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Division of Life Sciences, King’s College London, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by lymph node cells. Suppression of Th1-type cytokines was
also observed in SNF1 mice that were nasally administered
with H471 before intradermal injection with the peptide. Finally,
chronic nasal instillation of mice with the H471 peptide not only
suppressed the development of autoantibodies, but also altered the
severity of glomerulonephritis in lupus-prone SNF1
mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The efficacy of mucosal tolerance induction can be dependent on a
number of factors such as instilled Ag dose and solubility. It has been
shown that high doses of self Ag are able to delete Ag-specific
autoreactive T cells (7) or conversely, to break tolerance
that has been observed at lower doses (8). Several studies
of mucosal tolerance in animal models have demonstrated that this
hyporesponsiveness is T cell mediated and typically results in a switch
from an inflammatory Th1 T cell subset response (IFN-, IL-2) to a T
cell response associated with the production of suppressive or
noninflammatory cytokines (9, 10). T cell subsets that
have been associated with mucosal immune hyporesponsiveness include the
Th2 (IL-4, IL-10) (11), Th3 (TGF-
) (12), T
regulatory 1 (IL-10, TGF-) (13), and Th0 (IL-4, IL-10,
IFN-) cells (14) and CD4+
CD25+ T regulatory (T
reg)4 cells
(15). Ag-specific serum Ab levels may remain relatively
high after tolerization, but typically there is an IgG subclass switch
to an effector isoform less likely to promote an inflammatory response
(1). Finally, in animal models of mucosal tolerance in
which the subjects are typically tolerized before disease induction,
there is usually a significant reduction in the quality and measure of
pathology. This advantage of preimmunization tolerance is, however, not
applicable to the clinical situation in which researchers have
attempted, to date with mixed results, to tolerize patients who present
with chronic autoimmune disease, and this remains a significant
challenge.
Mononucleosomes, single units of the nucleosome, consist of 147-bp dsDNA wrapped around a core octamer of histone proteins: two of each histones H2a, H2b, H3, and H4 (16). Interest in the nucleosome as a notable autoantigen of systemic lupus erythematosus (SLE) has been brought about by a series of individuals with convergent observations, which suggest that the nucleosome is an important adjuvant in the early development of pathogenic anti-DNA autoantibody response associated with this syndrome (17, 18). Nucleosomes are major components of apoptotic bodies, and defective clearance mechanisms (19, 20) may result in their presence as autoantigens in lupus models several weeks before the appearance of anti-DNA Abs (18). Kaliyaperumal et al. (21) have demonstrated that a panel of pathogenic autoantibody-inducing Th clones derived from lupus-prone mice (22) can be induced to proliferate upon presentation of histone-derived peptides. Furthermore, upon i.p. challenge into young prenephritic female SNF1 mice, these peptides can induce and promote the production of pathogenic anti-DNA Abs and subsequently accelerate the onset of severe nephritis. In this study, we have synthesized one of the histone peptides identified by Kaliyaperumal et al., which we term H471 (derived from histone H4, position 71–93). In addition, mononucleosomes were purified from chicken RBCs. We have used the H471 peptide to induce tolerance to itself and mononucleosomes by nasal instillation of lupus-prone (NZB x SWR)F1 (SNF1) (H-2dxq) and nonautoimmune, but H-2-identical (BALB/c x SWR)F1 (BSF1) mice with the peptide. We report that tolerance was induced in mice nasally dosed with the H471 peptide. Nasal tolerance to the H471 peptide can be achieved even after Ag priming. Furthermore, nasal instillation of mice with the H471 peptide can suppress the immune response to mononucleosomes. Chronic nasal instillation of lupus-prone SNF1 mice with H471 can suppress the development of autoantibodies and reduce the incidence of severe glomerulonephritis. These findings demonstrate a clear potential for immune intervention in SLE with a peptide expressing a T cell epitope from a self protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SNF1 and BSF1 mice were bred and maintained in the animal facility at King’s College London. SNF1 mice used in the long-term study were purchased from Harlan (Bicester, Oxfordshire, U.K.) and maintained in our animal facility. Female mice between 8 and 10 wk old were used in all experiments, unless stated otherwise.
Antigens
Synthetic peptide H471 based on the amino acid sequences of histone protein H4 at positions 71–93 (TYTEHAKRKTVTAMDVVYALKRQ) was synthesized as a peptide amide using a Rink linker 4' methylbenzhydrylamine resin and Fmoc-based solid phase peptide synthesis automated by a PS3 peptide synthesizer (Anachem, Luton, U.K.). The purity of the H471 peptide was shown to be 96% by reverse-phase HPLC, and its composition was confirmed by mass spectrometry. Control peptides, OVA323–339 and H416 (positions 16–39 of H4), were synthesized in the same way, and shown to be >95% pure. Individual core histone proteins were purchased from Boehringer (Sussex, U.K.), and the manufacturer guaranteed 99% purity. Mononucleosomes were prepared using a protocol kindly given by K. E. van Holde (Oregon State University, Corvallis, OR). The protocol has also been published previously (23) and briefly, chicken RBC lysate was treated with micrococcal nuclease (45 IU ml-1; Sigma-Aldrich, Poole, U.K.) for 5 min at 37°C. The resulting nuclei were lysed by resuspension in 10 vol 0.25 mM EDTA, pH 7.5 (Sigma-Aldrich), followed by gentle stirring for 1 h in 35 vol 0.25 mM EDTA, and debris were eliminated by centrifugation for 20 min at 8000 x g. Histones H1 and H5 were removed by adding the supernatant containing chromatin to a Carboxymethyl Sephadex C25 column (Amersham Pharmacia, Buckinghamshire, U.K.). This was followed by redigestion of the chromatin with micrococcal nuclease, and mononucleosomes were obtained by running the redigested chromatin through an S-300 Sephacryl column (Pharmacia). Peaks containing mononucleosomes with 146-bp DNA were detected and retrieved by monitoring at A260. The presence of the four core histones (H2a, H2b, H3, and H4) in the mononucleosome preparation was confirmed on a 15% SDS-PAGE. The length of DNA in the mononucleosome was shown to be between 100 and 150 bp on a 2% agarose gel (data not shown).
Tolerance induction and immunization protocols
Mice were nasally dosed with peptide Ag dissolved in PBS for 5 consecutive days (days -12 to -8). In the control group, mice received OVA323–339 peptide in PBS. On day 0, each mouse was immunized intradermally with 100 µg peptide Ag or mononucleosomes emulsified in CFA (Sigma-Aldrich). Alternatively, each mouse was immunized intradermally with 100 µg peptide Ag emulsified in CFA, and from day 7–11, mice were nasally dosed with peptide Ag dissolved in PBS. In long-term disease studies, autoimmune but prenephritic SNF1 females at 4 wk old (25 mice per group) were nasally dosed with H471 or OVA323–339 peptide (20 µg divided equally over 5 consecutive days) at 2-wk intervals until they reached 32 wk of age.
ELISA for Ab production
For anti-peptide Abs, 96-well Falcon 3912 microtiter plates (Marathon, London, U.K.) were coated with 50 µl/well H471 at 10 µg/ml in deionized water by evaporation overnight at 37°C. For anti-dsDNA and anti-mononucleosome Abs, dsDNA or mononucleosomes at 20 µg/ml in PBS were coated onto poly(L-lysine) (Sigma-Aldrich)-sensitized Nunc Maxisorb 96-well plates (Life Technologies, London, U.K.). Nonspecific Ab binding sites were blocked by adding 50 µl/well 2% Marvel in PBS. A total of 50 µl/well mouse serum diluted 1/100 in PBS containing 0.05% Tween 20 (Sigma-Aldrich) and 1% Marvel (P-T-M) was coated on plates by incubation at 37°C for 1 h. For IgG Ab detection, 50 µl/well HRP-conjugated IgG goat anti-mouse Ab (Serotec, Oxford, U.K.) diluted 1/1000 in P-T-M was added and incubated for 1 h. Abs bound to Ags coated on the plate were detected by the addition of 50 µl/well o-phenylenediamine (BDH, Poole, U.K.) at 1 mg/ml in 0.01 M citrate phosphate buffer (pH 6) containing 0.001% H2O2 (Sigma-Aldrich). Finally, the enzyme reaction was stopped by the addition of 50 µl/well 0.5 M citric acid, and absorbance reading was taken at 450 nm.
For IgG subclass Ab detection, rat mAbs to mouse IgG1, IgG2a, IgG2b, and IgG3 (Serotec) diluted 1/750 in P-T-M were used. This was followed by the addition of goat anti-rat biotinylated Abs (Serotec) diluted 1/2000 in P-T-M. Finally, streptavidin complex with HRP (Sigma-Aldrich) diluted 1/1000 in P-T-M was added. Bound Abs were detected using o-phenylenediamine, and absorbance readings were measured as above.
Cell proliferation assay
Ten days following immunization, lymph nodes (LN; axial,
bronchial, and inguinal) were removed aseptically from each mouse. A
total of 3 x 105/200 µl/well LN cells was
cultured in triplicate with or without Ag in 96-well flat-bottom
microtiter plates (Life Technologies) for 96 h at 37°C with 5%
CO2 in a humid incubator. Tissue culture medium
used was IMDM supplemented with 1% L-glutamine, 2%
penicillin and streptomycin (all from Life Technologies), and 1%
normal mouse serum. Cultures were pulsed with 0.25 µCi tritiated
thymidine ([3H]d Thd; Amersham,
Buckinghamshire, U.K.) for the last 6 h.
[3H]d Thd incorporation was measured using a
liquid scintillation beta counter (Microbeta; Wallac, Milton Keynes,
U.K.). Cell proliferation was expressed in 
cpm or stimulation
index.
Cytokine analysis
LN cells (3 x 105/200 µl/well)
from mice of each experimental group were pooled and cultured in the
presence of Ag at 37°C in a humid incubator. In vitro production of
cytokines (IFN-, IL-2, IL-4, and IL-10) by cell cultures was
measured using CelELISA technique adopted from Beech et al.
(24). Briefly, 2 h before transfer of cell cultures,
50 µl/well anti-cytokine Abs (BD Biosciences, Oxford, U.K.) at 2
µg/ml in 0.05 M sodium bicarbonate buffer, pH 9.6, were coated onto
sterile 96-well flat-bottom microtiter plates at 37°C for 1 h.
This was followed by twice washing in PBS, then blocking of nonspecific
sites using PBS containing 1% BSA at 37°C for 1 h. Following
incubation, plates were washed twice with PBS, and 100 µl cell
cultures were transferred to anti-cytokine Ab-sensitized plates and
incubated for 24 h at 37°C. Two hours before harvest, doubling
dilutions of cytokine standards (BD Biosciences) starting from 1200
pg/ml were made on the plate and incubated at 37°C. This was followed
by washing in PBS containing 0.1% Tween 20 (PBS/Tween) and the
addition of 50 µl/well biotinylated detecting Abs at 2 µg/ml in 1%
BSA/PBS and incubation at 37°C for 1 h. Finally, bound Abs were
detected by the addition of streptavidin-conjugated HRP. The enzyme
reaction was developed using 100 µl/well tetramethylbenzene
(Sigma-Aldrich) at 1 mg/ml in citrate phosphate buffer and stopped by
the addition of 50 µl/well 2 M
H2SO4. Absorbance readings
were taken at 450 nm.
Histopathology
Mouse kidneys were processed through to paraffin wax. Kidney sections (5 µm) were mounted on glass slides, air dried, and dehydrated with 100% ethanol before being exposed to 1% periodic acid for 5 min. Sections were then stained with Schiff’s reagent (BDH) for 30 min, followed by brief washing with water and a gradient of ethanol (70–100%) before being mounted in xam. All slides were read by light microscopy (x400) as coded specimens by an independent observer. Glomerular lesions were graded as 1+, 2+, 3+, or 4+ (25). A 1+ lesion represents minor (>10%) thickening of the capillaries of the glomeruli; a 2+ lesion corresponded to focal or focal and diffuse thickening of the capillaries of 30–60% of the glomeruli; in a 3+ lesion, the capillaries of all glomeruli were affected; 4+ lesions were characterized by the preceding together with sclerosis of glomeruli, massive proliferation of epithelial cells, and numerous tubular casts. A grade of 0 was given to kidneys without glomerular lesions. Kidneys from 4-wk-old unmanipulated SNF1 mice were used as negative controls.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The tolerogenicity of the H471 peptide was tested in lupus-prone
SNF1 mice. Three dosages (5, 20, and 100 µg) of
the H471 peptide were tested. Mice in the control group were nasally
dosed with 20 µg OVA323–339. Mice were nasally
dosed with peptide Ags before receiving intradermal immunization with
H471. Results show that LN cells from mice nasally dosed with 20 µg
H471 produced significantly (p < 0.04,
t test) lower proliferative responses to 10 µg/ml H471 in
vitro than LN cells from control mice (Fig. 1
). However, LN
cell hyporesponsiveness was not seen with mice nasally dosed with 5
µg H471 and less apparent with mice nasally dosed with 100 µg H471.
Furthermore, the hyporesponsiveness of LN cells was overcome by
increasing Ag stimulation to 100 µg/ml (Fig. 1). Thus, the 20 µg
peptide dosage was the most effective in inducing hyporesponsiveness of
LN cells, and this was reiterated in a second experiment with groups of
six SNF1 mice (Fig. 2A). It is demonstrated in
this study that the mean proliferative response of the H471 nasally
dosed group was significantly reduced (p <
0.003, t test) compared with the control group
(OVA323–339). Again, this apparent reduction in
cell proliferation was overcome, but still significantly
(p < 0.02, t test) lower than the
control group, by increasing Ag stimulation in vitro (Fig. 2B). Therefore, a dose-dependent nasal tolerance was induced
in SNF1 mice, and the level of LN cell
hyporesponsiveness was dependent on the Ag concentration in vitro.
Experiments presented in this work were repeated twice in
SNF1 mice as well as BSF1
mice (data not shown), and similar observations were obtained on each
occasion.
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In subsequent experiments, we addressed the question of whether
tolerance can be induced in Ag-primed mice. Groups of six
SNF1 mice were immunized intradermally with 100
µg H471 in CFA before being nasally dosed with a total of 20 µg of
either H471 or OVA323–339 peptide. Mouse LN
cells were cultured with H471 or OVA323–339
peptide. After 96 h, LN cells from H471 nasally dosed mice
proliferated significantly (p < 0.02,
t test) less than LN cells from
OVA323–339 peptide nasally dosed mice (Fig. 3A). Like preimmunization
tolerance, LN cell hyporesponsiveness was overcome by increasing Ag
concentration in vitro (Fig. 3B; p < 0.003,
t test). Data shown in this work are highly reproducible in
repeat (3x) experiments in SNF1 as well as
BSF1 mice (data not shown). This is the first
recorded observation to show that ongoing autoimmune responses in
lupus-prone mice can be suppressed by nasal treatment with a self
Ag-derived peptide.
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The H471 peptide is derived from the core histone protein H4 of
mononucleosome. Therefore, we addressed the question of the effect of
nasal dosing of mice with H471 on the immune response to
mononucleosomes. Groups of six SNF1 mice were
nasally dosed with a total of 20 µg H471 or
OVA323–339 peptide before receiving intradermal
injection of 100 µg mononucleosomes emulsified in CFA. Mouse LN cells
were cultured in the presence of 10, 100, or 200 µg/ml
mononucleosomes, H471, or OVA protein (fraction V; Sigma-Aldrich).
Optimal cell proliferation was observed at 100 µg/ml mononucleosomes
in vitro. In fact, increasing Ag concentration to 200 µg/ml inhibited
cell proliferation (data not shown). Suppression of cell proliferative
response to mononucleosome in vitro was seen in the H471 nasally dosed
animals (Fig. 4A). Suppression
of cell proliferation was shown to be significant
(p < 0.05, t test) when the mean
proliferation counts of the experimental groups were plotted (Fig. 4B). Similar data were obtained in two repeat experiments.
Thus, nasal instillation of SNF1 mice with H471
can suppress the immune response to mononucleosomes that carry the H471
epitope.
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To examine the mechanism(s) of nasal tolerance, we measured the
level of cytokines produced in vitro by cells from mice nasally dosed
with H471 or OVA323–339. Therefore, LN cells
were pooled from mice nasally dosed with H471 or
OVA323–339 before being immunized with H471 or
mononucleosomes. The levels of IFN-, IL-2, IL-4, and IL-10 were
measured on day 3 of culture. Fig. 5A shows that cells from mice
nasally dosed with OVA323–339 before being
immunized with H471 produced significantly higher levels of IL-2
(p < 0.005, t test) and IFN-
(p < 0.001, t test) in response to
100 µg/ml H471 challenge compared with cells from mice nasally dosed
with H471. The levels of IL-4 and IL-10 produced by cells were similar
in both peptide nasal dose groups and not significantly above the
background levels. These findings were also observed at 10 µg/ml H471
stimulation in vitro (data not shown).
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B shows that a
significantly higher level of IL-10 (p <
0.001, t test) was produced by cells from mice nasally dosed
with H471, but not with OVA323–339 in response
to 100 µg/ml mononucleosome challenge. This was not seen at 10
µg/ml mononucleosomes in vitro (data not shown). Cells from mice
nasally dosed with OVA323–339 produced
predominantly IFN-. The levels of IL-2 and IL-4 produced by cells
from either peptide nasal dose group were similar and not significantly
above the background levels. Results in this study demonstrate that the
nature of the Ag encountered by lymphocytes and consequently the immune
response generated can determine the form of tolerance in terms of
cytokine production. Student’s t tests were performed on
triplicate data points. The experiments shown in this work were
repeated once in SNF1 mice, and similar results
were obtained. Total IgG and subclass anti-H471 peptide Abs
Blood was collected from mice nasally dosed with peptide before
being immunized with H471, and the levels of total IgG as well as IgG
subclass anti-H471 peptide Abs were examined. No significant
difference in the level of serum IgG anti-H471 peptide Abs was
found between the two experimental groups. However, mice nasally dosed
with H471 produced significantly (p < 0.03,
t test) higher level of IgG1 (Th2) anti-peptide Abs than
mice nasally dosed with OVA323–339, which
produced predominantly IgG2a (Th1) Abs (p <
0.05, t test) (Fig. 6).
Therefore, the IgG1-IgG2a ratios of the H471 and
OVA323–339 nasal treatment groups are 2 and 0.6,
respectively. This is a 3.3-fold shift in IgG subclass Ab response from
a Th1 to Th2 phenotype as a result of nasal dosing of
SNF1 mice with the H471 peptide.
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It was observed that nasal dosing of mice with H471 can suppress
not only cellular and Ab responses, but spontaneous autoimmunity was
also halted by chronic exposure of lupus-prone
SNF1 mice to H471. For each test Ag,
proliferation assays were performed on LN cells of five mice randomly
chosen from each peptide treatment group (Fig. 7). Cells from mice that received the
irrelevant OVA323–339 peptide intranasally
produced significantly (p < 0.001,
t test) higher response to mononucleosomes in vitro (Fig. 7A). This response was 7-fold higher then cells from mice
nasally dosed with H471 in the presence of 100 µg/ml mononucleosomes.
The suppression was not seen at 10 µg/ml mononucleosomes in vitro
(Fig. 7B). Proliferative responses to H471,
OVA323–339, and a peptide representing a second
T cell epitope in H4 (21, 26), which we named H416, were
minimal and did not reach statistically significant levels. The
presence of autoantibodies was also examined in mouse serum. Results
suggest that nasal dosing of mice with H471 can halt or at least delay
the appearance of IgG anti-mononucleosomes
(p < 0.03, t test) and
anti-dsDNA autoantibodies (p < 0.002,
t test) (Fig. 8A).
Examination of the IgG subclass anti-mononucleosome Abs did not
reveal significant differences between the two nasal treatment groups
(Fig. 8B). Data shown in this work demonstrate that chronic
nasal dosing of lupus-prone mice with the H471 peptide can suppress the
development of autoimmune responses in these mice.
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Mice were considered to have severe glomerulonephritis when their kidneys showed a 3+ to 4+ grade of nephritis by histopathology. Mice chronically exposed to the H471 peptide intranasally had a 10% incidence of severe glomerulonephritis at 32 wk of age, whereas mice nasally dosed with the OVA323–339 peptide had a 50% incidence of severe glomerulonephritis. This demonstrates that nasal tolerance induced with the H471 peptide can reduce the incidence of severe glomerulonephritis in lupus-prone SNF1 mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We attempted to induce tolerance in SNF1 mice by
delivering the H471 peptide across the nasal mucosa. Initially, we
tested three peptide dosages, 5, 20, and 100 µg, given intranasally
over 5 consecutive days. Initial results showed that a suboptimal dose
of 5 µg does not induce tolerance (Fig. 1) and in fact, in some
experiments, cells from mice that received this small dose of peptide
were shown to be more readily stimulated by the H471 peptide than cells
from control mice (data not shown). Derry et al. (27) and
others in our lab also recently showed that suppression of
collagen-induced arthritis by nasal instillation of DBA/1 mice with
type II collagen was dose dependent, and the lowest dose of type II
collagen (5 µg) in fact aggravated disease in mice. These findings
are inconsistent with previous observations made by Weiner et al.
(28), which suggested that low doses of Ag administered
across mucosal surfaces induce active suppression of immune responses
through the production of TGF- by Th3 cells. However, tolerogenicity
may vary greatly between different Ags, and this could be a reason for
the discrepancies observed in these studies.
The induction of tolerance was also less effective with a high dose of
peptide (100 µg) (Fig. 1). We analyzed the 100 µg peptide dosage
with scanning electron microscope. Although each peptide solution was
visually soluble, peptide aggregates were observed in the 100 µg
peptide solution, while no molecules were observed in the other (5 and
20 µg) peptide solutions or PBS (data not shown). This demonstrated
that the H471 peptide formed aggregates at high concentrations.
Therefore, when the high concentration H471 solution was applied across
the nasal mucosa, the peptide aggregates may obstruct peptide uptake or
even induce T cell activation. Hence, the physical properties of the
peptide Ag and its formulation are important factors that can influence
its tolerogenic efficacy.
Following nasal instillation of SNF1 mice with 20
µg H471, a marked lymphocyte hyporesponsiveness to in vitro H471
stimulation was shown. The reduction of cell proliferation was
apparently overcome by increasing the concentration of Ag in vitro.
This suggested that the underlying mechanism responsible for this form
of peptide-induced tolerance was most likely to be T cell anergy. By
definition, anergy represents a state of abolished proliferation and
cytokine responses that can be reversed by certain stimuli (29, 30) such as IL-2 (31). The ability of IL-2 to
reverse suppression of cell proliferation in mice nasally dosed with
H471 was not examined; however, the pattern of H471-induced suppression
was similar, in that reduction of cell proliferation was overcome by
increasing Ag stimulation in vitro. Similar to our findings, in the
study of IL-10-dependent suppression of the proliferative response of
PBMC from staphylococcal enterotoxin B-sensitized individuals to
staphylococcal enterotoxin B was not observed at low (0.04 ng/ml) or
high (3.3 ng/ml) Ag concentrations in vitro (31).
Thus, it is hypothesized that following H471 immunization in
SNF1 mice nasally dosed with H471, two distinct
H471-specific T cell populations, one anergic and the other pathogenic,
were primed. In the presence of a low level (10 µg/ml) of H471 in
vitro, competition for H471 and various cell growth factors such as
IL-2 between the two T cell populations meant that the reactivity of
pathogenic H471-specific T cells was obscured by the action of the
anergic T cells; hence, the net result was suppression. However, at a
high Ag concentration (100 µg/ml) in vitro, competition for H471 was
less, and both pathogenic and anergic H471-specific T cell populations
were activated, but the expression of hyporesponsiveness of
H471-specific anergic T cells was restricted; hence, reduction of cell
proliferation was overcome. There is no published data to support this
hypothesis; however, our analysis of cytokine production by cells from
mice tolerized to H471 indicates that anergic T cells are indeed
involved. It was revealed that while cells from mice nasally dosed with
OVA323–339 produced predominantly Th1 cytokines
(IFN- and IL-2), cells from mice nasally dosed with H471 did not
produce above background levels of any of the cytokines tested (Fig. 5A).
In animal models of mucosal tolerance in which the subjects are
typically tolerized before disease induction, there is usually a
significant reduction in the quality and measure of pathology. The
advantage of preimmunization tolerance is, however, not applicable to
clinical situations such as lupus because it is, as yet, impossible to
make a diagnosis before the patient has already developed disease.
Thus, suppressing autoimmune reactions in patients who have already
developed disease will be required if peptide-induced nasal tolerance
is to become a realistic form of therapeutic treatment for humans. With
this aim in mind, experiments were conducted to study nasal tolerance
in Ag-primed animals. Thus, SNF1 mice were primed
with H471 by intradermal injection before being nasally dosed with
H471. Similar to tolerance demonstrated in Ag-naive animals,
suppression of cell proliferation was also demonstrated in Ag-primed
animals (Fig. 3). This is the first recorded observation to show that
ongoing autoimmune responses in lupus-prone mice can be suppressed by
nasal instillation of a self Ag-derived peptide. The successful
demonstration of tolerance in Ag-experienced mice may have implications
for treatment of patients with SLE and other autoimmune conditions.
One of the major obstacles in developing treatments for systemic
autoimmune syndromes such as lupus has been to design a therapy that
allows the suppression or elimination of a widespread autoimmunity
against multiple self Ags and/or tissues. The early appearance of an
autoimmune response to nucleosomes in lupus has been suggested to
represent the initiation of a wider breakdown of immune regulation
against self Ags (18). If this is true, then the
suppression of cell proliferative response to nucleosomes induced by
nasal dosing of mice with the H471 peptide (Figs. 4 and 7) may prevent
or delay the development of autoreactivity against other Ags, hence a
reduction of disease pathology. This was indeed proven to be the case
in the histopathological study shown in this work.
SNF1 mice chronically treated with the H471
peptide intranasally have shown a significantly lower incidence of
severe glomerulonephritis (>3+) compared with mice treated with the
control OVA323–339 peptide (Table I). The significant improvement in
the severity of glomerulonephritis in SNF1
mice as a result of peptide nasal treatment is likely to be associated
with the suppression of autoantibody production shown in the disease
study (Fig. 8). A recent publication by Kaliyaperumal et al.
(26) also showed that suppression of disease in
SNF1 mice could be achieved by i.v. injection of
a peptide bearing a different autoepitope in histone H4 (16–39). They
showed that i.v. injection with the histone peptide in 3-mo-old
prenephritic SNF1 mice that were already
producing pathogenic autoantibodies markedly delayed the onset of
severe lupus nephritis. Furthermore, chronic i.v. therapy in mice with
established glomerulonephritis prolonged survival and even halted the
progression of renal disease. An important disadvantage associated with
tolerance induction by i.v. injection is its invasiveness. One would
argue that mucosal administration is superior to and less invasive than
i.v. as a potential route to reprogram the immune system in autoimmune
conditions.
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This hypothesis was supported by the cytokines produced by cells from
mice tolerized to mononucleosomes. It is shown in this study that
H471-induced nasal tolerance to mononucleosomes was associated with an
increased production of the antiinflammatory cytokine, IL-10 (Fig. 5B). This was not observed in control mice nasally dosed
with OVA323–339, which predominantly produced
IFN- when injected with mononucleosomes. Tolerance to the whole
protein by nasal instillation of a peptide derived from the protein was
also demonstrated in the study of diabetes in nonobese diabetes mice.
It was demonstrated in the study that repeated nasal instillation of
mice with a peptide derived from insulin protected them from developing
diabetes when challenged with insulin (33). The fact that
cytokine production by cells from SNF1 mice
nasally dosed with H471 is different in response to the peptide or the
whole protein suggests that the availability of the H471 epitope to T
cells determines the phenotype (anergic or regulatory) of such T cells
in terms of cytokine production and, consequently, the expression and
form of suppression of cellular and Ab responses in
SNF1 mice.
Recently, much attention has been placed on T cells that have
regulatory functions in both autoimmune diabetes (34) and
intestinal inflammatory disease (35, 36). These
functionally specialized T reg cells exist as part of the normal immune
repertoire and become anergic and/or suppressive (by producing TGF-
in vivo and IL-10 in vitro) when encountering self Ags. They may thus
control the appearance of autoimmunity by keeping the pathogenic
responses to self Ags in check (37). The T reg cells are
CD4+ and CD25+ and are
dependent on CTLA-4 for their suppressive function (36).
The CD4+ CD25+
CTLA-4-dependent T reg cells may bear similarities with T cells
involved in H471-induced nasal tolerance, in that both T cell subsets
are able to become anergic and/or produce suppressive cytokines.
Studies of CD4 and CD25 expression on H471-specific T cells from mice
nasally tolerized to H471 and the effect of CTLA-4 blockade on
H471-induced nasal tolerance are being conducted presently in our lab.
These studies will help to address whether nasal dosing of the
H471 peptide can activate and expand (38) the T reg cell
repertoire in lupus-prone mice.
In conclusion, we have demonstrated dose-dependent nasal tolerance to an autoreactive Th cell epitope and to a major autoantigen, nucleosome, in lupus. The induction of nasal tolerance can be achieved in both Ag-experienced and inexperienced mice. The mechanisms of nasal tolerance are likely to include active suppression by T reg cells as well as anergic T cells. Finally, chronic nasal instillation of a peptide representing an autoreactive Th cell epitope in histone H4 can suppress the development of autoantibodies and reduce the incidence of severe glomerulonephritis in lupus-prone mice. These intriguing observations may have important therapeutic implications on the future development of treatments for lupus.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Henry Yim Wu, Infection and Immunity Research Group, Division of Life Sciences, King’s College London, 150 Stamford Street, London SE1 9NN U.K. E-mail address: henry.wu{at}kcl.ac.uk
3 Current address: Department of Medicine and Therapeutics, University of Aberdeen, IMS, Foresterhill, Aberdeen AB25 2ZD U.K.
4 Abbreviations used in this paper: T reg, T regulatory; LN, lymph node; SLE, systemic lupus erythematosus.
Received for publication January 23, 2002. Accepted for publication May 17, 2002.
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secreting Th3 cells and oral administration of IL-4 enhances oral tolerance in EAE. Eur. J. Immunol. 28:2780.[Medline]
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) before intradermal
challenge with H471. Serum levels of total IgG and IgG subclass
anti-H471 Abs were examined. Negative controls (
) were sera
(n = 3) of 4-wk-old unmanipulated SNF1
mice. Mouse serum was diluted 1/100 in P-T-M. The crossbars represent
mean OD 450 nm readings for the six mice. Each symbol represents an
animal.
) were sera (n = 3) of
SNF1 mice previously injected with mononucleosomes in CFA.
Mouse serum was diluted 1/100 in P-T-M.