Mechanisms of Nasal Tolerance Induction in Experimental Autoimmune Myasthenia Gravis: Identification of Regulatory Cells

Mice

Wild-type (wt) B6 mice (CD4⁺8⁺), CD4 mutant mice (CD4^−/−), and CD8 mutant mice (CD8^−/−) were bred in the animal facilities of the Microbiology and Tumor Biology Center, Karolinska Institutet. The CD4^−/− and CD8^−/− mice were backcrossed ≥10 times to a B6 background. All of the mice used were female and were 8–10 wk of age at the beginning of the experiments. Animal experimental procedures were in compliance with institutional guidelines.

Antigens

Torpedo AChR was purified from the electric organs of Torpedo californica (Pacific Biomarine, Venice, CA) by affinity chromatography on an α-cobrotoxin-agarose resin (Sigma, St. Louis, MO) as described previously (18). The isolated product was pure as judged by SDS-PAGE analysis. The purified Torpedo AChR was used to induce EAMG and for stimulation in in vitro cultures. Muscle AChR extract from B6 mice was prepared as described previously (18) and used as Ag for the detection of anti-mouse AChR Abs. Myelin basic protein (MBP) used as control Ag was purified from normal mouse brains (19).

Nasal tolerance induction

A modified schedule described for nasal tolerance induction in rats was used (15). Briefly, each mouse was given a total amount of 150 μg of Torpedo AChR in 30 μl of PBS into each nostril. Control mice received PBS only. The administrations were performed daily for 10 consecutive days before immunization.

Induction and clinical evaluation of EAMG

Mice were immunized s.c. with 40 μg of AChR in CFA in a total volume of 100 μl and boosted twice on days 25 and 55 after primary immunization with 40 μg of AChR in CFA s.c. The mice were scored every other day after the second immunization for signs of muscle weakness that were characteristic of EAMG. The disease symptoms were graded between 0 and 3 (20): 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 an injection of neostigmine bromide and atropine sulfate (20).

RIA for muscle AChR content

Aliquots (2 pM) of ¹²⁵I-α bungarotoxin (α-BGT) (Amersham, Arlington Heights, IL) -labeled, Triton X-100-solubilized mouse muscle extract were mixed with standard pooled mouse anti-AChR antiserum in triplicate. After incubation, rabbit anti-mouse Ig (Dakopatts, Copenhagen, Denmark) was added. The precipitates were counted in a Packard gamma-counter (Meriden, CT). The percentage of loss of muscle AChR in test mouse muscle was calculated as described previously (21).

Measurements of serum anti-AChR Ab concentration, affinity, and IgG isotype

The serum Ab levels were measured by RIA (18). Briefly, 1 nM of muscle AChR was incubated with 2 nM α-BGT (Amersham). A total of 1 μl of serum was added to 1 ml of labeled muscle AChR, followed by rabbit anti-mouse Ig (Dakopatts). The samples were centrifuged, washed, and counted in a gamma-counter. The AChR precipitated minus the background value permitted calculation of the titer in moles of toxin-binding sites bound per liter of serum. After predetermination and adjustment of the anti-AChR IgG Ab levels, the relative affinity of anti-AChR IgG Abs in serum was determined by ELISA using thiocyanate (Sigma) elution (22). Isotypes of anti-AChR IgG Ab were detected using rabbit anti-mouse IgG1, IgG2a, or IgG2b (Dakopatts). Abs were detected by ELISA as described previously (23).

Culture medium

Cells were suspended in DMEM (Life Technologies, Paisley, U.K.) supplemented with 1% (v/v) minimum essential medium (Life Technologies), 2 mM glutamine (Flow Laboratories, Irvine, U.K.), 50 IU/ml penicillin, and 50 mg/ml streptomycin and 10% (v/v) FCS (both from Life Technologies). Supernatants to be assayed for TGF-β1 content were generated in Aim V serum-free medium (Life Technologies, Grand Island, NY).

Lymphocyte proliferation responses

Groups of mice were immunized s.c. with 40 μg of AChR in CFA at hindfoot and thigh regions and killed 14 days postimmunization (p.i.); mononuclear cell (MNC) suspensions from the popliteal and inguinal lymph nodes were prepared as described previously (23). Triplicate aliquots (200 μl) of MNC suspensions containing 4 × 10⁵ cells were applied in 96-well, round-bottom microtiter plates (Nunc, Copenhagen, Denmark). Aliquots (10 μl) of either AChR, MBP, or Con A (Sigma) were added into appropriate wells at a final concentration of 10 μg/ml (AChR or MBP) or 5 μg/ml (Con A). After 4 days of incubation, the cells were pulsed for 18 h with 10-μl aliquots containing 1 mCi of [³H]methylthymidine (specific activity of 42 Ci/mmol; Amersham). Cells were harvested onto glass fiber filters, and thymidine incorporation was measured.

Cytokine ELISA

Single-cell suspensions of draining LN cells from AChR-primed mice were cultured as described above. The supernatants were collected 48 h after in vitro boosting. IFN-γ and IL-4 production in culture supernatants were measured by optEIA kits (PharMingen, San Diego, CA). Biologically active TGF-β1 was measured with an ELISA kit (Promega, Madison, WI). The sensitivity of these ELISA assays was ∼50 pg/ml for IFN-γ and IL-4 and 30 pg/ml for TGF-β1.

In vivo anti-TGF-β treatment and in vitro suppression assay

The anti-TGF-β mAb 11D.16 (mouse IgG1) specific for TGF-β1, -2, and -3 (24) and 1410KG7 (mouse IgG1) isotype control mAb (25) were used to neutralize TGF-β in vivo. Group of mice were administered i.p. inoculations of 1 mg of anti-TGF-β in a total volume of 200 μl in PBS or control mAb at the time of immunization followed by 500-μg weekly administrations until the termination of experiments. In vitro suppressor cell activities were assayed as described previously (5).

Enumeration of AChR-reactive IFN-γ-secreting cells

A solid-phase enzyme-linked immunospot assay was used (26). Nitrocellulose-bottom microtiter plates were coated with 100 μl of IFN-γ capture Ab (Innogenetics, Zwijnaarde, Belgium) at 15 μg/ml. MNCs were cultured as described above. Wells were incubated with or without 4 ng/ml mouse rIL-2 (PharMingen). After 48 h of culture, secreted and bound IFN-γ was visualized by a sequential application of biotinylated detector Ab IFN-γ (Innogenetics) and avidin-biotin complex (Dakopatts). After peroxidase staining, the red-brown immunospots corresponding to the cells that had secreted IFN-γ were enumerated in a dissection microscope.

Statistical analysis

Differences between groups were evaluated by ANOVA. Differences between the groups with respect to disease incidence were analyzed by Fisher’s exact test.

Nasal AChR administration prevents development of clinical EAMG and reduces muscle AChR loss in wt and CD8^−/− mice

To establish a protocol for nasal tolerization with AChR in B6 mice, different dosages of AChR were administered nasally to mice that were subsequently immunized s.c. with AChR in CFA and scored for signs of myasthenia gravis (MG). Nasal administration of a total amount of 150, 300, or 600 μg of AChR per mouse divided in 10 consecutive daily administrations was equally effective in preventing the development of EAMG (data not shown). A total of 150 μg of AChR per mouse was adopted as a standard dose and used throughout the present study.

To study the role of CD8⁺ T cells in the generation of nasal tolerance, wt and CD8^−/− mice were administered AChR nasally and subsequently immunized with AChR in CFA three times. The mice were monitored for the muscle weakness characteristic of EAMG. Of the wt mice, 26 of 31 developed muscle weakness, whereas only 6 of 34 wt mice receiving AChR nasally before immunization with AChR developed muscle weakness (p < 0.01). The onset of disease was delayed in the group of wt mice receiving AChR nasally (Table I⇓). The disease incidence was relatively lower in CD8^−/− mice than in wt mice. In total, 18 of 35 AChR-immunized CD8^−/− mice developed muscle weakness. In contrast, only 7 of 34 CD8^−/− mice receiving AChR nasally before immunization with AChR developed muscle weakness (p < 0.01). The onset of disease was also delayed in the group of CD8^−/− mice receiving AChR nasally (Table I⇓). The mean values of muscle AChR loss in control and AChR-treated wt mice were 68.8 ± 15.5% and 21.8 ± 3.2%, respectively (p < 0.05). The mean values of muscle AChR loss in the control and AChR-treated CD8^−/− mice were 42.4 ± 7.2% and 25.5 ± 6.8%, respectively (p < 0.05). Thus, nasal tolerance to AChR can still be effectively induced in the absence of CD8⁺ T cells.

Nasal AChR administration alters anti-AChR IgG Ab isotype repertoire and affinity

The anti-AChR Abs in MG and EAMG consist mainly of IgG Abs of all subtypes (27, 28). These Abs are responsible for the functional loss of AChR in the neuromuscular junctions (27). Anti-AChR Ab levels were not significantly different in wt and CD8^−/− mice, irrespective of nasal AChR administration before immunization (Fig. 1⇓). However, the affinity of the anti-AChR IgG Abs was lower in both wt and CD8^−/− mice that had received nasal administrations of AChR (Fig. 1⇓). In particular, this was the case for the IgG2a and IgG2b isotypes, whereas affinity levels of IgG1 isotypes were largely unaltered (Fig. 2⇓).

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FIGURE 1.

Anti-AChR IgG Ab responses on day 90 p.i. Serum anti-AChR Ab concentrations were measured by RIA and expressed as moles of α-BGT binding sites per liter of serum (upper panel). The anti-AChR IgG Ab affinity index was measured by potassium thiocyanate ELISA (lower panel). Results are expressed as mean values ± SD. The samples tested in this figure are from the mice indicated in Table I⇑. For mice receiving PBS or AChR, n = 10. For mice receiving AChR in conjunction with treatment with anti-TGF-β or control mAb, n = 4. ∗, p < 0.05; #, p < 0.01. Comparisons were between tolerized and nontolerized PBS-treated mice, respectively.

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FIGURE 2.

Anti-AChR IgG Ab isotypes measured by ELISA. The serum samples used were the same as those used in Fig. 1⇑. No differences in levels of anti-AChR IgG Ab isotypes in tolerized mice and in tolerized mice treated with isotype control Ab were observed (data not shown). Results are expressed as mean values ± SD. ∗, p < 0.05; #, p < 0.01. Comparisons were made between tolerized and nontolerized mice, respectively.

Nasal AChR administration suppresses AChR-reactive lymphocyte proliferation

Proliferative responses to AChR were suppressed in wt as well as in CD8^−/− mice receiving AChR nasally (Fig. 3⇓). The suppression was Ag-specific, because T cells from AChR-treated wt and CD8^−/− mice as well as control wt and CD8^−/− mice proliferated at similar levels in response to the control Ag MBP and to Con A (Fig. 3⇓).

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FIGURE 3.

Effects of nasal administration of AChR on in vitro proliferative responses to AChR and effects of neutralizing TGF-β. Mice were first treated with AChR or PBS nasally and subsequently immunized with AChR in CFA. A fraction of the mice receiving AChR nasally were given 1 mg of anti-TGF-β or isotype control Ab at the time of immunization followed by 500 μg of mAbs weekly until the termination of experiments. Mice were killed at 14 days p.i., draining LN cells were isolated and cultured with different Ags, and responses to stimulation were measured. ∗, p < 0.05; #, p < 0.01. Comparisons were made between tolerized and nontolerized mice, respectively. Results represent one of two independent experiments and are expressed as mean values ± SD. There were four mice in each group.

Nasal AChR administration alters cytokine responses

Upon activation, Th cells differentiate into Th1, Th2, and Th3 functional subgroups that are characterized by their ability to produce IFN-γ, IL-4, and TGF-β, respectively (29). The production of anti-AChR Abs in EAMG and MG is regulated by these cytokines (27). We have shown that the suppression of EAMG in Lewis rats by nasal administration of AChR correlates with decreased numbers of IFN-γ and increased numbers of TGF-β mRNA-expressing cells (30, 31). Thus, the altered anti-AChR IgG Ab repertoire and affinity in the tolerized mice should theoretically be determined by the altered cytokine profile in these mice. To demonstrate this, we determined IFN-γ, IL-4, and TGF-β1 production in the culture supernatants of draining LN cells from the tolerized and nontolerized wt and CD8^−/− mice, respectively. On day 14 p.i., IFN-γ production was reduced both in wt and CD8^−/− mice receiving AChR nasally compared with control wt and CD8^−/− mice, respectively (p < 0.01 for both comparisons, Table II⇓). IL-4 levels were unaltered in the tolerized mice. In contrast, wt and CD8^−/− mice that had received AChR nasally had significantly elevated levels of TGF-β compared with control mice (p < 0.05 for both comparisons). A similar pattern of cytokine profile was also observed on day 90 p.i. (Table II⇓). Thus, the current study demonstrates that the tolerance induction in B6 mice was associated with decreased IFN-γ production and increased TGF-β production.

Effects of neutralizing TGF-β in nasal tolerance induction to AChR

To determine whether the tolerance induced by nasal administration of AChR can in fact be attributed to the enhanced production of TGF-β, we injected mice with anti-TGF-β and isotype control mAbs at the time of immunization. Neutralization of TGF-β abrogated the tolerance effects induced by nasal administration of AChR in both B6 and CD8^−/− mice (Table I⇑). Compared with corresponding control mice, the tolerized wt and CD8^−/− mice treated with anti-TGF-β but not isotype control Ab had similar anti-AChR IgG Ab affinity (Fig. 1⇑) as well as similar levels anti-AChR IgG2a and IgG2b isotypes (Fig. 2⇑). Similarly, the suppression of AChR-specific proliferation and IFN-γ production were nearly completely reversed by anti-TGF-β Ab treatment (Table II⇑, Fig. 3⇑).

Splenic MNCs from wt and CD8^−/−, but not CD4^−/− mice, that have received AChR nasally suppress AChR primed lymphocyte proliferation

To further investigate the role of CD4⁺ vs CD8⁺ T cells in the generation of active suppression after nasal administration of AChR, splenic MNCs were obtained from wt, CD8^−/−, and CD4^−/− mice that had received AChR nasally before immunization. A suppression of AChR-induced proliferation was observed in the cocultures with spleen MNCs derived from wt and CD8^−/− mice (p < 0.05, for both comparisons), but not CD4^−/− mice (Table III⇓). This suppressive effect could be blocked by anti-TGF-β Ab treatment in vivo and in vitro (Table III⇓). In contrast, CD4^−/− mice that had received AChR nasally before immunization with AChR had similar levels of lymphocyte proliferative responses and IFN-γ, IL-4, and TGF-β1 production compared with control CD4^−/− mice (data not shown). Thus, these data suggest that CD4⁺ Th3 TGF-β-producing cells, even in the absence of CD8⁺ T cells, generate active suppression upon nasal administration of AChR.

Suppression of IFN-γ-secreting cells in wt as well as in CD8^−/− mice receiving AChR cannot be reversed by addition of IL-2

Nasal administration of AChR suppressed Th1 cytokine IFN-γ production. To further determine whether this T cell subset was selectively anergized, we enumerated the IFN-γ-secreting cells among MNCs in the presence and absence of IL-2 in the cultures. Consistent with cytokine ELISA data, the numbers of AChR-reactive IFN-γ-secreting cells were lower in wt and CD8^−/− mice that had received AChR nasally (Fig. 4⇓). IL-2 preincubation increased the numbers of IFN-γ-secreting cells in control mice; however, it did not increase the numbers of IFN-γ-secreting cells in the tolerized mice. Thus, the present findings do not support induction of anergy as a possible explanation for the observed results, although this possibility cannot be formally excluded in our system.

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FIGURE 4.

Numbers of IFN-γ-secreting cells and effects of preincubation with IL-2. Mice were first treated with AChR or PBS nasally and subsequently immunized with AChR in CFA. Mice were killed at 14 days p.i., and LN MNCs were harvested and cultured in the presence or absence of AChR (10 μg/ml). rIL-2 (4 ng/ml) was applied to every second well in parallel. ∗, p < 0.05. Comparisons were made between tolerized and nontolerized mice, respectively. Results represent one of two independent experiments and are expressed as mean values ± SD. There were four mice in each group. The numbers of IFN-γ-secreting cells in response to control Ag MBP or no Ag at all were <3.