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*
Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden;
Division of Neurology, Huddinge University Hospital, Karolinska Institutet, Stockholm, Sweden; and
Section of Cytokine Biology, Biomedical Primate Research Center, Rijswijk, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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responses, whereas levels
of IL-4 were unaltered. Splenocytes from AChR-pretreated wt and
CD8-/- mice, but not from CD4-/- mice,
suppressed AChR-specific lymphocyte proliferation. This suppression
could be blocked by Abs against TGF-ß. Thus, our results demonstrate
that the suppression induced in the present model is independent of
CD8+ T cells and suggest the involvement of Ag-specific
CD4+ Th3 cells producing TGF-ß. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Other forms of mucosal tolerance have been investigated recently, in particular the administration of Ag via the nasal route. This route appears equally efficient and, in some instances, even more effective than oral tolerance induction in suppressing autoimmune diseases in animal models (10, 11, 12, 13, 14). We have studied nasal tolerance induction as a strategy to prevent EAMG in Lewis rats and found that acetylcholine receptor (AChR) administration by the nasal route, although using only 1/500 of the dose of AChR needed for oral tolerance induction, is still as effective as oral administration (15, 16, 17). However, the mechanisms underlying nasal tolerance remain largely unexplored.
In the present study, we investigate the effects of nasal administration of AChR on the development of EAMG in C57BL/6 (B6) mice. We demonstrate tolerance induction against EAMG in these mice and analyze the mechanisms behind this effect.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 
-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- bungarotoxin (-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 -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-ß1 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 x 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- and IL-4 production in
culture supernatants were measured by optEIA kits (PharMingen, San
Diego, CA). Biologically active TGF-ß1 was measured with an ELISA kit
(Promega, Madison, WI). The sensitivity of these ELISA assays was 
50
pg/ml for IFN- and IL-4 and 30 pg/ml for TGF-ß1.
In vivo anti-TGF-ß treatment and in vitro suppression assay
The anti-TGF-ß mAb 11D.16 (mouse IgG1) specific for TGF-ß1, -2, and -3 (24) and 1410KG7 (mouse IgG1) isotype control mAb (25) were used to neutralize TGF-ß in vivo. Group of mice were administered i.p. inoculations of 1 mg of anti-TGF-ß 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--secreting cells
A solid-phase enzyme-linked immunospot assay was used (26).
Nitrocellulose-bottom microtiter plates were coated with 100 µl of
IFN- 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- was visualized by a sequential application
of biotinylated detector Ab IFN- (Innogenetics) and avidin-biotin
complex (Dakopatts). After peroxidase staining, the red-brown
immunospots corresponding to the cells that had secreted IFN- 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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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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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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Upon activation, Th cells differentiate into Th1, Th2, and Th3
functional subgroups that are characterized by their ability to produce
IFN-, IL-4, and TGF-ß, 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- and
increased numbers of TGF-ß 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-, IL-4, and
TGF-ß1 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- 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-ß
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- production and increased TGF-ß production.
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To determine whether the tolerance induced by nasal administration
of AChR can in fact be attributed to the enhanced production of
TGF-ß, we injected mice with anti-TGF-ß and isotype control
mAbs at the time of immunization. Neutralization of TGF-ß 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-ß 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- production were
nearly completely reversed by anti-TGF-ß 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-ß 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-, IL-4, and TGF-ß1 production
compared with control CD4-/- mice (data not shown). Thus,
these data suggest that CD4+ Th3 TGF-ß-producing cells,
even in the absence of CD8+ T cells, generate active
suppression upon nasal administration of AChR.
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-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-
production. To further determine whether this T cell subset was
selectively anergized, we enumerated the IFN--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--secreting cells were lower in wt and CD8-/- mice
that had received AChR nasally (Fig. 4).
IL-2 preincubation increased the numbers of IFN--secreting cells in
control mice; however, it did not increase the numbers of
IFN--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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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responses, whereas
levels of IL-4 were unaltered. Splenocytes from wt as well as from
CD8-/- mice, but not from CD4-/- mice,
suppressed AChR-primed lymphocyte proliferation. This suppression could
be blocked by Abs against TGF-ß. Thus, the present results extend our
previous observations in rats (15, 16, 17) and suggest that active
suppression by the sensitization of CD4+ Th3 cells
producing TGF-ß plays a major role in the generation of nasal
tolerance. Nasal tolerance induction is sometimes associated with immune deviation from a Th1 to a Th2 phenotype in T cell-mediated autoimmune diseases (12) and is associated with AChR peptides in at least one study of nasal tolerance induction against EAMG (32). However, in the present study, Th2 cytokine IL-4 responses were neither enhanced nor suppressed by the nasal administration of AChR, suggesting that the suppression of Th1 cytokines is a result of up-regulation of Th3 cytokines rather than Th2 cytokines in this system. Previous studies on the tolerance induction by nasal administration of AChR in the rat model of EAMG have indicated that the suppression of disease development was likely due to TGF-ß-secreting cells (30, 31). The present study provides evidence in support of active suppression by the sensitization of Th3 cells producing TGF-ß in nasal tolerance induction. TGF-ß production was augmented, and the effects of in vitro suppression of AChR-primed lymphocyte proliferation could be blocked by anti-TGF-ß Abs.
The role of CD8+ T cells in the pathogenesis of EAMG has recently been investigated. Shenoy et al. (33) reported that ß2-microglobulin-/- mice with deficient MHC class I expression and a reduced number of CD8+ cells showed a more severe EAMG than corresponding wt mice. In contrast, Zhang et al. (26, 34) reported that the depletion of CD8+ T cells by either Abs or gene targeting reduces the severity of EAMG in Lewis rats and in B6 mice. Differences in in vivo systems and in the antigenic properties of AChR preparations may account for the discrepancies observed in these studies. The present results indicate that disease development was relatively mild in CD8-/- mice compared with wt mice. In part, this could be explained by the ability of CD8+ T cells to help autoreactive B cells by secreting an array of cytokines and by expressing the CD40 ligand (35). However, it is unlikely that CD8+ T cells function as effector cells in EAMG pathogenesis. B cell-deficient mice have normal CD8+ T cell cytotoxic functions but remain completely free from EAMG because no anti-AChR Ab is produced (36, 37). The numbers of infiltrating CD8+ T cells in the neuromuscular junctions are very sparse in patients with MG as well as in animals with EAMG (37, 38).
There is much controversy regarding whether CD8+ T cells actively participate in the induction of oral tolerance. CD8+ T cells were identified as "the suppressor cells" in early studies (39, 40). Recent studies have shown that CD8+ T cells alone are not sufficient to mediate the active suppression in oral tolerance induced in the T cell-mediated experimental autoimmune encephalomyelitis and experimental autoimmune uveitis (9, 41). The mechanisms of nasal tolerance induction have been suggested to be analogous to those of oral tolerance (42). However, there are a number of structural differences between the upper respiratory tract and gastrointestinal mucosa. For example, the ratio of CD4+ vs CD8+ cells, cytokine milieu, and Ag presentation and costimulation requirements differ (43, 44). These differences suggest that the mechanisms operating in the generation of peripheral tolerance at these two different mucosal surfaces might differ.
Our study has shown that nasal tolerance can be readily established in CD8-/- mice. Because both CD4+ and CD8+ T cells contribute to the production of TGF-ß (24, 29), the establishment of tolerance against EAMG in CD8-/- mice suggested that nasal administration of AChR before immunization regulates the CD4+ Th3 subset, to compensate for the absence of CD8+ T cells, and mediates nasal tolerance to AChR. Therefore, the cellular requirements for CD4+ and CD8+ T cells in the generation of EAMG and the generation of nasal tolerance are distinctly different.
Under certain circumstances, when the suppression of autoaggressive T
cells does not always parallel the suppression of autoreactive B cells,
sensitization of Th2 cells and augmented Ab production can occur (45, 46), which may be detrimental. This could be one of the confronting
problems in applying mucosal tolerance induction in the treatment of
MG. In the present study, in accordance with the differential
regulation of Th subsets, IgG2a and IgG2b Abs and affinity were
selectively reduced, whereas the total anti-AChR Ab levels were
similarly high in the tolerized mice compared with the control mice.
Thus, nasal tolerance does not appear to significantly alter the
production of anti-AChR Abs, but rather changes their isotype
repertoire. At present, the mechanisms underlying this consistent
observation are not clear (15, 23). Nasal tolerance induction in EAMG
may be associated with the suppression of certain pathogenic Ab
subtypes of high affinity to AChR. IFN--dependent anti-AChR
IgG2a and IgG2b subtypes were suggested to be pathogenic in B6 mice in
several recent studies (47, 48, 49, 50). In contrast, IL-4 is not required for
the development of EAMG in B6 mice (51). Thus, the suppression of
IFN- responses and of IgG2a and IgG2b and other productions of
pathogenic Ab subtypes may, in part, be responsible for the tolerance
induction achieved by nasal administration of AChR in the EAMG model.
Although the current therapy of MG with immunosuppressive drugs is reasonably effective, such treatment must be continued indefinitely and may result in global suppression of the immune system, with increased risks of infection and neoplasia (25). A clinical trial of Ag-specific therapy for MG has not been initiated, but is currently the subject of intensive investigations. The present study provides insight into the mechanisms of nasal tolerance induction and should facilitate the design of an ideal treatment of MG.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Fu-Dong Shi, Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 77, Stockholm, Sweden. E-mail address:
3 Current address: Division of Immunology, Department of Pathology, New York University Medical Center, 555 First Avenue, New York, NY 10016.
4 Abbreviations used in this paper: EAMG, experimental autoimmune myasthenia gravis; AChR, acetylcholine receptor; B6, C57BL/6; wt, wild type; MBP, myelin basic protein; -BGT, 125I- bungarotoxin; p.i., postimmunization; MNC, mononuclear cell; LN, lymph node; MG, myasthenia gravis.
Received for publication November 2, 1998. Accepted for publication February 22, 1999.
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Z.-m. Chen, M. J. O'Shaughnessy, I. Gramaglia, A. Panoskaltsis-Mortari, W. J. Murphy, S. Narula, M. G. Roncarolo, and B. R. Blazar IL-10 and TGF-{beta} induce alloreactive CD4+CD25- T cells to acquire regulatory cell function Blood, June 15, 2003; 101(12): 5076 - 5083. [Abstract] [Full Text] [PDF]
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