences. J. Clin. Invest. 100:3027.[Medline]
16. Wu, B., C. Deng, E. Goluszko, P. Christadoss. 1997. Tolerance to a dominant T cell epitope in the acetylcholine receptor molecule induces epitope spread and suppresses murine myasthenia gravis. J. Immunol. 159:3016.[Abstract]
17. Karachunski, P. I., N. S. Ostlie, D. Okita, R. Garman, B. M. Conti-Fine. 1999. Subcutaneous administration of T epitope sequences of the acetylcholine receptor prevents experimental myasthenia gravis. J. Neuroimmunol. 93:108.[Medline]
18. Karachunski, P. I., N. S. Ostlie, D. Okita, B. M. Conti-Fine. 1999. Interleukin-4 deficiency facilitates development of experimental myasthenia gravis and precludes its prevention by nasal administration of CD4⁺ epitope sequences of the acetylcholine receptor. J. Neuroimmunol. 95:73.[Medline]
19. Im, S.-H., D. Barchan, S. Fuchs, M. C. Souroujon. 1999. Suppression of ongoing experimental myasthenia by oral treatment with an acetylcholine receptor recombinant fragment. J. Clin. Invest. 104:1723.[Medline]
20. Shi, F. D., H. Li, H. Wang, X. Bai, P. H. van der Meide, H. Link, H. G. Ljunggren. 1999. Mechanisms of nasal tolerance induction in experimental autoimmune myasthenia gravis: identification of regulatory cells. J. Immunol. 162:5757.[Abstract/Free Full Text]
21. Constant, S. L., K. Bottomly. 1997. Induction of Th1 and Th2 CD4⁺ T cell responses: the alternative approach. Annu. Rev. Immunol. 15:297.[Medline]
22. Hagenbaugh, A., S. Sharma, S. M. Bubinett, S. H. Y. Wei, R. Aranda, H. Cheroutre, D. J. Fowell, S. Binder, B. Tsao, R. M. Locksley, K. W. Moore, M. Kronenberg. 1997. Altered immune responses in interleukin 10 transgenic mice. J. Exp. Med. 185:2101.[Abstract/Free Full Text]
23. Pauza, M. E., H. Neal, A. Hagenbaugh, H. Cheroutre, D. Lo. 1999. T cell production of an inducible IL-10 transgene provides limited protection from autoimmune diabetes. Diabetes 48:1998.
24. Bellone, M., N. Ostlie, S. Lei, B. M. Conti-Tronconi. 1991. Experimental myasthenia gravis in congenic mice: sequence mapping and H-2 restriction of T helper epitopes on the subunits of Torpedo californica and murine acetylcholine receptors. Eur. J. Immunol. 21:2303.[Medline]
25. Lowry, O., N. Rosebrough, A. Farr, R. Randall. 1981. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265.
26. Bellone, M., N. Ostlie, P. Karachunski, B. M. Conti-Tronconi. 1993. Cryptic epitopes on nicotinic acetylcholine receptor are recognized by autoreactive CD4⁺ cells. J. Immunol. 151:1025.[Abstract]
27. Karachunski, P. I., N. Ostlie, M. Bellone, A. J. Infante, B. M. Conti-Fine. 1995. Mechanisms by which the I-A^bm12 mutation influences susceptibility to experimental myasthenia gravis: a study in homozygous and heterozygous mice. Scand. J. Immunol. 42:215.[Medline]
28. Jouvin-Marche, E., M. Goncalves Morgado, C. Leguern, D. Voegtle, F. Bonhomme, P.-A. Cazenave. 1989. The mouse Igh-1^a and Igh-1^b H chain constant regions are derived from two distinct isotopic genes. Immunogenetics 29:92.[Medline]
29. Martin, R. M., A. Silva, A. M. Lew. 1997. The Igh-1 sequence of the non-obese diabetic (NOD) mouse assigns it to the IgG2c isotype. Immunogenetics 46:167.[Medline]
30. Infante, A. J., P. A. Thompson, K. A. Krolick, K. A. Wall. 1991. Determinant selection in murine experimental autoimmune myasthenia gravis: effect of the bm12 mutation on T cell recognition of acetylcholine receptor epitopes. J. Immunol. 146:2977.[Abstract]
31. Oshima, M., A. R. Pachner, M. Z. Atassi. 1994. Profile of the regions of acetylcholine receptor chain recognized by T-lymphocytes and by antibodies in EAMG-susceptible and nonsusceptible mouse strains after different periods of immunization with the receptor. Mol. Immunol. 31:833.[Medline]
32. Bellone, M., N. Ostlie, S. Lei, X-D. Wu, B. M. Conti-Tronconi. 1991. The I-A^bm12 mutation, which confers resistance to experimental myasthenia gravis, drastically affects the epitope repertoire of murine CD4⁺ cells sensitized to nicotinic acetylcholine receptor. J. Immunol. 146:2253.[Abstract]
33. Berman, P. W., J. Patrick. 1980. Experimental myasthenia gravis: a murine system. J. Exp. Med. 151:204.[Abstract/Free Full Text]
34. Briere, F., C. Servet-Delprat, J. M. Bridon, J. M. Saint-Remy, J. Banchereau. 1994. Human interleukin 10 induces naive surface immunoglobulin D⁺ (sIgD⁺) B cells to secrete IgG1 and IgG3. J. Exp. Med. 179:757.[Abstract/Free Full Text]
35. Briere, F., J. M. Bridon, D. Chevet, G. Souillet, F. Bienvenu, C. Guret, H. Martinezvaldez, J. Banchereau. 1994. Interleukin 10 induces B lymphocytes from IgA-deficient patients to secrete IgA. J. Clin. Invest. 94:97.
36. Burdin, N., C. van Kooten, L. Galibert, J. S. Abrams, J. Wijdenes, J. Banchereau, F. Rousset. 1995. Endogenous IL-6 and IL-10 contribute to the differentiation of CD40-activated human B lymphocytes. J. Immunol. 154:2533.[Abstract]
37. Rousset, F., S. Peyrol, E. Garcia, N. Vezzio, M. Andujar, J. A. Grimaud, J. Banchereau. 1995. Long-term cultured CD40-activated B lymphocytes differentiate into plasma cells in response to IL-10 but not IL-4. Int. Immunol. 7:1243.[Abstract/Free Full Text]
38. Malisan, F., F. Briere, J. M. Bridon, N. Harindranath, F. C. Mills, E. E. Max, J. Banchereau, H. Martinezvaldez. 1996. Interleukin-10 induces immunoglogulin G isotype switch recombination in human CD40-activated naive B lymphocytes. J. Exp. Med. 183:937.[Abstract/Free Full Text]
39. Cerutti, A., H. Zan, A. H., L. Schaffer, N. Bergsagel, E. E. Harindrananth, E. E. Max, P. Casali. 1998. CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plasmacytoid phenotypic differentiation in a human monoclonal IgM⁺IgD⁺ B cell line. J. Immunol. 160:2145.[Abstract/Free Full Text]
40. Silvy, A., C. Lagresle, C. Bella, T. Defrance. 1996. The differentiation of human memory B cells into specific Ab-secreting cells is CD40 independent. Eur. J. Immunol. 26:517.[Medline]
41. Kindler, V., R. H. Zubler. 1997. Memory, but not naive, peripheral blood B lymphocytes differentiate into Ig-secreting cells after CD40 ligation and costimulation with IL-4 and the differentiation factors IL-2, IL-10 and IL-3. J. Immunol. 159:2085.[Abstract/Free Full Text]
42. Agematsu, K., H. Nagumo, Y. Oguchi, T. Nakazawa, K. Fukushima, K. Yasui, S. Ito, T. Kobata, C. Morimoto, A. Komiyama. 1998. Generation of plasma cells from peripheral blood memory B cells: synergistic effect of interleukin-10 and CD27/CD70 interaction. Blood 91:173.[Abstract/Free Full Text]
43. Schilizzi, B. M., R. Boonstra, T. H. The, L. F. de Leij. 1997. Effect of B-cell receptor engagement on CD40-stimulated B cells. Immunology 92:346.[Medline]
44. Ding, L., P. S. Linsley, L. Y. Huang, R. N. Germain, E. M. Shevach. 1993. IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression. J. Immunol. 151:1224.[Abstract]