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International Immunology, Vol. 12, No. 10, 1381-1388, October 2000
© 2000 Japanese Society for Immunology

Tumor necrosis factor receptor-1 is critically involved in the development of experimental autoimmune myasthenia gravis

Hua-Bing Wang1, Hulun Li1, Fu-Dong Shi2,3, Benedict J. Chambers2, Hans Link1 and Hans-Gustaf Ljunggren2

1 Experimental Neurology Unit, Division of Neurology, Karolinska Institutet, Huddinge University Hospital, 14186 Stockholm, Sweden
2 Microbiology and Tumor Biology Center (MTC), Karolinska Institutet, 17177 Stockholm, Sweden

Correspondence to: H.-B. Wang


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Tumor necrosis factor receptor-1 (TNFR1, CD120a) has been implicated in the pathogenesis of several experimental models of T cell-mediated autoimmune disorders, but its role in antibody-mediated autoimmune diseases has not been addressed. Experimental autoimmune myasthenia gravis (EAMG), an autoantibody-mediated T cell-dependent neuromuscular disorder, represents an animal model for myasthenia gravis in human. To investigate the role of TNFR1 in the pathogenesis of EAMG, TNFR1–/– and wild-type mice were immunized with Torpedo acetylcholine receptor (AChR) in complete Freund's adjuvant. TNFR1–/– mice failed to develop EAMG. Lymphoid cells from TNFR1–/– mice produced low amounts of Th1 (IFN-{gamma}, IL-2 and IL-12)-type cytokines, but elevated levels of Th2 (IL-4 and IL-10)-type cytokines compared with lymphoid cells of wild-type mice. Accordingly, the levels of anti-AChR IgG2 antibodies were severely reduced and the level of anti-AChR IgG1 antibodies were moderately reduced. Co-injection of recombinant mouse IL-12 with AChR in adjuvant restored T cell responses to AChR and promoted development of EAMG in TNFR1–/– mice. These results demonstrate that the TNF/TNFR1 system is required for the development of EAMG. The lack of a functional TNF/TNFR1 system can, at least in part, be substituted by IL-12 at the stage of initial priming with AChR and adjuvant.

Keywords: autoantibody, experimental autoimmune myasthenia gravis, IL-12, Th cell, tumor necrosis factor receptor-1


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Tumor necrosis factor (TNF)-{alpha} and lymphotoxin-{alpha} are key mediators of inflammatory and autoimmune diseases by engagement of two distinct receptors, TNFR1 and TNFR2 (1,2). TNFR1, the major mediator of TNF, has been implicated in the pathogenesis of T cell-mediated experimental autoimmune models including encephalomyelitis (3,4), diabetes mellitus (5,6) and collagen-induced arthritis (7).

Experimental autoimmune myasthenia gravis (EAMG) is an antibody-mediated T cell-dependent animal model for myasthenia gravis (MG) in humans. In MG and EAMG, autoantibodies against nicotinic acetylcholine receptor (AChR) of the neuromuscular junction are responsible for the functional loss of AChR and impaired muscle contraction (8). TNF-{alpha} has been implicated in the development of MG. MG patients showed increased numbers of TNF-{alpha} mRNA expressing cells among blood mononuclear cells (MNC) compared to healthy subjects (9). In addition, spontaneous production of TNF-{alpha} in blood MNC cultures has been demonstrated to be associated with disease severity (10). TNF-{alpha} binding capacity of CD4+ T cells has been demonstrated to be increased in MG patients compared with healthy subjects (11). Linomide, a synthetic compound that inhibits systemic TNF-{alpha} production, suppressed clinical EAMG in Lewis rats (12). Early detection of TNF-{alpha} in muscle sections also implies its importance in EAMG (13). However, the role of the TNF/TNFR pathway in MG and EAMG remains to be clarified.

To determine the role of TNFR1 in the development of a B cell-mediated autoimmune disease, we studied EAMG in TNFR1–/– mice. Our results clearly demonstrate that TNFR1–/– mice do not develop MG after repeated immunization. Furthermore, the results suggest that addition of IL-12 at the stage of initial priming promotes the development of EAMG in the absence of a functional TNF/TNFR1 system.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
TNFR1–/– mice were generated by gene targeting in embryonic stem cells and have been previously described (14,15). Germ-line transmitter of the mutated TNFR1 allele was backcrossed onto an EAMG-susceptible C57BL/6 (B6) genetic background for nine generations. The resulting heterozygous mice were interbred to yield homozygous mutant offspring. Mice were bred and maintained under pathogen-free conditions in the animal facilities of the Microbiology and Tumor Biology Center, Karolinska Institutet. Female mice between 8 and 10 weeks of age were used. Animal experimental procedures were in compliance with institutional guidelines.

Antigens and synthetic peptide
Torpedo AChR was purified from the electric organs of Torpedo californica (Pacific Biomarine, Venice, CA) by affinity chromatography on a {alpha}-cobrotoxin–agarose resin (Sigma, St Louis, MO) as described previously (16). The isolated product was pure as judged by SDS–PAGE. The purified AChR was used to induce EAMG and for stimulation of in vitro cultures. Keyhole limpet hemocyanin (KLH) and concanavalin A (Con A) were purchased from Sigma. Myelin basic protein (MBP) to be used as control antigen was purified from normal mouse brains (17). The AChR {alpha} chain immunodominant peptide {alpha}146–162 (L-G-I-W-T-Y-D-G-T-K-V-S-I-S-P-E-S) was synthesized and used for in vitro culture. An unrelated peptide (K-A-I-V-E-L-A-F-T-Y-R-S-D-S-F-E-N) derived from Ku protein was synthesized as control (18). Purified protein derivative (PPD) (Statens Seruminstitute, Copenhagen, Denmark) was used as internal control.

Induction and clinical evaluation of EAMG
Mice were immunized s.c. with 20 µg Torpedo AChR in complete Freund's adjuvant (CFA) in a total volume of 100 µl, along the shoulders and back. Mice were boosted twice at 1 month interval with 20 µg of AChR in CFA at four sites on the shoulders and thighs. The mice were observed every other day in a blinded fashion for signs of muscle weakness characteristic of EAMG. The disease symptoms were graded between 0 and 3 (19): 0, no definite muscle weakness; 1+, normal strength at rest but weak with chin on the floor and inability to raise the head after exercise consisting of 20 consecutive paw grips; 2+, as grade 1+ and weakness at rest; and 3+, moribund, dehydrated and paralyzed. Clinical EAMG was confirmed by injection of neostigmine bromide and atropine sulfate (19). In some experiments, 0.1 or 1 µg of recombinant mouse IL-12 (rmIL-12; R & D Systems, Abingdon, UK) was incorporated in the emulsion at the first immunization according to a previous report (20).

Mononuclear cell (MNC) suspensions
Mice were immunized with 20 µg Torpedo AChR in CFA s.c. in the hind footpads and thighs and killed 7 days post primary immunization (p.i.). MNC suspensions from the popliteal and inguinal lymph nodes (PILN) or spleen were prepared by grinding through a wire mesh. Erythrocytes in spleen cell suspension were osmotically lyzed. Cells were washed 3 times in DMEM (Gibco, Paisley, UK) supplemented with 1% (v/v) MEM (Gibco), 2 mM glutamine (Flow, Irvine, UK), 50 IU/ml penicillin and 50 µg/ml streptomycin (Gibco), and 10% (v/v) FCS (Gibco). The cells were then adjusted to 2x106/ml.

Lymphocyte proliferation responses
Triplicate aliquots (200 µl) of MNC suspensions, derived from lymph nodes or spleen, containing 4x105 cells were cultured at 96-well round-bottomed microtiter plates (Nunc, Copenhagen, Denmark) in the presence or absence of AChR, AChR {alpha} chain peptide {alpha}146–162, Ku peptide, PPD or MBP (all preparations 10 µg/ml). KLH and Con A were used at 50 and 5 µg/ml respectively. rmIL-12 and rmIL-2 (R & D Systems) were used for in vitro stimulation. After 4 days of incubation, the cells were pulsed for 18 h with 10 µl of aliquots containing 1 µCi of [3H]methylthymidine (sp. act. 42 Ci/mmol; Amersham, Arlington Heights, IL). Cells were harvested onto glass fiber filters and thymidine incorporation was measured. The results were expressed as c.p.m.

Cytokine ELISA
Lymphoid cells from either PILN or spleen were cultured in the presence or absence of AChR or {alpha}146–162 for 48 h. Supernatants were harvested and assessed for IFN-{gamma}, IL-2 and IL-4 using optEIA kits (PharMingen, San Diego, CA). IL-10 was measured with an ELISA kit according to the manufacturer's instruction (R & D Systems). IL-12 was measured with an ELISA kit from Endogen (St Woburn, MA).

Assays of anti-AChR IgG antibodies
To enumerate anti-AChR IgG antibody-secreting cells among PILN MNC, solid-phase ELISPOT assays were used with some modifications (21). Briefly, wells of microtiter plates with nitrocellulose bottoms were coated with 100 µl of AChR or the control antigen MBP (10 µg/ml in PBS). Aliquots of 100 µl suspension containing 2x105 MNC were added to individual wells in triplicate. After incubation for 24 h, the wells were emptied, followed by addition of rabbit anti-hamster IgG (Sigma), biotinylated swine anti-rabbit IgG (Dakopatts, Copenhagan, Denmark) and avidin–biotin peroxidase complex (ABC; Dakopatts). After peroxidase staining, the red/brown immunospots corresponding to cells that had secreted anti-AChR IgG were counted and standardized to numbers per 105 MNC.

Isotypes of anti-AChR IgG antibodies were detected as described (22). Microtiter plates (Costar, Corning, NY) were coated with 100 µl/well of AChR (2 µg/ml) at 4°C overnight. Uncoated sites were blocked with 10% FCS (Gibco). Sera (diluted 1:200 for IgG2a, and 1:1000 for IgG1, IgG2b and IgG) with a predetermined amount of anti-AChR antibodies was added and incubated for 2 h at room temperature. Then, plates were incubated for 2 h with biotinylated rabbit anti-mouse IgG1, IgG2a or IgG2b (Cymbus Biotechnology, Hants, UK), followed by alkaline phosphatase-conjugated avidin–biotin complex (Dakopatts). The color was developed with p-nitrophenyl phosphate and expressed as optical density (OD) at 405 nm. IgG2b:IgG1 ratios were calculated based on OD readings at 1:1000 dilution of sera.

Determination of the antibody responses to KLH
Mice were primed with 100 µg of KLH in CFA on day 0 and boosted on day 15 p.i. Sera taken at day 30 p.i. were examined for anti-KLH IgG and isotype antibodies as described (23). The results were expressed as OD values.

Statistical analysis
Differences between groups were analyzed by two-tailed Student's t-test. Clinical scores were analyzed using the non-parametric Mann–Whitney U-test. Differences between the groups with respect to disease incidence were analyzed by Fisher's exact test. The level of significance was set at P = 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
TNFR1 deficiency renders resistance to EAMG
To evaluate the contribution of the TNFR1 in the immune response leading to the development of EAMG, we immunized TNFR1–/– mice and wild-type mice with 20 µg AChR in CFA. On days 30 and 60 p.i., mice were rechallenged with 20 µg AChR in CFA. Three separate experiments were conducted and the clinical course of EAMG was followed up to 150 days p.i. (Table 1Go). Following the second immunization, 60–86% of the wild-type mice showed progressive MG characterized by muscular weakness with a mean onset on day 38 p.i. In contrast, all of the TNFR1–/– mice remained without MG up to the termination of the experiments. These results indicate that TNFR1 is critically required in EAMG.


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  		Table 1. TNFR1–/– mice are resistant to EAMG.
 
TNFR1–/– mice exhibit markedly reduced T cell responses to AChR and its {alpha}146–162 sequence
TNF-{alpha} stimulates T cell proliferation through the IL-2/IL-2R pathway by up-regulating IL-2R{alpha} (24). To evaluate whether absence of TNFR1 alters the generation of AChR and immunodominant peptide {alpha}146–162-specific T cell responses, lymphoid cells from either PILN or spleen derived from TNFR1–/– and wild-type mice 7 days p.i. with AChR were re-stimulated in vitro with antigens. Lymphoid cells from TNFR1–/– mice showed reduced proliferation, and produced reduced amounts of IL-2 in response to AChR and {alpha}146–162 compared to wild-type mice (Fig. 1A and BGo). In contrast, proliferative responses to either control Ku peptide, internal control PPD or Con A did not differ between two groups. To address whether the reduction in proliferation observed in AChR-immunized TNFR1–/– mice could be due to T cell anergy, T cell proliferation was determined after stimulation with AChR in the presence of IL-2. IL-2 supplementation to in vitro cultures did not restore the proliferative responses in TNFR1–/– mice as compared to wild-type mice (Fig. 1CGo), excluding clonal anergy as an explanation for the inability of the T cells to proliferate.



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  		Fig. 1. T cell immune responses from AChR immunized TNFR1–/– and wild-type (wt) mice. PILN or spleen were taken 7 days p.i. with 20 µg of AChR in CFA and cultured in the presence or absence of antigen or mitogen. Each group consisted of four mice. Lymphocyte proliferation was measured by 3H incorporation and expressed as c.p.m. (A). IL-2 concentrations were determined after 48 h of culture (B). Addition of recombinant mouse IL-2 failed to restore the AChR-specific proliferation in TNFR1–/– mice relative to that of wild-type mice (C). Symbols refer to mean values and bars to SD. **P < 0.01. Data are representative of three independent experiments utilizing PILN MNC showing similar patterns as data derived from spleen MNC.

 
In MG and EAMG, production of anti-AChR antibodies depends on T cell help (25). T cells differentiate into distinct Th cell subsets that produce characteristic cytokine profiles upon activation (26). To assess the cytokine profiles in the absence of TNFR1, supernatants were collected from AChR- or {alpha}146–162-stimulated cell cultures obtained from TNFR1–/– and wild-type mice, and analyzed for cytokine concentrations by ELISA. As shown in Fig. 2Go, TNFR1 gene disruption led to substantially reduced levels of IFN-{gamma} and IL-12 production, but enhanced AChR-specific IL-4 and IL-10 production by lymphoid cells.



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  		Fig. 2. Cytokine profiles in TNFR1–/– and wild-type mice. MNC from PILN or spleen were cultured in the presence or absence of AChR (10 µg/ml) or {alpha}146–162 (10 µg/ml) under conditions similar to that in Fig. 1Go. Culture supernatants were collected 48 h later. Lower levels of IFN-{gamma} (A) and IL-12 (B), but higher levels of IL-4 (C) and IL-10 (D) were produced by MNC from TNFR1–/– compared to wild-type mice. Low levels of IFN-{gamma} (TNFR1–/– < 36 pg/ml and wild-type < 15 pg/ml), IL-10 (TNFR1–/– < 10 pg/ml and wild-type < 8 pg/ml), and undetectable IL-12 and IL-4 were produced by MNC cultured in medium alone, or by antigen stimulated MNC derived from naive or CFA immunized wild-type mice. Symbols refer to mean values and bars to SD. *P < 0.05, **P < 0.01. Data are representative of three independent experiments utilizing PILN MNC showing similar patterns as data derived from spleen MNC.

 
Anti-AChR IgG and IgG2 antibodies were substantially reduced in TNFR –/– mice
Anti-AChR antibody production is a hallmark of disease development in MG and EAMG (8,25). To assess the influence of a TNFR1 gene deficiency on humoral immune responses, TNFR1–/– and wild-type mice were bled at days 30, 45 and 75 p.i. Sera were tested for anti-AChR IgG, IgG1, IgG2a and IgG2b antibody levels by ELISA. TNFR1–/– mice displayed lower anti-AChR IgG and isotype antibody levels, but enhanced serum IgG1:IgG2b ratios compared with wild-type mice (Fig. 3A and BGo), suggesting the generation of a polarized Th2 response and induction of a resistant phenotype. This is in accordance with previous findings that mice resistant to EAMG or tolerized by a recombinant fragment of human AChR {alpha} subunit produced predominantly Th2 cytokines and IgG1 antibodies (27,28). ELISPOT assays confirmed decreased anti-AChR IgG production by TNFR1–/– mice at the single-cell level (Fig. 3CGo).



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  		Fig. 3. Anti-AChR antibodies are markedly reduced in TNFR1–/– mice compared with wild-type mice. Sera were taken from mice indicated in experiment 1 (Table 1, Gon = 8–12), and anti-AChR IgG and isotype antibodies were analyzed by ELISA. Results are expressed as optical density (OD) at 405 nm (A). Anti-AChR IgG1:IgG2b antibody ratios were calculated based on data that represent sera taken at day 45 p.i. (B). PILN MNC obtained from TNFR1–/– and wild-type mice 7 days p.i. were cultured for 24 h and assayed by ELISPOT assays. Results were expressed as numbers of antigen-specific IgG antibody-secreting cells per 105 MNC (C). Symbols refer to mean values and bars to SD. *P < 0.05, **P < 0.01, #P < 0.001. Data are representative of two independent experiments.

 
TNFR1 gene deficiency does not impair T cell proliferation in response to KLH and anti-KLH antibody response
To compare immune responses to AChR with those to other T cell-dependent antigens, mice were immunized with KLH plus CFA (see Methods) and sacrificed on day 7 p.i. Surprisingly, T cells from TNFR1–/– and wild-type mice proliferated to a similar extent in response to KLH (Fig. 4AGo). Accordingly, both TNFR1–/– and wild-type mice mounted significant amounts of anti-KLH IgG antibodies. However, among the IgG isotypes, IgG2b antibodies were decreased in TNFR1–/– mice compared with wild-type mice, confirming an association between TNFR1 and IgG2b responses (Fig. 4BGo).



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  		Fig. 4. Immune responses to KLH are not significantly affected relative to those against AChR in TNFR1–/– mice. PILN were obtained 7 days p.i. with KLH in CFA and cultured in the presence or absence of antigens or mitogen. Each group consisted of four mice. Lymphocyte proliferation was measured by [3H]methylthymidine incorporation and expressed as c.p.m. (A). Sera taken at day 30 p.i., i.e. after the boost immunization, were analyzed for anti-KLH antibody responses at OD 592 nm (B). Symbols refer to mean values and bars to SD. *P < 0.05. Data are representative of two independent experiments.

 
Co-injection of rmIL-12 with AChR in CFA restores T cell responses to AChR and sensitivity to EAMG in TNFR1–/– mice
IL-12 is an obligatory factor for Th1 cell generation and involved in the induction of EAMG in B6 mice (20). As shown in Fig. 2Go, secretion of IL-12 by lymphoid cells in TNFR1–/– mice was diminished by 95% of that secreted by wild-type mice, suggesting a causal relationship between the depolarization of Th1 cells and the resistance to EAMG. To investigate this possibility, TNFR1–/– mice and wild-type mice were immunized with AChR plus CFA, together with 0.1 or 1 µg of rmIL-12 in PBS, or with PBS alone, and monitored for the development of EAMG. Consistent with a recent study in B6 mice employing larger amounts of rmIL-12 (20), co-injection of IL-12 with AChR plus CFA moderately increased the severity of EAMG compared with PBS treatment. In contrast, it was striking that three of six TNFR1–/– mice treated with 0.1 µg of IL-12 and four of six TNFR1–/– mice treated with 1 µg of IL-12 died of severe EAMG after the second boost (Table 2Go). Surviving mice remained moribund until termination of the experiment (data not shown). The difference in the mortality between TNFR1–/– mice treated with IL-12 and those treated with PBS were associated with dramatically enhanced levels of anti-AChR antibodies and reduced levels of anti-AChR IgG1:IgG2b ratios (Table 2Go).


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  		Table 2. Co-injection of IL-12 induces fulminating EAMG in TNFR1–/– mice and enhances anti-AChR antibodies in a dose-dependent manner
 
To further investigate the effect of IL-12, MNC derived from AChR-immunized TNFR1–/– mice were propagated after in vitro supplementation with IL-12. Addition of IL-12 reversed the suppression of T cell proliferation and IFN-{gamma} production in TNFR1–/– mice, suggesting that the decrease in IL-12 content in cultures was responsible for the decrease in proliferation and IFN-{gamma} production (Fig. 5A and BGo). Therefore, the conversion of resistance to a state of high susceptibility by addition of IL-12 indicates that IL-12 is essential in deviating immune responses and disease phenotype in TNFR1–/– mice.



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  		Fig. 5. PILN MNC obtained on day 7 p.i. with AChR in CFA were re-stimulated with AChR in the presence or absence of different concentrations of IL-12. Shown data were obtained with the dose that gives the maximal effects (100 pg/ml). Addition of 100 pg/ml of recombinant mouse IL-12 restored the suppressed T cell responses, i.e. IFN-{gamma} production (A) and proliferation (B) in TNFR1–/– mice. Symbols refer to mean values and bars to SD. **P < 0.01. Data are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the present study, we investigated the role of TNFR1 in the pathogenesis of EAMG in mice genetically deprived of TNFR1. TNFR1–/– mice mounted only low AChR-specific Th1 and humoral responses, and did not develop clinical EAMG. T cell proliferation and Th1 cell responses against whole AChR or its immunodominant {alpha}146–162 sequence were lower in TNFR1–/– compared to wild-type mice, whereas levels of IL-4 and IL-10 were concomitantly increased. Co-injection of rmIL-12 in TNFR1–/– mice restored T cell responses to AChR and susceptibility to EAMG. The results directly demonstrate that TNFR1 regulates the development of EAMG and that IL-12 may play a critical role in this process.

The TNFR1 mediates many of the pleiotropic effects of TNF-{alpha} in host defense and autoimmunity (1,2). Transgenic mice expressing human TNF transgenes developed spontaneous inflammatory arthritis (29). Non-obese diabetic (NOD) mice expressing soluble TNFR1 were protected from spontaneous or accelerated insulin-dependent diabetes mellitus (5). A clear beneficial role of anti-TNF therapy has been documented in established human rheumatoid arthritis and chronic inflammatory bowel disease (30,31). Paradoxically, chronic administration of TNF-{alpha} suppressed the function of mature T cells by altering TCR signaling in adult animals (32). TNF-{alpha} injections have either positive or negative effect on the progression to diabetes in NOD mice depending on the age when administered. This discrepancy may reflect differences in involvement of local versus systemic immune responses, duration of cytokine exposure and effector cells involved (33). Our results suggest that altered Th1/Th2 cytokine profiles, probably due to reciprocal regulation between Th1 and Th2 subsets, resulted in the resistance to EAMG in TNFR1–/– mice.

TNFR1-related signaling is not essential for lymphoid organ genesis but rather for the cellular and structural organization of B cell follicles in all secondary lymphoid tissues (34). We cannot exclude that altered formation of B cell follicles in TNFR1–/– mice may influence antibody production, although this is less likely because TNFR1–/– mice primed with KLH mounted similar levels of cellular responses and anti-KLH antibodies compared with wild-type mice, as previously shown in IFN-{gamma}–/– mice (23). Abnormalities of other antigen-presenting cells (APC) are not ruled out. For example, it has been reported that TNF-{alpha} is important in the recruitment and differentiation of dendritic cells (35,36).

There is no general consensus on the phenotype (Th1 or Th2) of pathogenic T cells in EAMG. Studies from our laboratory and others showed that IFN-{gamma} and IFN-{gamma}R were required for the development of EAMG (23,27). With respect to IL-4, conflicting results have been reported. In one study, it was demonstrated that mice lacking IL-4 still developed MG with similar morbidity and mortality as wild-type mice (37). In contrast, another study has indicated that an IL-4 deficiency facilitates development of EAMG (38). Elevated levels of circulating Th1 cells producing IFN-{gamma}, TNF-{alpha} and perforin were found in MG patients (10,39). TNF-{alpha} is mainly produced by macrophages, which are activated early after sensitization. Production of TNF-{alpha} stimulates production of cytokines, possibly involving IL-1 and IL-12, which then act in concert to drive a pathogenic Th1 response. Becher et al. showed that TNFR–IgG fusion protein inhibited IL-12 expression by systemic APC and reduced IFN-{gamma} production by T cells responding to the same APC (40). However, the regulation and function of an individual cytokine in EAMG might be more complex in the context of other cytokines within the cytokine network. Our results demonstrate that the requirement of TNFR1 is not absolute for the pathogenesis of EAMG. Supplementation of IL-12 allowed induction of EAMG in TNFR1–/– mice by reversing T cell responses to AChR, and promoting the sequential release of cytokines and anti-AChR antibodies. Therefore, IL-12-driven Th1 responses, or other components activated by IL-12, could effectively bypass TNFR1 and promote the development of EAMG.

It was reported that absence of TNFR1 enhanced the kinetics and incidence of virus-induced diabetes. In that study, it was postulated that TNFR1 exerted local inflammatory effects on establishing organ-specific autoimmune disease in a pathogen-dependent fashion (41). We have shown that NK cells, a component of innate immunity, determine the outcome of murine MG by promoting AChR-specific Th1 responses (42). Reduction in IL-12 production may impair NK cell activation and subsequently reduce Th1 activity in TNFR1–/– mice or, alternatively, it may origin from abnormal numbers of NK cells in TNFR1–/– mice (43). The possible relationship between NK cell and TNFR signaling warrants further investigations.

In summary, TNFR1 is critically involved in the development of EAMG by inhibition of autoreactive T cell responses, which in turn results in abrogation of autoantibodies important in the pathogenesis of this disease. Predictably, this phenomenon provides a therapeutic potential of anti-TNF-{alpha} therapy for MG patients. However, the absence of TNFR1 can be compensated by addition of IL-12. This scenario provides plausible mechanisms of counteraction of various cytokine pathways in MG.


   Acknowledgments
 
We thank Dr Tak W. Mak (Department of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, Canada) for kindly providing the TNFR1–/– mice; Drs M. Levi and B. Wahren for help with peptide synthesis. This study was supported by grants from the Swedish Medical Research Council (MFR), the Neurologiskt Handikappades Riksförbund (NHR), the Swedish Cancer Society, the Petrus and Augusta Hedlund foundation, the Lars Hierta foundation, the Magnus Bergwall foundation, the Åke Wiberg foundation, and the Karolinska Institutet.


   Abbreviations
 
AChR acetylcholine receptor
APC antigen-presenting cells
CFA complete Freund's adjuvant
Con A concanavalin A
EAMG experimental autoimmune myasthenia gravis
KLH keyhole limpet hemocyanin
MBP myelin basic protein
MG myasthenia gravis
MNC mononuclear cells
NOD non-obese diabetic
p.i. post primary immunization
PILN popliteal and inguinal lymph nodes
PPD purified protein derivative
TNF tumor necrosis factor
TNFR1 tumor necrosis factor receptor-1

   Notes
 
3 Present address: The Scripps Research Institute, IMM-23, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Back

Transmitting editor: L. Steinman

Received 28 April 2000, accepted 12 June 2000.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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