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*
Department of Immunology, IMM-23, The Scripps Research Institute, La Jolla, CA 92037; and
Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, produced by NK, NKT
and 
T cells (1). Abundant NO production occurs
mostly during the adaptive phase of the immune response induced by
IFN--producing T cells. NOS2/NO is now believed to affect not only
antimicrobial activities but also NK cell functions (1, 2). Less is known about a role for NOS2/NO in the generation and
regulation of adaptive immune responses, in particular adaptive
response of an autoimmune nature. Because infection is often associated
with autoimmune diseases in humans, and bacterial adjuvant is required
to induce experimental autoimmune diseases in rodents (3, 4), innate immunity with NO involvement has been suggested to
participate in the development of such diseases (5). The
contributions of NOS2/NO in autoimmune disease have been investigated
by pharmacological and genetic blockade of NOS2, although these
approaches have yielded contrasting results in some experimental
systems (reviewed in Ref. 5). Nevertheless, these studies
have suggested that NOS2/NO may play a dual role as cytotoxic or
regulatory molecule in some T cell-mediated experimental models of
autoimmune disease, including diabetes (reviewed in Ref.
6), encephalomyelitis (7, 8), uveitis
(9), and arthritis (10).
In myasthenia gravis (MG), the target Ag acetylcholine receptor
(AChR) has been defined, and the physiological consequences of AChR
loss from the cell membrane in the neuromuscular junction is fully
appreciated (11). Studies of molecular mechanisms leading
to the development of MG have been greatly aided by the availability of
an excellent animal model: experimental autoimmune MG (EAMG), induced
by immunization of rodents with the AChR and bacterial adjuvant
(12). Preclinical studies using EAMG (e.g., the
recognition of pathogenic autoantibodies) have been successfully
applied to clinical treatment (reviewed in Ref. 12). MG
and EAMG have served as a model for the elucidation of mechanisms
underlying other autoimmune disorders (11, 12). In both MG
and EAMG, autoreactive CD4+ T cells provide help
for B cells to produce anti-AChR Abs (11). Although
the Th1 cytokines IFN- and IL-12 have been demonstrated to be
important for the generation of EAMG in C57BL6 (B6) mice, the
production of both Th1 and Th2 cytokines may be required for the
development of full-blown EAMG (reviewed in Ref. 12).
According to prevailing notions, MG follows a pathogenic course in
which autoantibodies to AChR interfere with neuromuscular
transmission (11). Focal collections of lymphocytes,
including macrophages and CD4+ T cells, have been
observed in muscle of individuals with MG and EAMG (12, 13). Direct toxicity by inflammatory mediators such as NO has
also been suggested, but not directly demonstrated. Here, we
investigate the role of NOS2/NO in autoimmunity through
characterization of the immune response to AChR and the development of
clinical EAMG in NOS2-deficient (NOS2-/-) mice.
Our results reveal that NO-mediated inhibition of autoreactive T cells
diversification provides a novel pathway for counterregulation of the
autoimmune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeding pairs of B6 and NOS2-/- (14) (B6 x 129SvEv, backcrossed to B6 for 5 generations and intercrossed to obtain homozygous breeders) were obtained from Taconic Farms (Germantown, NY). B6 OT-II (OVA TCR-transgenic mice; Ref. 15), F2 (B6 x 129SvEv), and B6/SCID mice were purchased from The Jackson Laboratory (Bar Harbor, ME).
Ags synthetic peptide and Abs
AChR was purified from the electric organs of Torpedo
californica (Pacific Biomarine, Venice, CA) by affinity
chromatography on a 
-cobrotoxin-agarose resin (Sigma, St. Louis, MO)
(16). Myelin basic protein was purified from mouse brains
(17). Keyhole limpet hemocyanin (KLH) and Con A were
purchased from Sigma. The AChR peptides
146–162 (L-G-I-W-T-Y-D-G-T-K-V-S-I-S-P-E-S),
182–198 (R-G-W-K-H-W-V-Y-Y-T-C-C-P-D-T-P-Y),
111–126 (D-K-T-G-K-I-M-W-T-P-P-A-I-F-K-S),
122–138 (A-I-F-K-S-Y-C-E-I-I-V-T-H-F-P-F-D)
(18, 19, 20, 21), and a control Ku peptide
(K-A-I-V-E-L-A-F-T-Y-R-S-D-S-F-E-N) (22) were synthesized
at Swedish Institute for Infectious Disease Control, (Stockholm,
Sweden). OVA323–339
(I-S-Q-A-V-H-A-A-H-A-E-I-N-E-A-G-R) (23) was provided by
S. Webb (The Scripps Research Institute, La Jolla, CA).
Conjugated Abs to mouse DX5, TCR
, CD4, CD8, CD40, CD54, and CD106
were purchased from BD PharMingen (La Jolla, CA).
Induction and clinical evaluation of EAMG
An inoculum of 20 µg of AChR in CFA in a total volume of 100
µl was used to immunize mice s.c. along the shoulders and back. Mice
were boosted once after 1 month with 20 µg of AChR in CFA s.c. at
four sites on the shoulders and thighs. In some experiments, mice were
immunized s.c. with 20 µg of 146–162 in CFA
and boosted once as described above. Clinical manifestation of EAMG was
graded between 0 and 3 with a standard criteria (13): 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; 3, moribund, dehydrated, and paralyzed. Clinical disease has been
assessed by three individual observers in the group, and was confirmed
by injection of neostigmine bromide and atropine sulfate
(13).
Cytotoxicity assay
NK cell-mediated cytotoxicity was assayed using a standard 51Cr release assay (24).
Cell isolation, sorting
Mononuclear cell suspensions from the lymph nodes or spleen of
test mice were ground through a wire mesh. CD4+ T
cells from spleen were purified using anti-CD4 MicroBeads
(Miltenyi Biotech, Auburn, CA). Spleen DX5+
TCR- cells (NK cell) were sorted using a
FACStarPlus (BD Biosciences, Mountain View, CA).
Spleen and peritoneal macrophages from AChR and CFA-primed mice were
obtained as described (25).
Lymphocyte proliferation
Triplicate aliquots (200 µl) of mononuclear cell suspensions
containing 4 x 105 cells were applied in
96-well round-bottom microtiter plates (Nunc, Copenhagen, Denmark).
Aliquots (10 µl) of Ags (10 µg/ml unless indicated) or Con A (5
µg/ml; Sigma) were added in triplicate into appropriate wells. In
other experiments, 2 x 105 wild-type (wt)
or NOS2-deficient macrophages, treated with mitomycin-C (Sigma) at 25
µg/ml for 40 min and then washed thoroughly, were seeded with 1
x 105 CD4+ cells from
AChR-primed wt mice or OT-II mice, in 200 µl medium into the plates
in the presence of AChR 146–162 or
OVA323–339 peptide, respectively. After 4 days
of incubation, the cells were pulsed for 18 h with 10-µl
aliquots containing 1 mCi of
[3H]methylthymidine (sp. act. of 42 Ci/mmol;
Amersham, Arlington Heights, IL). The results were expressed as
stimulation index or cpm.
Cytokine ELISA and ELISPOT
IFN-, IL-12 (p40), and IL-4 production in supernatants after
48 h of culture was measured by optEIA kits (BD PharMingen). The
sensitivity was 30 pg/ml for IFN- and IL-12, and 8 pg/ml for IL-4.
IL-4 ELISPOT assay was described previously (24).
Assays of anti-AChR IgG Abs
Single Ab-producing B cell was enumerated by ELISPOT (24). Levels of anti-AChR IgG Abs were detected by ELISA (13).
Confocal microscopy for anti-AChR IgG bound to muscle AChR
Muscle sections (5 µm) from SCID mice transplanted with
AChR-primed spleen cells were prepared. The sections were incubated in
PBS for 10 min and stained for 1 h at room temperature with
FITC-labeled -bungarotoxin (Sigma) and rhodamine-labeled rabbit
anti-mouse IgG (Molecular Probes, Eugene, OR), diluted 1/1000 in
PBS containing 2% BSA.
Statistical analysis
Differences between groups were analyzed by Student’s t test. Clinical scores were analyzed using the nonparametric Mann-Whitney U test. Disease incidence was analyzed by Fisher’s exact test. The level of significance was set at p = 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the role of NOS2 in the development of the B
cell-mediated autoimmune disease EAMG, B6 wt and
NOS2-/- mice were immunized twice with AChR in
CFA. Both wt and NOS2-/- mice exhibited
progressive muscle weakness following the second immunization. However,
the percentage of diseased mice and the severity of muscle weakness
were significantly higher in NOS2-/- mice than
in wt mice (Table I
, experiments 1 and 2,
p < 0.05 for all comparisons). Both wt and
NOS2-/- mice immunized with CFA alone exhibited
no sign of muscle weakness.
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). Evidence implicating NOS/NO-mediated destruction of CNS and pancreas has been reported (reviewed in Ref. 5). However, no NOS2 expression could be detected by immunostaining in muscle tissues of NOS2-/- or wt mice immunized with AChR and CFA (data not shown), implying no detectable effect of NO on muscles in EAMG.
NOS2-deficient splenocytes have higher myasthenogenic potential
MG can be induced in SCID mice by reconstitution with lymphocytes
from MG patients (27). Similarly, AChR-primed spleen cells
can also induce MG when transferred into SCID mice (F.-D. Shi and N.
Sarvetnick, unpublished data). Following transfer, muscle
weakness becomes visible after 40 days. Anti-AChR IgG Abs can be
detected in the sera and neuromuscular junctions of recipient mice
(Fig. 1, neuromuscular junctions of two
recipients). To test the ability of NOS2-deficient splenocytes to
induce MG, we transferred AChR-sensitized spleen cells into B6/SCID
mice. Transfer of 10 x 107 spleen cells
from either NOS2-/- or wt mice caused MG at a
100% incidence of similar severity in B6/SCID mice (Table II). Transferred 5 x
107 or 2.5 x 107
spleen cells from NOS2-/- mice still induced MG
at a similar incidence and severity in B6/SCID mice as transfer of
10 x 107 spleen cells (Table II, and data
not shown). However, transferring this number of spleen cells from wt
mice induced MG in B6/SCID mice at a much lower severity (Table II).
These experiments indicate that AChR-reactive T and B cells become more
autoaggressive when primed in the absence of NOS2.
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The production of anti-AChR Abs in MG is T cell dependent.
Therefore, we measured T cell-proliferative responses to AChR and a
number of determinants on its subunit. Interestingly, T cells from
NOS2-/- mice showed significantly enhanced
proliferative responses to AChR and the immunodominant determinant
146–162 (21) compared with T
cells from wt mice (Fig. 2a).
Additionally, T cells from NOS2-/- mice, but
not from wt mice, also recognized the subdominant epitopes
111–126, 182–198 on
Torpedo AChR (19), and
122–138, a conserved determinant on both
Torpedo and mouse AChR (18, 20) (Fig. 2b).
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subunit. To investigate this
possibility, NOS2-/- and wt mice were immunized
twice with the 146–162 peptide in CFA. As
expected, by day 15 p.i., T cells from both
NOS2-/- and wt mice recognized the
146–162 peptide (albeit the levels were
relatively lower in wt mice), but T cells from both strains failed to
recognize the 111–126,
182–198, and
122–138 epitopes (data not shown).
Significantly, by day 75 p.i., T cells from
NOS2-/- mice, but not from wt mice, could
clearly recognize the subdominant epitopes
111–126, 182–198,
and 122–138 (Fig. 2c). Because
reactivity to T cell determinants other than the one used for primary
immunization was greatly enhanced by NOS2 deficiency, it appears that
these gene products limited T cell determinant spreading to the AChR.
When immunized with KLH, NOS2-/- mice also
demonstrated greater T cell proliferation to KLH than wt mice did, but
only during the first week following immunization (Fig. 2d).
This implies that NOS2 negatively regulates the immune responses to Ags
other than AChR. Diversification of autoantibody repertoire
The pathogenic anti-AChR Abs in MG and EAMG, responsible for
the functional loss of AChRs and impaired neuromuscular transmission,
are predominantly IgG Abs (11). To determine how
anti-AChR Ab responses were influenced by the absence of NOS2, we
measured the AChR-specific IgG in serum by ELISA at 15 days p.i. There
was no significant difference in the levels of anti-AChR Ab at this
time point (data not shown). However, the ELISPOT analysis revealed
that the numbers of IgG-secreting cells within lymph node were higher
in NOS2-/- mice than in wt mice (Fig. 2e), indicating that more B cells acquired the capacity to
produce autoantibodies in NOS2-/- mice. By day
65 p.i., levels of anti-AChR IgG and IgG2b were nearly 2-fold
higher in NOS2-/- mice than in wt mice (Fig. 2f). Abs to the four AChR determinants were also induced in
NOS2-/- mice, but not in wt mice (Fig. 2g). The proportion of B220-positive lymph node cells
stained for CD19 and CD21 were similar in
NOS2-/- and wt mice (data not shown), implying
that the bulk B cell population was not altered by the loss of
NOS2.
To further associate the exacerbated clinical MG in
NOS2-/- mice to determinant spreading, we
immunized wt and NOS2-/- mice twice with the
146–162 peptide in CFA. In a previous study,
this determinant failed to induce clinical EAMG because of its
inability to induce a sustained level of pathogenic autoantibody
(19). Nevertheless, the 146–162
peptide-immunized NOS2-/- mice, but not wt
mice, developed clinical MG after a prolonged period (45 days) of
immunization (Table I).
NOS2 deficiency leads to the enhanced cytokine release
The production of anti-AChR autoantibodies requires the
participation of cytokines derived from Th cells. To ascertain how NOS2
affects AChR-induced cytokine responses, we measured IFN- and IL-4
production in culture supernatants. NOS2-/-
mice produced markedly more AChR-specific IFN- than wt mice (Fig. 3a). IL-4 production was below
the ELISA detection level both in NOS2-/- and
wt mice, indicating that the immunization of AChR in CFA had mainly
directed naive T cells to differentiate into the Th1 phenotype. We also
used a sensitive ELISPOT assay to enumerate a single T cell that
produces IL-4. Fig. 3b indicates no significant increase in
numbers of IL-4-producing cells in NOS2-/- mice
compared with wt mice.
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NOS2 has been reported to regulate the innate immune response, in
part, by affecting NK cell function (1, 2). Because NK
response may be important only during initial T cell priming
(24), we compared NK cell number and function at day
15 p.i. with AChR in NOS2-/- and wt mice.
Both groups had similar NK cell numbers in the spleens (data not
shown). The capacity of NK cells to kill YAC-1 cells, proliferate, and
produce IFN- (data not shown), were also similar in
NOS2-/- and wt mice. Therefore, NK cell
functions seem unaltered in AChR-primed
NOS2-/- mice.
Enhanced ability of NOS2-deficient macrophages to stimulate autoreactive T cells
To elucidate the mechanisms underlying the enhanced T lymphocyte
responses following immunization with AChR in CFA in
NOS2-/- mice, we purified AChR-primed spleen
and peritoneal macrophages and compared their immunophenotype. The
numerical yields of macrophages were similar in
NOS2-/- and age- and gender-matched wt mice
(data not shown). The basal level of ICAM-I, VCAM, CD40, and MHC class
II in the nonimmunized wt and NOS2-/- mice did
not differ from each other (data not shown). The levels of ICAM-I and
VCAM expression were similar in immunized
NOS2-/- and wt mice (Fig. 4); however, the expression of CD40 and
MHC class II were markedly enhanced in NOS2-/-
mice (Fig. 4). Macrophages from NOS2-/- mice
also produced significantly more IL-12 (p40) than those from wt mice
(Fig. 5a). These differences
were not observed between dendritic cells or B cells from wt and
NOS2-/- mice (data not shown).
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146–162 peptide. The
proliferation of T cells after supplementation with macrophages from
NOS2-/- mice was significantly more vigorous
than that after supplementation with macrophages from wt mice (Fig. 5b). NOS2-/- macrophages were also
more efficient in presenting OVA (data not shown) and
OVA323–339 peptide to OT-II T cells than wt
macrophages (Fig. 5c).
To compare the in vivo function of macrophages from
NOS2-/- and wt mice, we performed cotransfer
experiments. Peritoneal macrophages and spleen cells from AChR-primed
NOS2-/- or wt mice were collected and
transferred to B6/SCID mice. Cotransfer of 5 x
105 macrophages from
NOS2-/- or wt mice with AChR-reactive
splenocytes did not produce significant difference in disease
development (Table III). Interestingly,
following the transfer of macrophages from
NOS2-/- mice mixed with AChR-reactive
splenocytes, five of six recipients exhibited MG. In contrast,
following transfer of macrophages from wt mice with AChR-reactive T
cells, only two of seven mice developed muscle weakness (Table III).
Previous studies demonstrated that cotransfer of the same number of
purified DX5+ TCR-
NK cells from either NOS2-/- or wt mice results
in similar disease incidence and severity (data not shown). Therefore,
NOS2-deficient macrophages, and not NK cells, appear to elicit highly
aggressive autoreactive T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the binding of autoantibodies to muscle AChR causes a
progressive weakening of muscles in MG, autoreactive T cells provide
the essential interface with B cells that leads to autoantibody
production. The current study provides evidence that NOS2/NO is one of
the likely factors that limit T cell determinant spreading during the
induction phase of EAMG. T cell reactivity is skewed to the AChR
subunit in both MG and EAMG (18). In B6 mice,
146–162-reactive CD4+ T
cells interact with AChR-specific B cells to produce pathogenic
anti-AChR Abs in vitro and in vivo (21), whereas
immunization with this peptide fails to break self-tolerance (18, 21). Our study demonstrated that immunization of
NOS2-/- mice with
146–162 induced vigorous T cell responses not
only to the 146–162 peptide, but also to the
111–126, 122–138,
and 182–198 peptides. The diversified AChR
autoantibody repertoire, as a consequence of T cell determinant
spreading, is probably responsible for the profound clinical MG in the
NOS2-/- mice.
The close relationship between macrophages and NO suggests that macrophages, as opposed to dendritic cells or B cells, may serve as a primary inducer of T cell determinant spreading in the NOS2-/- mice. In fact, in the absence of NOS2, macrophages exhibit enhanced expression of Ag presentation molecules CD40 and class II, produce more IL-12, and exhibit enhanced ability to activate AChR-reactive T cells in vitro and in vivo. The enhanced capacity in Ag presentation by NOS2-deficient macrophages is also observed with other Ags, as indicated by OVA peptide presentation to OVA-specific T cells. Although the molecular mechanisms require further investigation, the present study revealed that the absence of NOS2 could lead to a lowered threshold for maximal macrophage activation. Because NOS2-/- mice have poor ability to clear pathogens (1, 2), the enhanced immune responses involving T and B cells are required in controlling infection. This process may consequently result in the expansion of autoreactive T cells.
In mice infected with intracellular bacteria or viruses, several types
of interactions between NOS2-derived NO and T cells have been reported,
including inhibition of T cell proliferation and cytokine induction.
The mechanisms underlying these effects are not clear. Involvement of
NK cells (1) or macrophages (28), or
direct activity of NO on T cells (29), are the recently
suggested explanations. In the present study, NK cell functions, e.g.,
cytotoxic activity and production of IFN-, are not altered in the
absence of NOS2 in the current model. Although NK cells are involved in
initial T cell priming in response to AChR (24, 30), the
present results signify that NK cells were not directly involved in the
ability of NOS2 to control autoimmune responses to AChR.
The suppressive effects on autoreactive T cells by NOS2 suggest a
functional similarity with two other molecules: CTLA4 and TGF-
(31, 32), both of which serve as negative
regulators in EAMG (24, 32) and other autoimmune diseases
(31). NOS2 exerts its effect early when a host receives
infectious signals before T cell activation. CTLA4 controls T cells
during TCR ligation, whereas TGF- regulates committed T cells and
inhibits T cell activation. Interestingly, the control of T cell
response by these molecules all seems to be achieved through APCs
(Refs. 32, 33 , and this study), and they may act
synergistically (34). Thus, the immune system has evolved
a serial, multistep means to silence autoreactive T cell proliferation
to counterregulate other positive signals to avoid the development of
autoimmune diseases.
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Acknowledgments |
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Footnotes |
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2 This is manuscript 13572-IMM from The Scripps Research Institute.
3 Address correspondence and reprint requests to Dr. Nora Sarvetnick, Department of Immunology, IMM-23, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: noras{at}scripps.edu
4 Abbreviations used in this paper: NOS2, type 2 NO synthase; AChR, acetylcholine receptor; MG, myasthenia gravis; EAMG, experimental autoimmune MG; B6, C57BL6; wt, wild type; KLH, keyhole limpet hemocyanin; p.i., postimmunization.
Received for publication March 19, 2001. Accepted for publication July 3, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by NK cell. J. Immunol. 165:3099.-cell dysfunction and damage in IDDM. Diabetes Metab. Rev. 13:293.[Medline]
, nitric oxide, and apoptosis. J. Exp. Med. 189:219.- and -chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76:34.[Medline]
146–162 of acetylcholine receptor. Clin. Immunol. Immunopathol. 66:230.[Medline]
146–162 of Torpedo californica nicotinic acetylcholine receptor. J. Immunol. 157:3192.[Abstract]
chain of Torpedo californica acetylcholine receptor. Eur. J. Immunol. 17:1697.[Medline]
subunit is exposed at neuromuscular junction and induces experimental autoimmune myasthenia gravis, T-cell immunity, and modulating autoantibodies. Proc. Natl. Acad. Sci. USA 82:8805.1 alters APC preference, polarizing islet antigen responses toward a Th2 phenotype. Immunity 8:601.[Medline]
(TGF-) production by murine CD4+ T cells. J. Exp. Med. 18:1894.This article has been cited by other articles:
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) and
NOS2-/- mice (
) were collected 15 days after
AChR-immunized mice (n = 4) and exposed to AChR and
several epitopes on AChR 
