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(The FASEB Journal. 2001;15:2140-2148.)
© 2001 FASEB

Suppression of experimental myasthenia gravis, a B cell-mediated autoimmune disease, by blockade of IL-18

SIN-HYEOG IM, DORA BARCHAN, PRASANTA KUMAR MAITI, LILY RAVEH1, MIRIAM C. SOUROUJON* and SARA FUCHS2

Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel; and
* The Open University, Tel-Aviv 61392, Israel

2Correspondence: Department of Immunology, The Weizmann Institute of Science, Wolfson Bldg., Room 404, Rehovot 76100, Israel. E-mail: sara.fuchs{at}weizmann.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin-18 (IL-18) is a pleiotropic proinflammatory cytokine that plays an important role in interferon gamma (IFN-{gamma}) production and IL-12-driven Th1 phenotype polarization. Increased expression of IL-18 has been observed in several autoimmune diseases. In this study we have analyzed the role of IL-18 in an antibody-mediated autoimmune disease and elucidated the mechanisms involved in disease suppression mediated by blockade of IL-18, using experimental autoimmune myasthenia gravis (EAMG) as a model. EAMG is a T cell-regulated, antibody-mediated autoimmune disease in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Th1- and Th2-type responses are both implicated in EAMG development. We show that treatment by anti-IL-18 during ongoing EAMG suppresses disease progression. The protective effect can be adoptively transferred to naive recipients and is mediated by increased levels of the immunosuppressive Th3-type cytokine TGF-ß and decreased AChR-specific Th1-type cellular responses. Suppression of EAMG is accompanied by down-regulation of the costimulatory factor CD40L and up-regulation of CTLA-4, a key negative immunomodulator. Our results suggest that IL-18 blockade may potentially be applied for immunointervention in myasthenia gravis.—Im, S.-H., Barchan, D., Maiti, P. K., Raveh, L., Souroujon, M. C., Fuchs, S. Suppression of experimental myasthenia gravis, a B cell-mediated autoimmune disease, by blockade of IL-18.


Key Words: autoimmunity • interleukin 18 • cytokines and costimulatory factors • immunotherapy • MG


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
INTERLEUKIN-18 (IL-18), AN interferon gamma (IFN-{gamma}) -inducing factor (IGIF), is similar to interleukin 1ß (IL-1ß) in structure, but has a distinct role as a costimulatory factor on the activation of T helper type 1 (Th1) but not of Th2 cells (for a review, see ref 1 ). Like IL-1ß, IL-18 is synthesized as a biologically inactive precursor molecule, pro-IL-18, which is cleaved by the IL-1ß-converting enzyme to produce active IL-18 (2) . IL-18 plays a significant role in the induction and activation of Th1 cells and serves as a strong costimulator in IL-12-driven Th1 development. It also induces the production of IFN-{gamma}, IL-2, granulocyte macrophage colony stimulating factor, and tumor necrosis factor alpha in T cells (3) . In addition, IL-18 promotes Fas-mediated killing of natural killer (NK) cells, induces CXC and CC chemokines in monocytes and NK cells, and stimulates ICAM-1 expression in monocytic cell lines (4) . IL-18 acts synergistically with IL-12 in IFN-{gamma} production via an IL-12-induced up-regulation of IL-18 receptor expression (5) . Elevated expression levels of IL-18 have been observed in the plasma of leukemia patients (6) , in mucosal samples of Crohn’s disease patients (7) , in synovial fluids of rheumatoid arthritis (RA) patients (8) , in the spinal cord of rats presenting experimental autoimmune encephalomyelitis (EAE) (9) , and in pancreatic cells of mice in which insulin-dependent diabetes mellitus (IDDM) has been induced (10) , suggesting its importance in immune regulation.

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are autoimmune disorders mediated by autoantibodies against the nicotinic acetylcholine receptor (AChR) in skeletal muscle. EAMG in rats mimics human MG in its clinical and immunopathologic manifestations and is a reliable model for the investigation of therapeutic strategies for myasthenia (11) . The production of immunopathogenic self-reactive antibodies at the neuromuscular junction in both MG and EAMG is dependent on T cells. Activated T cells help autoreactive B cell proliferation and differentiation by producing pathogenic proinflammatory cytokines (IL-12 and IFN-{gamma}) and delivering costimulatory signals.

Since IFN-{gamma} is involved in the induction of EAMG (12 13 14 15) , IL-18, known as an IGIF, may play a role in the pathogenesis of this disease. Indeed, IL-18-/- mice were recently shown to be resistant to induction of EAMG (16) . In this study we address the question whether antibodies to IL-18, given at different stages of EAMG, have an effect on disease progression, and attempt to study the underlying mechanism of this treatment. We show that IL-18 blockade is effective when treatment is given before induction of disease as well as during the acute or chronic phases of ongoing EAMG. The underlying mechanism of disease suppression by anti-IL-18 treatment seems to be down-regulation of proinflammatory Th1-type cytokines and CD40L levels and by up-regulation of AChR-reactive immunosuppressive responses mediated by TGF-ß and CTLA-4.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and antigen preparation
Female Lewis rats, 6–7 wk of age, were purchased from the Animal Breeding Center of the Weizmann Institute of Science (Rehovot, Israel). Torpedo AChR was purified from Torpedo californica by affinity chromatography as described previously (17) . A recombinant rat AChR fragment, R{alpha}1–205, corresponding to residues 1–205 of the extracellular domain of the {alpha} subunit and cloned in pET8C-derived vector (18) , was used to determine AChR-specific IgG isotype levels. Trx-H{alpha}1–210 is a human AChR {alpha} subunit fragment corresponding to residues 1–210 and fused to thioredoxin. Trx-H{alpha}1–210 is similar in its conformation to native AChR, as assessed by its reactivity with {alpha}-bungarotoxin and with anti-AChR mAbs, specific for conformation-dependent epitopes (19) . Both recombinant AChR fragments were expressed in Escherichia coli and purified as described previously (18 , 19) . The purified proteins appeared as a single band on SDS-PAGE stained with Coomassie blue and were estimated to be at least 95% pure (19) .

Induction and clinical evaluation of EAMG
Rats were immunized once in both hind foot pads by subcutaneous (s.c.) injection of Torpedo AChR (40 µg/rat) emulsified in complete Freund’s adjuvant (CFA) containing additional Mycobacterium tuberculosis (0.5 mg/rat; Difco, Detroit, MI). Clinical severity of EAMG was graded between 0 and 4, in which 0 stands for healthy normal rats and 4 stands for dead rats as described previously (20) . Animals were evaluated weekly for 8–9 wk after immunization with Torpedo AChR.

Antibody treatment
Recombinant rat IL-18 was expressed and purified as described (21) . Polyclonal rabbit antibodies to recombinant rat IL-18 or to BSA were prepared by two immunizations with a 4 wk interval, each with 25 µg of antigen in CFA. Immunoglobulins were fractionated from anti-IL-18 or anti-BSA serum by ammonium sulfate precipitation and intraperitoneally (i.p.) administered to rats (600 µg/dose/ml) four times/wk, and continued until the end of the experiments. In some experiments, we used anti-IL-18 antibodies (100 µg/dose/ml) purified on a Sepharose-IL-18 column. For prevention of EAMG, treatment was started 2 days before induction of disease. For treatment of ongoing EAMG, treatment started 1 wk after AChR immunization in the acute-phase treatment protocol and 4 wk after AChR injection in the chronic phase treatment protocol.

Antibody assays
Antibodies to rat muscle AChR were measured by radioimmunoassay with crude rat muscle extract labeled by 125I-{alpha}-bungarotoxin and results were expressed as nmol antibody/l serum (22) . Determination of AChR-specific IgG isotypes was performed as described previously (20) . Microtiter plates were coated with recombinant rat AChR fragment (R{alpha}1–205) and reacted with tested serum samples at proper dilutions (1:50 for IgG1; 1:300 for IgG2a; 1:10 for IgG2b and IgG2c). Biotinylated mouse mAbs to rat IgG isotypes (1:1000; Caltag Laboratories Inc., Burlingame, CA) were added and bound Abs were detected by the activity of alkaline phosphatase monitored at 405 nm.

Delayed-type hypersensitivity
Assessment of delayed-type hypersensitivity (DTH) was performed in rats in which EAMG was induced by injection of Torpedo AChR. Starting on day 4 after the induction of disease, rats were given three i.p. injections of anti-IL-18 or anti-BSA on 3 consecutive days. The test was initiated by s.c. injection of Torpedo AChR (20 µg) into the contralateral ear. The difference in ear lobe thickness before challenge and 30 h after challenge was recorded for each animal. Results are expressed as the mean of four animals for each experimental group ± SE.

Lymphocyte proliferation assay
Proliferation of lymph node cells (LNCs) from treated rats was performed essentially as described (19) . Draining LNCs were removed 8 to 9 wk after disease induction and cultured (5x105/well) in RPMI 1640 medium supplemented with HEPES, sodium pyruvate, glutamine, 2-mercaptoethanol, antibiotics, nonessential amino acids, and 0.5% normal rat serum either alone or in the presence of Torpedo AChR (0.25 µg/ml). Proliferation was assessed by measuring [3H]-thymidine (0.5 µCi/well) incorporation during the last 18 h of a 4 day culture period. Results are expressed as {Delta}cpm after subtracting the background counts of unstimulated cultures from that of stimulated LNCs. To test the effect of TGF-ß on lymphocyte proliferation, anti-TGF-ß (20 µg/ml; R&D Systems, Inc., Minneapolis, MN) or recombinant human TGF-ß (250 ng/ml; R&D Systems) were added to the culture medium. The test for T lymphocyte anergy was performed as described previously (20) . Essentially, single cell suspensions of draining LNCs were incubated for 5 days with or without recombinant rat IL-2 (rIL-2, 10 ng/ml; R&D Systems). The cells were then transferred to 96-well plates (5x105 cells/well) and incubated with Torpedo AChR. Proliferation was assessed by measuring [3H]-thymidine uptake as described above.

B cell proliferation assay based on alkaline phosphatase activity
B cell proliferation was assayed as described previously (19) . Draining LNCs (1x106/ml) were cultured in the medium used for lymphocyte proliferation supplemented by 10% FCS. The cells were stimulated in vitro with Trx-H{alpha}1–210 (50 µg/ml) alone or in the presence of either anti-IL-18 Ig (200 µg/ml) or anti-BSA Ig (200 µg/ml). LPS (5 µg/ml) was used as a positive control. After 4 days in culture, cells were harvested, washed, and diluted in PBS. For alkaline phosphatase assay, 100 µl cell suspensions containing different cell concentrations were transferred to 96-well plates into which 100 µl/well of substrate solution (p-nitrophenyl phosphate, disodium; 1 mg/ml) was added. The plates were incubated for 2–4 h at 37°C in 5% CO2. Data are expressed as optical density at 405 nm.

Adoptive transfer of splenocytes
Adoptive transfer experiments were performed as described previously (18) . Donor rats were treated for 4 consecutive days with anti-IL-18 Ig (600 µg/ml) or anti-BSA Ig (600 µg/ml), and their spleens were removed 1 day after the last treatment. T cell enrichment was performed by removal of B cells and plastic-adherent APC: single cell suspensions of splenocytes were incubated for 1 h in T-75 cell culture flasks precoated with rabbit anti-rat IgG (100 µg/ml) and the procedure was repeated twice. The enriched T cell suspension (2x108 cells consisting of 80–85% T cells as assessed by FACS analysis) was injected i.p. into naive syngeneic recipient rats irradiated with 260 Gy/2 min. Recipients were immunized with Torpedo AChR (40 µg) in CFA 1 h after cell transfer and evaluated for clinical symptoms of EAMG up to 9 wk after immunization.

Determination of cytokines and costimulatory factors
PCR-ELISA was used to assess the levels of mRNA specific for cytokines (IL-2, IL-4, IL-10, IL-12, IFN-{gamma}, and TGF-ß) and costimulatory factors (CD40, CD40L, CD28, CTLA4, B7–1, and B7–2). RNA extraction, cDNA synthesis, and RT-PCR in the presence of digoxigenin (DIG) -dNTP were performed as described (20) by using a PCR-ELISA kit (Roche Molecular Biochemicals, Mannheim, Germany).

The sequences of primer pairs and internal primers specific for cytokines and costimulatory factors were the same as previously reported (18) . The internal primers were all biotinylated by Biotin-Chem-Link (Roche Molecular Biochemicals) according to the manufacturer’s protocol. The amplified DIG-labeled PCR products were quantified with a peroxidase (POD) -conjugated anti-DIG Ab. PCR products were viewed with the POD substrate ABTS and monitored by absorbance at 405 nm (20) .

Statistical analyses
Mann-Whitney U was used for the analysis of EAMG clinical scores. Student’s two-tailed t test was used to determine the significance of differences between group means for all other parameters.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pretreatment with anti-IL-18 protects against EAMG
IL-18 is a multifunctional regulatory cytokine for IL-12-mediated Th1 differentiation and an inducing factor for IFN-{gamma} production. Since IFN-{gamma} was previously reported to be involved in the pathogenesis of EAMG, we decided to test the effect of IL-18 blockade at different stages of EAMG on disease progression. Anti-IL-18 antibodies were administered either before induction of disease or after disease induction, at the acute or chronic phase of EAMG.

We first tested the ability of anti-IL-18 pretreatment to protect rats against EAMG induction. Anti-IL-18 or anti-BSA Ig (600 µg/dose) was administered to rats on 2 consecutive days; 1 day later, EAMG was induced by Torpedo AChR immunization. Subsequent treatment with either anti-IL-18 or anti-BSA was continued four times a week until the end of the experiments and rats were followed clinically for up to 8 wk after AChR injection. As shown in Table 1 and Fig. 1A , rats treated with anti-IL-18 were protected against EAMG induction. At all time points tested, the mean clinical score of the anti-IL-18-treated group was lower than that of the anti-BSA-treated group (Fig. 1A ). Eight weeks after induction of EAMG, all seven rats in the control (anti-BSA-treated) group were sick (mean clinical score: 2.4), whereas among rats treated with anti-IL-18, three of seven rats were healthy and did not present any symptoms of EAMG; the remaining anti-IL-18-treated rats showed mild symptoms compared with the control group. The mean clinical score of the anti-IL-18-treated group was 0.8.


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  		Table 1. Effect of anti-IL-18 antibody treatment on EAMG



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  		Figure 1. Suppression of EAMG by anti-IL-18 treatment. Antibody treatment was initiated 2 days before induction of EAMG (A, prevention protocol) or 1 wk (B, acute-phase treatment) or 4 wk (C, chronic phase treatment) after induction of EAMG by immunization with Torpedo AChR. The treatment was continued 4 times a week until the end of the experiments. Representative of two independent experiments for each protocol. *P < 0.05., **P < 0.016.

Treated rats were further evaluated by weight changes as well as by cellular and humoral immune responses. Rats in the control, anti-BSA-pretreated group lost 17 ± 22 g per rat between 4 and 8 wk after induction of EAMG, whereas rats in the anti-IL-18-treated group gained at the same time 22 ± 8 g per rat (Table 1) . Treatment with anti-IL-18 led to reduced AChR-specific humoral and cellular immune responses measured 8 wk after immunization with Torpedo AChR (Table 1) .

Treatment with anti-IL-18 suppresses ongoing EAMG
To investigate whether blockade of IL-18 is also potentially suitable for treatment of already existing myasthenia, we tested its effect on ongoing EAMG, at the acute and chronic phase of disease. For treatment at the acute-phase of EAMG, administration of anti-IL-18 Ig or anti-BSA Ig (600 µg/dose) was initiated 1 wk after induction of EAMG and was continued four times a week for 7 wk. For treatment at the chronic phase of EAMG, administration with anti-IL-18 or anti-BSA Ig was initiated 4 wk after AChR injection, when clinical symptoms of the chronic disease usually appear, and continued four times a week for 5 wk. In the acute-phase treatment protocol, all eight rats in the control anti-BSA-treated group got sick; 8 wk after disease induction, five of eight died of EAMG. On the other hand, in the anti-IL-18-treated group, three of eight rats were healthy (clinical score 0) and only one of eight died (Table 1) . When assessed 8 wk after AChR injection, the mean clinical score of anti-IL-18-treated rats was 1.5 compared with 3.4 in the anti-BSA-treated rats (Table 1 and Fig. 1 B). In the chronic phase treatment protocol, there was also a suppressive effect on disease severity (Fig. 1C and Table 1 ). Suppression of EAMG symptoms was observed ~2–3 wk after treatment (Fig. 1C , wk 6–7 after disease induction). This suppressive effect was diminished with time (Fig. 1C , wk 8–9 after disease induction). At 9 wk after AChR injection, the mean clinical score of the anti-IL-18-treated group was 1.7 and the mean clinical score of the anti-BSA-treated group was 2.6. These observations suggest that anti-IL-18 treatment alone may not be sufficient to suppress chronic myasthenia and should be used in combination with other treatments. In both acute and chronic phase treatment protocols, rats in the control anti-BSA-treated group (all presenting EAMG) lost weight between 4 and 8 or 9 wk after induction of disease, whereas rats in the anti-IL-18-treated group gained weight during the same period (Table 1) . The effect of anti-IL-18 treatment on clinical symptoms of EAMG was also accompanied by suppression of humoral and cellular AChR-specific immune responses. Rats treated with anti-IL-18 had decreased levels of anti-self AChR antibodies and AChR-specific proliferative T cell responses (Table1).

Suppression of DTH response to AChR by anti-IL-18 treatment
AChR-specific DTH, a known Th1-regulated response, was measured in anti-IL-18 or anti-BSA-treated rats. Torpedo AChR was injected into rats; 4 days later they were treated with either anti-IL-18 or anti-BSA Abs for 3 consecutive days. Torpedo AChR (20 µg) was then injected s.c. into the ear lobes and DTH responses were evaluated 30 h later. Anti-IL-18-treated rats had a reduced DTH response to AChR (46±5 mmx10-2) compared with anti-BSA-treated rats (81±4 mmx10-2).

Effect of anti-IL-18 treatment on cytokines and costimulatory factors
To analyze the possible mechanisms involved in suppression of EAMG by anti-IL-18 treatment, we monitored the levels of cytokines and costimulatory factors in treated rats. Draining LNCs from rats treated with anti-IL-18 or anti-BSA, starting at the acute-phase of disease, were removed 5 to 8 wk after EAMG induction and cultured for 40 h in the presence of Torpedo AChR. Total RNA was then prepared from the cells and subjected to PCR-ELISA with cytokine- or costimulatory factor-specific primers. As shown in Fig. 2 , anti-IL-18 treatment resulted in down-regulation of Th1-type cytokines (IL-2, IL-12 and IFN-{gamma}). On the other hand, levels of the tested Th2-type cytokines (IL-4 and IL-10) did not change and levels of the immunosuppressive Th3-type cytokine TGF-ß increased significantly (P<0.005) when compared with their respective levels in anti-BSA-treated rats. These observations suggest that anti-IL-18 treatment induces a shift from a Th1-mediated proinflammatory response to a Th3-mediated immunosuppressive response to AChR.



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  		Figure 2. Effect of anti-IL-18 treatment on cytokines. LNCs from rats treated with anti-IL-18 or anti-BSA, starting at the acute-phase of EAMG, were cultured for 40 h in the presence of AChR and their total RNA was isolated. The expression level of cytokines (and of ß-actin as control) was determined by PCR-ELISA. mRNA levels of the cytokines were first normalized with respect to ß-actin. The bars represent relative normalized amount of expression for each cytokine in the anti-IL-18-treated group compared with the anti-BSA-treated group. *P < 0.005. The experiment was repeated three times.

To test whether the observed changes in the cytokine profile from Th1 to Th3 are also accompanied by changes in costimulatory factors, we measured expression levels of the costimulatory factors CD40, CD40L, B7–1, B7–2, CD28, and CTLA-4 by performing PCR-ELISA on the RNA preparations, using costimulatory factor-specific primers. As shown in Fig. 3 , anti-IL-18 treatment induced a significant reduction in CD40L levels, some reduction of CD28, and an increase in CTLA-4 levels (P<0.005). Other costimulatory factors tested were similarly expressed in anti-IL-18- and anti-BSA-treated rats.



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  		Figure 3. Effect of anti-IL-18 treatment on costimulatory factors. The expression levels of costimulatory factors was determined by PCR-ELISA on total RNA isolated from LNCs after stimulation with AChR for 40 h. mRNA levels of the costimulatory factors were first normalized with respect to ß-actin. The data are expressed for each costimulatory factor as the relative normalized value in the anti-IL-18-treated group compared with that of the anti-BSA-treated group. *P < 0.005. The experiment was repeated three times.

Effect of anti-IL-18 treatment on AChR-specific IgG isotypes
The analysis of the cytokine profile showed that anti-IL-18 treatment down-regulates the Th1 type immune response and has no significant effect on the Th2 type response (Fig. 2) . The analysis of IgG isotypes of anti-AChR antibodies elicited after treatment with anti-IL-18 confirmed these observations. The IgG isotype levels of AChR-specific antibodies were measured 6–8 wk after induction of EAMG. R{alpha}1–205, a recombinant rat AChR fragment representing syngeneic muscle-type AChR, was used as antigen in this assay. As shown in Fig. 4 , in the acute and chronic phase treatment protocols, IgG1, IgG2b, and IgG2c isotype levels were all reduced in anti-IL-18-treated rats compared with control anti-BSA-treated rats. These data are concordant with the observed cytokine profile (Fig. 2) and indicate again that anti-IL-18 treatment does not induce a shift from Th1 to Th2-regulation of the anti-AChR response, since such a shift would lead to an increase in IgG1 that is regulated in rats by Th2 cells (20 , 23) .



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  		Figure 4. Effect of anti-IL-18 treatment on IgG isotype levels. Rats were immunized with Torpedo AChR and 1 wk later treatment was initiated with either anti-IL-18 or anti-BSA. Rats were bled 8 wk after AChR injection and their pooled sera were used to determine AChR-specific IgG isotype levels by ELISA. The experiment was repeated three times.

In vitro effect of anti-IL-18 treatment on B and T cell proliferation
The suppression of ongoing EAMG by anti-IL-18 treatment seems to be mediated by down-regulation of proinflammatory Th1 type cytokines (Fig. 2) and reduction of anti-self AChR antibody levels (Table 1) . To examine whether the latter is directly mediated by suppression of B cell proliferation, we tested the in vitro effect of anti-IL-18 treatment on B cell proliferation in response to AChR. LNCs were removed from myasthenic rats at their chronic stage of disease (mean clinical score 2–3) and cultured for 4 days in the presence of Trx-H{alpha}1–210, a human AChR fragment with conformational similarity to native AChR (19) . Alkaline phosphatase activity, which is specific for activated B cells (24 , 25) , was used to determine B cell proliferation in LNCs cultured with Trx-H{alpha}1–210 alone or with either anti-IL-18 or anti-BSA. As shown in Fig. 5 A, the presence of anti-IL-18 did not affect in vitro B cell proliferation. However, addition of anti-IL-18 strongly suppressed T cell proliferation to AChR compared with the effect of anti-BSA Abs (Fig. 5B ). This may suggest that the effect of anti-IL-18 treatment on the humoral response to self AChR is not executed by direct suppression of B cell proliferation, but rather via T cell suppression.



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  		Figure 5. Effect of anti-IL-18 treatment on in vitro B and T cell proliferation. Proliferation of B and T cells from myasthenic rats in response to Trx-H{alpha}1–210 (AChR) alone or in the presence of anti-IL-18 or anti-BSA was determined as described in Materials and Methods. The level of B cell proliferation was determined by alkaline phosphatase activity (A) and proliferation of T cells was determined by measuring [3H]-thymidine incorporation (B). *P < 0.0001 compared with proliferation in the presence of anti-BSA-Abs. The experiment was repeated twice.

Mechanism of T cell suppression
The suppressed T cell proliferation in response to AChR in anti-IL-18-treated rats (Table 1) could be mediated by several mechanisms. To test the possible involvement of anergy in suppressed T cell proliferation in anti-IL-18-treated rats, LNCs from anti-IL-18 and anti-BSA-treated rats were preincubated for 5 days in the presence or absence of rIL-2 (10 ng/ml) and proliferation in response to Torpedo AChR was assessed. Preincubation with exogenous rIL-2 did not restore the suppressed AChR-specific proliferative response (data not shown), suggesting that the suppressed AChR-specific T cell response in anti-IL-18-treated rats is not mediated by induction of anergy.

To assess the possible contribution of TGF-ß to T cell suppression, we tested whether antibodies to TGF-ß reverse this suppressed AChR-specific T cell proliferation. LNCs from rats treated with anti-IL-18 or anti-BSA were removed 9 wk after EAMG induction and lymphocyte proliferation in response to Torpedo AChR was performed in the presence or absence of anti-TGF-ß Abs (up to 20 µg/ml). As shown in Fig. 6 , anti-TGF-ß partially reversed the suppressed T cell proliferation in the anti-IL-18-treated group and led to increased proliferation in the anti-BSA-treated rats. In addition, recombinant TGF-ß (250 ng/ml) abolished T cell proliferation in response to AChR in both groups (Fig. 6) . These results, together with the up-regulation of TGF-ß observed after anti-IL-18 treatment (Fig. 2) , indicate the role of TGF-ß in suppressing both AChR-specific T cell response and disease progression.



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  		Figure 6. Effect of anti-TGF-ß and rTGF-ß on T cell proliferation. LNCs were removed from rats treated with anti-IL-18 or anti-BSA 9 wk after EAMG induction. Lymphocyte proliferation in response to Torpedo AChR was measured in the absence or presence of anti-TGF-ß (20 µg/ml) or of recombinant TGF-ß (250 ng/ml). *P < 0.005 proliferation in response to Torpedo AChR in the anti-IL-18-treated group vs. proliferation in the presence of anti-TGF-ß or rTGF-ß. **P < 0.005 same as above but for the anti-BSA-treated group. The experiment was repeated three times.

We then tested whether anti-IL-18 treatment induced regulatory T cells that can adoptively transfer protection against EAMG. Splenocytes from rats treated for 4 days by anti-IL-18 or anti-BSA were depleted of APC and B cells and transferred i.p. (2x108/rat) into naive syngeneic recipient rats irradiated by 260 Gy/2 min. Recipient rats were injected 1 h later with Torpedo AChR to induce EAMG. A protective effect of the splenocytes transferred from anti-IL-18-treated rats was observed in the recipient rats, and was especially evident at the chronic phase of disease (Fig. 7 ). At 9 wk after transfer, recipients of splenocytes from anti-IL-18-treated rats had a mean clinical score of 1.1 vs. 2.3 in rats that received splenocytes from control, anti-BSA-treated donors.



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  		Figure 7. Protection against EAMG is adoptively transferred by splenocytes from anti-IL-18-treated rats. Splenocytes from anti-IL-18 or anti-BSA-treated rats were transferred i.p. to naive recipients that were subsequently immunized with Torpedo AChR to induce EAMG. Clinical symptoms in the recipient rats were followed for 9 wk after immunization. *P < 0.005 compared with control, anti-BSA-treated rats. The experiment was repeated twice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To understand the role of IL-18 in the pathogenesis of EAMG, an antibody-mediated autoimmune disease, we studied the effect on disease progression of anti-IL-18 treatment initiated at different stages of EAMG. We demonstrate that in vivo blockade of IL-18 activity by anti-IL-18 antibodies has a suppressive effect on EAMG even when treatment is initiated at the chronic phase of the disease. Our results suggest that the underlying mechanism of disease suppression involves a down-regulation of Th1-type cytokines and up-regulation of the immunosuppressive Th3-type cytokine TGF-ß, but no change in Th2-type cytokines. This shift from a Th1 to a Th3 response was accompanied by down-regulation of the costimulatory factor CD40L and up-regulation of CTLA-4, a key negative immunonoregulator.

Anti-IL-18 treatment down-regulated the pathogenic cytokines IL-12 and IFN-{gamma} (Fig. 2) and suppressed IL-2-induced T cell proliferation in response to AChR. This could contribute to the observed reduced anti-self AChR antibody titers in the anti-IL-18-treated rats. The down-regulated IL-12 levels inhibit Th1 differentiation and may favor differentiation of Th2/Th3 cells (26) . It should be noted that in EAE, a T cell-mediated animal model for multiple sclerosis, anti-IL-18 treatment induced a shift in T cell responses from Th1 to Th2 (21) . However, in our study, the levels of Th2-type cytokines (IL-4 and IL-10) were not affected by anti-IL-18 treatment. On the other hand, the Th3-type cytokine TGF-ß was significantly increased in anti-IL-18-treated rats. This is in concordance with the observation in IL-18-/- mice, in which the resistance to EAMG induction was associated with a defective Th1 response and increased TGF-ß production (16) . TGF-ß is a potent immunomodulator that polarizes CD4+ responses toward a Th2 phenotypes (27) and blocks the effect of IL-12-driven differentiation of Th1 cells (28) via down-regulation of IL-12R ß2-chain (29) . The increased TGF-ß levels after treatment with anti-IL-18 may be mediated either by induction of TGF-ß-secreting regulatory T cells or by blocking of IL-18-mediated Th1 polarization without the induction of specific regulatory T cells and possibly by both.

We have demonstrated that AChR-specific T cell proliferation could be partially restored by anti-TGF-ß (Fig. 6) and that the protective effect induced by anti-IL-18 treatment could be transferred to naive syngeneic rats (Fig. 7) . These observations indicate that anti-IL-18-treatment is associated with TGF-ß production and induces TGF-ß-secreting regulatory cells. Activation of costimulatory molecules is crucial for T cell activation and B cell proliferation, differentiation, and survival. T cells provide help for B cell proliferation and Ab production by cell–cell contact and by releasing soluble factors. Anti-IL-18 treatment reduced CD40L level but increased CTLA-4 levels; other costimulatory factors did not change (Fig. 3) . CD40L is involved in contact-dependent T cell help and is a predominant B cell costimulatory molecule expressed on T cells upon activation (30) . CD40L-CD40 interaction induces increased IFN-{gamma} and IL-12 production, and intervention with this interaction significantly reduces IFN-{gamma} production by T cells (31) and inhibits B cell expansion in vivo (32) . CD40L knockout mice (CD40L-/-) are completely resistant to EAMG induction (33) and anti-CD40L treatment in chronic EAMG suppresses disease progression (34) . The effect of CD40L-blockade on disease in either CD40L-/- or rats treated with anti-CD40L was associated with diminished Th1 and Th2 responses as well as with severely impaired T cell-dependent, AChR-reactive B cell responses. We expected the reduced CD40L level induced by anti-IL-18 treatment (Fig. 3) to lead to a comparable reduction of the anti-self Ab levels. However, the marked effect on CD40L expression in the acute-phase treatment protocol resulted in just a mild reduction in the anti-AChR antibody titer (Table 1) .

Anti-IL-18 treatment resulted in increased CTLA-4 expression levels. CTLA-4, present on CD4+ or CD8+ T cells, acts as a key negative immunomodulator of immune responses by blocking CD28-dependent T cell activation (35) . CTLA-4 is also involved in the induction of peripheral T cell tolerance in vivo (36) . It normally acts as a negative regulator of T cell activation; therefore, the observed up-regulation of CTLA-4 levels by anti-IL-18 treatment may be responsible for the attenuated Th1-type cell activation (Fig. 3) (37) . The unchanged levels of costimulatory factors, other than CD40L and CTLA-4, suggest that T cells are not in an anergic state in anti-IL-18-treated rats. This was confirmed by failure of exogenous IL-2 to reverse the suppressed AChR-specific T cell responses.

Although Ab treatments seem to be effective in blocking IL-18 activity, it should be kept in mind that repeated Ab administrations could lead to undesirable side effects. To overcome these limitations, blockade of IL-18 activity could be achieved by a soluble inhibitory receptor for IL-18, such as IL-18 binding protein (IL-18BP) (38) . Our attempts to affect EAMG at its chronic phase resulted in significant suppression during the first period after the initiation of treatment, yet this suppression later diminished. These observations may suggest that anti-IL-18 treatment alone may not be sufficient to suppress myasthenia and should be used to augment other treatment modalities involving antigen-specific immunomodulation (18 19 20 , 39) . This may be applicable especially for severely affected patients in which antigen-specific treatment itself may not be sufficient. Thus, a combined therapy consisting of Ag-specific suppression together with IL-18 blockade by anti-IL-18 treatment or by IL-18BP may be potentially useful for future immunotherapy of myasthenia gravis and other T cell-regulated antibody-mediated autoimmune diseases.


   ACKNOWLEDGMENTS
 
This research was supported by grants from The Muscular Dystrophy Association of America, The Association Française contre les Myopathies, and the Abramson Family Foundation.


   FOOTNOTES
 
1 Permanent address: Israel Institute for Biological Research, Ness-Ziona 74100, Israel.

Received for publication February 16, 2001. Revision received May 25, 2001.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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