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Escherichia coli heat-labile enterotoxin B subunit prevents autoimmune arthritis through induction of regulatory CD4+ T cells.

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Vol. 46, No. 6, June 2002, pp 1671–1682
DOI 10.1002/art.10328
© 2002, American College of Rheumatology
Escherichia coli Heat-Labile Enterotoxin B Subunit
Prevents Autoimmune Arthritis Through Induction of
Regulatory CD4⫹ T Cells
Jeffrey A. Luross, Tricia Heaton, Timothy R. Hirst, Michael J. Day, and Neil A. Williams
Objective. The receptor-binding B subunit of
Escherichia coli heat-labile enterotoxin (EtxB) is a
highly stable, nontoxic protein that is capable of modulating immune responses. This study was conducted to
determine whether mucosal administration of EtxB can
block collagen-induced arthritis (CIA) and to investigate the mechanisms involved.
Methods. Clinical arthritis in DBA/1 mice was
monitored following mucosal administration of EtxB on
4 occasions. The dependence of disease prevention on
receptor binding by EtxB and the associated alterations
to the immune response to type II collagen (CII) were
assessed. Adoptive transfer experiments and lymph
node cell cocultures were used to investigate the underlying mechanisms.
Results. Both intranasal and intragastric delivery
of EtxB were effective in preventing CIA; a 1-␮g dose of
EtxB was protective after intranasal administration. A
non–receptor-binding mutant of EtxB failed to prevent
disease. Intranasal EtxB lowered both the incidence and
severity of arthritis when given either at the time of
disease induction or 25 days later. EtxB markedly
reduced levels of anti-CII IgG2a antibodies and
interferon-␥ (IFN␥) production while not affecting levels of IgG1, interleukin-4 (IL-4), or IL-10. Disease
protection could be transferred by CD4ⴙ T cells from
treated mice, an effect that was abrogated upon depletion of the CD25ⴙ population. In addition,
CD4ⴙCD25ⴙ T cells from treated mice were able to
suppress anti-CII IFN␥ production by CII-primed
lymph node cells.
Conclusion. Mucosal administration of EtxB can
be used to prevent or treat CIA. Modulation of the
anti-CII immune response by EtxB is associated with a
reduction in Th1 cell reactivity without a concomitant
shift toward Th2. Instead, EtxB mediates its effects
through enhancing the activity of a population of CD4ⴙ
regulatory T cells.
Rheumatoid arthritis (RA) is a chronic, systemic
inflammatory disease that manifests predominantly in
synovial tissues of articular joints. Disease progression is
frequently linked with extraarticular complications that
are associated with increased patient morbidity and
mortality (1). The current approach to treatment of RA
tends to focus on managing chronic inflammation and
pain. These methods have little effect on the autoimmune processes that are thought to underlie disease
and are of limited use in altering disease progression.
Therefore, developing novel treatments that target the
autoimmune component of the disease is highly desirable. Progress in this area has been hampered by uncertainty concerning the specific antigens that trigger the
autoimmune response. Consequently, use of antigenindependent therapies that invoke natural regulatory
processes within the immune system is an attractive
Collagen-induced arthritis (CIA) is a welldefined animal model of RA that has provided considerable insights into disease pathogenesis and has been
used extensively to test the potential of novel therapies
(2). Like RA, CIA is characterized by the presence of
fibrin deposition, hyperplasia of synovial cells, periosteal
Supported by grants from the Arthritis Research Campaign
and The Wellcome Trust. Dr. Luross’s work was supported by a joint
Arthritis Research Campaign/Glaxo SmithKline research grant. Dr.
Williams is a Wellcome Trust Research Leave Fellow.
Jeffrey A. Luross, PhD, Tricia Heaton, PhD (current address:
TVW Telethon Institute for Child Health Research, Subiaco, Western
Australia, Australia); Timothy R. Hirst, PhD, Michael J. Day, PhD,
MRCPath, Neil A. Williams, PhD: University of Bristol School of
Medical Sciences, Bristol, UK.
Address correspondence and reprint requests to Neil A.
Williams, PhD, University of Bristol, Department of Pathology and
Microbiology, School of Medical Sciences, University Walk, Bristol
BS8 1TD, UK. E-mail:
Submitted for publication August 27, 2001; accepted in
revised form February 21, 2002.
bone formation, mononuclear infiltrates, pannus formation, and eventual ankylosis of 1 or more articular joints
(3,4). Moreover, susceptibility to both CIA and RA is
strongly associated with expression of specific major
histocompatibility complex (MHC) class II molecules
(5,6), and potential roles for non-MHC loci have been
described (7,8). Generation of interferon-␥ (IFN␥)–
producing CD4⫹ Th1 cells is thought to be critical for
the induction of CIA (9). Indeed, IFN ␥ and
interleukin-12 (IL-12) dominate the anti–type II collagen (CII) immune response in the acute disease phase,
while remission coincides with reduced production of
IFN␥ and increased production of IL-10 (10,11).
IFN␥ promotes generation of complement-fixing
IgG2a anti-CII antibodies, the deposition of which is
thought to trigger early inflammatory events, allowing
immune cell entry into the joint (12). In addition, the
activity of IFN␥ in stimulating myeloid cells to produce
IL-1 and tumor necrosis factor ␣ likely contributes to the
localized tissue inflammation and subsequent joint destruction characteristic of CIA (13). Importantly, administration of exogenous Th1-associated cytokines has
been shown to exacerbate CIA (14), while counterregulation of the Th1 response by administration of Th2
cytokines has been shown to protect against disease (15).
The nontoxic B subunit of Escherichia coli heatlabile enterotoxin (EtxB) and its structural homolog
from cholera toxin (CtxB) are highly stable proteins that
mediate binding of their respective enterotoxins to
membrane receptors on mammalian cells. The principal
receptor to which both EtxB and CtxB bind is the
ganglioside GM1. In addition, both molecules bind to
GD1b, and EtxB has some affinity for asialo-GM1,
lactosylceramide, and certain galactoproteins (16). Recent reports have indicated that the nontoxic B subunits
may be useful agents for prevention and treatment of
Th1-mediated autoimmune diseases (16). Mucosal administration of CtxB-autoantigen conjugates has been
shown to prevent experimental allergic encephalomyelitis (17), diabetes in NOD mice (18), and CIA (19). The
observation that this effect requires direct coupling of
autoantigen with CtxB led to the suggestion that the B
subunit acts as a carrier molecule, directing antigen into
natural mucosal tolerance-inducing pathways. However,
there is clear evidence indicating a more active immune
modulatory role for the B subunits.
Intravenous or intraperitoneal delivery of CtxB
alone can delay the onset of diabetes in the NOD mouse
(20), and subcutaneous administration of EtxB in
Freund’s incomplete adjuvant (IFA) can prevent CIA
(21). The fact that these effects can be achieved follow-
ing parenteral delivery shows that B subunit–mediated
protection is not dependent on features of the mucosal
immune system. Moreover, the observation that CtxB
and EtxB are active in the absence of conjugated autoantigen shows that they are not acting simply as
delivery vehicles.
The precise mechanisms of immune modulation
by the B subunits remain to be established. Prevention of
CIA following subcutaneous administration of EtxB/IFA
was associated with altered T cell reactivity to CII, as
demonstrated by a reduction in IFN␥ production concomitant with increased release of IL-4 (21). Although
these data suggest that EtxB can mediate a change from
Th1 to Th2 responsiveness, measurement of serum
anti-CII antibodies revealed an overall suppression of
the response, indicating that other immunomodulatory
mechanisms may be involved. The purpose of the
present study was to investigate the basis of the protective effect of EtxB in CIA. We report that disease
prevention can be mediated by mucosal administration
of EtxB alone, and that this effect involves the enhanced
activity of a population of splenic CD4⫹ regulatory T
Mice. Age-matched male DBA/1 mice were purchased
from Harlan Olac (Bicester, UK) and were used for experimentation at 7 to 13 weeks of age. Animals were used in
accordance with institutional and Home office guidelines.
Antigens, immunizations, and induction of arthritis.
Recombinant EtxB, EtxB(G33D) (a non–receptor-binding
mutant of EtxB), and CtxB were synthesized and purified as
previously reported (22). Levels of endotoxin were ⬍30
units/mg in all samples, as determined using a Kinetic-QCL
chromogenic limulus amebocyte lysate assay (BioWhittaker,
Walkersville, MD). Groups of mice were given various doses of
EtxB, EtxB(G33D), CtxB, or chicken CII (Sigma-Aldrich,
Poole, UK) by intranasal (20-␮l volume) or intragastric (200-␮l
volume) routes at 24-hour intervals for 4 days, unless otherwise
stated. On the final day of treatment (day 0), mice were
challenged with 100 ␮g of CII in Freund’s complete adjuvant
(CII/CFA) or 100 ␮g of keyhole limpet hemocyanin (KLH;
Calbiochem, La Jolla, CA) in CFA, given subcutaneously at
the base of the tail.
Disease assessment. Mice were examined regularly for
clinical evidence of arthritis, characterized by a combination of
erythema and swelling of the tarsal joints, digits, and foot pads.
Each paw was assigned a score of 0 (normal appearance), 1
(mild erythema and/or swelling), or 2 (significant erythema
and/or swelling), and the cumulative score (maximum of 8) was
then calculated. Data are presented as the mean ⫾ SEM
clinical score for each group of mice. Histologic sections of
stifle joints were examined for pathology using an established
4-point scoring system (21). All scoring of disease was performed under blinded conditions.
Antibody responses. Fifteen days after CII immunization, serum samples were obtained and analyzed by enzymelinked immunosorbent assay (ELISA) for the presence of
anti-CII antibodies. The distribution of CII-specific IgG1 and
IgG2a antibody subclasses was determined using specific detecting antibodies for mouse IgG1 and IgG2a (Serotec, Oxford, UK). Antibody concentrations were calculated against
defined concentrations of myeloma-derived IgG1 and IgG2a
(Serotec) by weighted probit analysis (23).
Adoptive transfer. Spleens from mice that were treated
with intranasal EtxB or phosphate buffered saline (PBS) and
challenged with CII/CFA were harvested 24 days after immunization. To enrich for CD4⫹ cells, splenocytes were separated
using CD8, CD11b, and CD45R magnetic-activated cell sorting (MACS) beads (Miltenyi Biotec, Bisley, UK). In experiments in which CD4⫹ or CD25⫹ cells were depleted, splenocytes were labeled with anti-CD4 MACS beads or biotinconjugated anti-CD25 (7D4; BD PharMingen, San Diego,
CA), followed by streptavidin-conjugated MACS beads (Miltenyi Biotec). Resulting purities were a minimum of 82% for
CD4⫹ cells, 97% for CD4⫹ depleted cells, and 98% for
CD25⫹ depleted cells, as assessed by flow cytometry. Purified
cell populations were suspended in Hanks’ balanced salt
solution for intravenous adoptive transfer of 1 ⫻ 107 cells into
naive recipients. Groups of control mice received 200 ␮l of
T cell proliferative and cytokine responses. Mice were
killed 25 days after immunization, and spleen and/or inguinal
lymph node (ILN) cell populations were derived for culture
(2.5 ⫻ 106 and 2–3 ⫻ 106 viable cells/ml, respectively, in 2-ml
culture). Fluorescently labeled antibodies to CD4 (H129.19;
Sigma) and CD25 (PC61; BD PharMingen) were used for
fluorescence-activated cell sorting (FACS) of spleen cells for
coculture experiments. Cell purities were a minimum of 94%.
Cells were cultured under previously described conditions (24)
in the presence or absence of either 50 ␮g/ml of CII or 10
␮g/ml of KLH. At select time points, duplicate or triplicate
100-␮l samples were transferred into 96-well Costar plates
(Corning, Corning, NY) for assessment of proliferation and
cytokine production using 3H-thymidine (3H-TdR) incorporation and cell-based ELISA (25) techniques, respectively. Concentrations of IFN␥, IL-4, and IL-10 were calculated against
standard curves produced with recombinant cytokine (BD
PharMingen). Limits of detection for each cytokine assayed
were 20 pg/ml, 10 pg/ml, and 40 pg/ml, respectively.
Statistical analysis. For normally distributed data,
statistical comparisons were performed using Student’s unpaired t-test. For nonparametric data, the Mann-Whitney U
test or Kruskal-Wallis analysis of variance was used. Differences in disease incidence were tested by chi-square analysis.
Prophylactic and therapeutic effect of mucosally
delivered EtxB on CIA. We previously reported that
subcutaneous injection of EtxB in IFA can prevent CIA
(21). Although injectable therapies may be appropriate
for the treatment of RA, it would be advantageous if a
noninvasive method of delivery could be used. There-
Figure 1. Effects of mucosal administration of Escherichia coli heatlabile enterotoxin B subunit (EtxB) on collagen-induced arthritis
(CIA). A, Scores for clinical disease in mice receiving 100 ␮g of EtxB
intranasally (i.n.) (䡲; n ⫽ 8) or intragastrically (i.g.) (䡺; n ⫽ 5) or 1 ␮g
of EtxB i.n. (Œ; n ⫽ 8) or i.g. (‚; n ⫽ 7), or left untreated (✖; n ⫽ 10)
prior to induction of CIA. Values are the mean ⫾ SEM disease score
for each group. Similar observations were made in 3 separate experiments. B, Histopathologic scores in mice receiving 100 ␮g or 1 ␮g of
EtxB i.n. or i.g. Values are the mean and SEM disease score for each
group. C and D, The incidence of arthritis and the severity of clinical
disease, respectively, in affected animals. The data are pooled from
animals assessed in 3 separate experiments in which mice were either
untreated (✖; n ⫽ 26) or treated with EtxB intranasally (■; n ⫽ 33)
prior to type II collagen/Freund’s complete adjuvant challenge. ⴱ ⫽ P
⬍ 0.005 compared with untreated controls.
fore, we investigated whether mucosal delivery of EtxB
could prevent CIA. Groups of mice were either left
untreated or were treated using inhalation or gastric
lavage with 100 ␮g or 1 ␮g of EtxB for 4 days before
disease induction. Untreated mice developed a severe
arthritis (Figure 1A), which was evident by day 25 and
became progressively worse during the following 20
days. In contrast, mice that received 100 ␮g of EtxB,
either intranasally or intragastrically, had markedly
lower clinical disease scores (P ⬍ 0.05 versus controls on
days 30–45). Strong disease protection following intranasal administration of EtxB was also observed when the
dose was reduced to 1 ␮g (P ⬍ 0.05 versus controls on
days 30 and 35; P ⬍ 0.001 versus controls on days 40 and
45). However, intragastric EtxB treatment at this dose
was not effective. Mice that received four 10-␮g doses of
CII intranasally before CII/CFA challenge (a regimen
similar to that previously reported to induce mucosal
tolerance) (26) exhibited an intermediate level of disease protection (data not shown).
Histopathologic analysis of the stifle joints of
mice on day 45 correlated well with clinical disease
scores (Figure 1B). Clinically effective doses of EtxB
reduced the severity of joint pathology. However, differences in cellular infiltration and joint destruction between treated mice and untreated mice were statistically
significant only when the 100-␮g dose of EtxB was given
intranasally (P ⬍ 0.005).
To further investigate the process of protection
against CIA following mucosal administration of EtxB,
data from several similar experiments in which EtxB was
given intranasally were pooled, and the incidence and
severity of disease in affected mice were analyzed separately. This analysis (Figures 1C and D) clearly showed
that intranasal EtxB treatment had a dual effect on
disease progression. A marked difference in the incidence of CIA between treated and untreated mice was
evident on day 25. Furthermore, during the following 3
weeks, increasing numbers of untreated animals developed signs of arthritis, while only a slight increase in the
proportion of affected mice was observed in the EtxBtreated group. The difference in the incidence of arthritis between the PBS-treated and EtxB-treated groups
was significant at each time point tested (P ⬍ 0.005).
Although the severity of clinical disease in affected mice
in the untreated group increased over time, progression
of arthritis was markedly attenuated in EtxB-treated
animals (P ⬍ 0.005 at every time point studied after
day 25).
To test whether prevention of CIA by mucosally
delivered EtxB was dependent on receptor interaction,
groups of mice were treated intranasally with an intermediate, 10-␮g dose of either EtxB or EtxB(G33D)
before disease induction. The mean clinical score for
arthritis in the EtxB-treated group remained low
throughout the course of the experiment (Figure 2A).
This effect differed from that in EtxB(G33D)-treated
mice, which developed severe arthritis (P ⬍ 0.02 on days
28 and 32; P ⬍ 0.001 on days 35–46). Further experiments tested whether 10 ␮g of intranasally administered
CtxB could prevent CIA. At this dose, CtxB failed to
Figure 2. A, Data showing that receptor binding by EtxB is critical for
modulation of CIA. Mice were given four 10-␮g intranasal doses of
EtxB (■; n ⫽ 10) or EtxB(G33D) (E; n ⫽ 10), and CIA was induced.
Data are the mean ⫾ SEM clinical score of arthritis. ⴱ ⫽ P ⬍ 0.02;
ⴱⴱ ⫽ P ⬍ 0.001. B, Lack of modulation of CIA with intranasal
administration of the structural homolog from cholera toxin (CtxB).
Mice (n ⫽ 7) were left untreated (E) or were given 10 ␮g of CtxB (■;
n ⫽ 10) before disease induction. Values are the mean ⫾ SEM clinical
score of arthritis (P ⬎ 0.7 at all time points studied). C and D, Effects
of EtxB on recent-onset arthritis. Mice were immunized with type II
collagen/Freund’s complete adjuvant on day 0 to induce arthritis. On
days 25, 27, 30, and 32, mice were treated intranasally with 10 ␮g of
EtxB (■; n ⫽ 14) or EtxB(G33D) (E; n ⫽ 13). Arrows indicate days of
treatment. C, Incidence of clinical arthritis. D, Severity of disease in
affected animals. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.03. See
Figure 1 for other definitions.
alter the clinical arthritis score in treated mice compared
with untreated mice (Figure 2B).
In RA, it is not possible to predict the onset of
disease. Therefore, effective therapies must be able to
limit progression of established arthritis. Accordingly,
we investigated the effects of administering intranasal
EtxB after CIA was established. Mice challenged with
CII/CFA to induce arthritis were randomized on day 25
to receive treatment with four 10-␮g doses of either
EtxB or EtxB(G33D) over 7 days. During the treatment
period, the incidence of CIA in the 2 groups was nearly
identical (Figure 2C). However, disease incidence then
declined in the EtxB-treated group, from a peak at day
32, and remained lower than that in the EtxB(G33D)treated group for the remainder of the experiment (P ⬍
0.03 on days 35, 42, and 46). Among affected mice, the
severity of CIA was also consistently lower in mice given
EtxB (Figure 2D), although the difference did not reach
statistical significance. Taken together, these data show
that receptor binding following mucosal delivery of EtxB
can prevent the onset of CIA and actively limit progression of established disease.
Alteration of the anti-CII immune response by
EtxB treatment. Production of complement-fixing
IgG2a anti-CII autoantibodies is an important component of the pathologic immune response in CIA (9).
Thus, serum was obtained from mice that were treated
with EtxB intranasally or intragastrically before disease
induction, in order to determine whether protection
against CIA correlated with changes in the amount and
isotype of anti-CII antibodies. Sera from mice that
received CII intranasally were included for comparison.
As expected, IgG2a was the most abundant isotype
detected in sera from untreated mice, which is indicative
of a Th1-dominated response (Figure 3). Importantly,
the amounts of anti-CII IgG2a antibodies were markedly
suppressed in all treatment groups (P ⬍ 0.01 compared
with the untreated group), with the lowest levels present
in mice that had received either 1 ␮g or 100 ␮g EtxB
intranasally (P ⬍ 0.001). However, mice that received a
disease-protecting dose (100 ␮g) of EtxB intragastrically
had amounts of CII-specific IgG2a similar to those of
mice that received an ineffective dose of 1 ␮g. The
amounts of anti-CII IgG1 antibodies within treatment
groups were comparable with those detected in untreated mice, with the exception of the group that
received 100 ␮g of EtxB intranasally, in which an
⬃2.3-fold increase was detected (P ⬍ 0.01). No anti-CII
antibodies were measured in the sera of mice that were
not challenged with CII/CFA.
In further investigations, we determined whether
mucosal administration of EtxB altered the anti-CII T
cell response. Spleen cells from mice treated with EtxB
or PBS intranasally before being immunized with CII/
CFA were cultured in the presence or absence of CII.
The proliferative and cytokine responses in the cultures
were monitored over a 5-day period. Figure 4 shows data
from 1 of 5 identical experiments on day 3 of culture (the
time at which responses were maximal). Spleen cells
from both EtxB-treated and PBS-treated mice proliferated strongly in response to CII. In the experiment
shown, the level of proliferation was lower in the culture
Figure 3. Changes in the anti–type II collagen (anti-CII) antibody
response following mucosal delivery of EtxB. Mice were left untreated
(⫹) or were treated intranasally or intragastrically with 100 ␮g or 1 ␮g
of EtxB or with 10 ␮g of CII intranasally, for 4 days prior to induction
of CIA (day 0). Each animal was bled on day 14 to determine the
presence of anti-CII–specific IgG1 and IgG2a serum immunoglobulin.
Sera from unchallenged mice were used as a negative control (⫺).
Data for individual mice are shown. Bars show the median antibody
concentration. ⴱ ⫽ P ⬍ 0.01; ⴱⴱ ⫽ P ⬍ 0.001. NS ⫽ not significant. See
Figure 1 for other definitions.
from EtxB-treated mice. However, when the results of 5
identical experiments were analyzed, the difference in
levels of spleen cell proliferation between cultures from
EtxB-treated and untreated mice was not significant
(mean ⫾ SEM proliferative response 7,753 ⫾ 1,467
counts per minute in PBS-treated mice and 7,712 ⫾
1,791 cpm in EtxB-treated mice; P ⬍ 0.98).
Analysis of the cytokines produced in the spleen
cell cultures revealed that EtxB treatment dramatically
altered the nature of the T cell response. Spleen cells
from mice receiving EtxB produced lower levels of
IFN␥. The mean ⫾ SEM reduction was 42 ⫾ 11% in 5
Figure 4. Altered splenic T cell responses to type II collagen (CII)
after Escherichia coli heat-labile enterotoxin B subunit (EtxB) treatment. Mice (n ⫽ 10) were given 20 ␮g of EtxB or phosphate buffered
saline (PBS) intranasally and were then immunized with type II
collagen (CII)/Freund’s complete adjuvant. Splenocytes, harvested 24
days later, were cultured for 5 days in the presence (䊐) or absence (■)
of 50 ␮g/ml of heat-denatured CII for the analysis of cellular proliferation (3H-thymidine [3H-TdR] incorporation [cpm]) and the production of the cytokines interferon-␥ (IFN␥), interleukin-4 (IL-4), and
IL-10. Data are the mean and SEM value of triplicate samples from
the day at which maximum proliferation was recorded (day 3). The
results shown are from 1 of 5 identical experiments.
identical experiments. The decrease in IFN␥ production
following administration of EtxB was not associated with
increased levels of IL-4 or IL-10 (the higher background
release of IL-10 in cultures from EtxB-treated animals
was not a consistent feature of the 5 experiments).
Protection against CIA following adoptive transfer of CD4ⴙ T cells from EtxB-treated mice. The
observation that mucosal administration of EtxB modulated the differentiation of anti-CII–responsive T cells
led us to hypothesize that the B subunit may enhance the
activity of a regulatory T cell population. To investigate
this theory, we administered 1 ⫻ 107 splenic CD4⫹ cells
(isolated from either EtxB-treated or PBS-treated mice
24 days after CII/CFA challenge) intravenously to naive
recipients, 3 days before induction of CIA. Additional
groups were either given intravenous vehicle only or
were left untreated before CII/CFA challenge. Clinical
signs of arthritis were monitored, and the results of 1 of
3 identical experiments are shown in Figure 5A.
CIA developed normally in mice that were not
manipulated and in those that had been given vehicle
only. In contrast, animals that received CD4⫹ cells from
EtxB-treated mice developed an attenuated arthritis.
The mean clinical score in this group was significantly
lower than that in any of the control groups from day 26
onward (P ⬍ 0.01 on days 26 and 29; P ⬍ 0.05 on the
remaining days studied). The ability of transferred
CD4⫹ cells to prevent CIA was dependent on whether
donors had been treated with EtxB; recipients of CD4⫹
cells from PBS-treated mice developed an arthritis that
was similar to that seen in control animals. The decreased mean clinical score in mice that received CD4⫹
cells from EtxB-treated donors was reflected in both a
lower incidence of arthritis and decreased disease severity in affected animals. Data from these transfer experiments clearly demonstrate that mucosal administration
of EtxB leads to the presence of CD4⫹ cells in the
spleen that are capable of suppressing CIA.
Further experiments were performed to determine whether CD4⫹ cells were the only population of
spleen cells from EtxB-treated mice capable of conferring protection against CIA. As in previous adoptive
Figure 5. A, Attenuation of CIA development by adoptive transfer of
CD4⫹ cells from mice given EtxB intranasally. Mice (n ⫽ 10) were
given 20 ␮g of EtxB intranasally or phosphate buffered saline (PBS)
and immunized with type II collagen/Freund’s complete adjuvant
(CII/CFA). Twenty-four days later, 1 ⫻ 107 CD4⫹ enriched splenocytes from EtxB-treated mice (■; n ⫽ 8) or PBS-treated mice (Œ; n ⫽
9), or an equal volume of the vehicle (Hanks’ balanced salt solution; 䉬;
n ⫽ 10) was inoculated intravenously into naive recipients. Three days
after adoptive transfer, recipient mice were immunized with CII/CFA
to induce arthritis (day 0). An additional group of untreated mice
received CII/CFA immunization at this time (E; n ⫽ 9). Values are the
mean ⫾ SEM clinical score of arthritis, the incidence, and the severity
of disease in affected animals. Similar results were observed in 2
additional identical experiments. ⴱ ⫽ P ⬍ 0.05, ⴱⴱ ⫽ P ⬍ 0.01 versus
other groups. B, Spleen cells were isolated 25 days following administration of 4 doses of 10 ␮g EtxB intranasally on consecutive days and
were either left unselected (■; n ⫽ 10) or were depleted of CD4⫹ cells
(Œ; n ⫽ 9) before transfer into recipient mice. Disease progression was
compared with that seen in untreated controls (E; n ⫽ 10). Values are
the mean ⫾ SEM clinical score of arthritis, the incidence, and the
severity of disease in affected animals. ⴱ ⫽ P ⬍ 0.05, ⴱⴱ ⫽ P ⬍ 0.01
versus untreated controls. See Figure 1 for other definitions.
transfer experiments, spleen cells were isolated following intranasal administration of EtxB and were then left
unselected or were depleted of CD4⫹ cells before being
injected intravenously into naive recipients. Arthritis was
induced in the recipients, as well as in an additional
group of mice that was left untreated. The results
(Figure 5B) demonstrate that although unselected
spleen cells from EtxB-treated mice are capable of
potently suppressing CIA, depletion of the CD4⫹ subset
abrogates this ability. Both the mean clinical arthritis
score and the disease incidence were markedly lower in
mice receiving unselected cells, and no statistically significant differences were observed between the groups
receiving CD4⫹ cell–depleted splenocytes and those left
Suppression of IFN␥ production by CD4ⴙCD25ⴙ
T cells from EtxB-treated mice. Coexpression of CD25 on
CD4⫹ T cells defines both activated cells and naturally
occurring T regulatory cells (27,28). Activated effector
cells may be capable of mediating immune deviation
(29), and CD4⫹CD25⫹ cells from naive mice can
suppress T cell responses in vitro (30) and prevent
Th1-associated diseases following adoptive transfer
(31,32). Two approaches were taken to test whether
CD4⫹CD25⫹ cells were involved in immune regulation
following mucosal EtxB treatment. First, spleen cells
were isolated from either EtxB-treated or PBS-treated
mice 24 days after CII/CFA challenge, and the
CD4⫹CD25⫹ population was purified by FACS.
CD4⫹CD25⫹ cells were added to cultures of CII/CFAprimed ILN cells from untreated mice at a ratio of 1:10.
During 5 days of culture, cell proliferation and cytokine
production were monitored in the presence or absence
of CII.
Results at the time of peak response (day 4) are
shown in Figure 6A. In the presence of CII, ILN cells
from CII/CFA-primed mice responded vigorously, producing high levels of IFN␥ but no IL-4 or IL-10 above
background levels. Addition of CD4⫹CD25⫹ cells from
PBS-treated mice had minimal effect on the response;
there was a low-level increase in 3H-TdR incorporation
and a slight reduction in IFN␥ secretion. However, in
cultures of ILN cells to which CD4⫹CD25⫹ cells from
EtxB-treated mice were added, IFN␥ was markedly
suppressed. A similar level of suppression was seen in
identical experiments (mean IFN␥ concentration ⫾
SEM [ng/ml]: ILN cells alone, 3.60 ⫾ 0.49; ILN cells
with CD4⫹CD25⫹ cells from EtxB-treated mice, 1.00 ⫾
0.17 [P ⬍ 0.01; n ⫽ 4]). The addition of CD4⫹CD25⫹
cells from EtxB-treated mice did not affect either levels
Figure 6. Suppression of IFN␥ production in vitro by CD4⫹CD25⫹
cells from Escherichia coli heat-labile enterotoxin B subunit (EtxB)–
treated mice. Purified CD4⫹CD25⫹ splenocytes from mice (n ⫽ 10
per group) treated with four 20-␮g intranasal doses of EtxB or PBS
intranasally and immunized with CII (A) or keyhole limpet hemocyanin (KLH) (B) were harvested 24 days after challenge and added at a
ratio of 1:10 into cultures of antigen-primed inguinal lymph node
(ILN) cells derived from mice immunized with corresponding antigen/
Freund’s complete adjuvant on day 0. Cells were cultured in the
presence or absence of either 50 ␮g/ml heat-denatured CII or 10 ␮g/ml
KLH for 5 days. Cellular proliferation (3H-TdR [cpm]) and the
production of IFN␥, IL-4, and IL-10 are shown for the day at which
maximum proliferation was observed in cultures of antigen-primed
ILN cells (white bars), or cocultures of antigen-primed ILN cells and
CD4⫹CD25⫹ splenocytes from PBS-treated mice (grey bars) or
EtxB-treated mice (black bars). All samples were assayed in duplicate.
Horizontal lines represent the maximum background activity detected
in each assay. Data are representative of 4 experiments. See Figure 4
for other definitions.
of cellular proliferation or production of IL-4 or IL-10 in
ILN cell cultures.
The ability of EtxB to trigger the modulatory
activity of CD4⫹CD25⫹ T cells in the spleen may be
related to the fact that CII is an autoantigen. Thus, EtxB
could be activating CII-reactive CD4⫹CD25⫹ regulatory T cells that are present as a result of normal
tolerance-inducing pathways. To test this possibility, we
investigated whether similar regulatory activity could be
detected in experiments in which a foreign antigen was
Figure 7. CIA in mice following adoptive transfer of EtxB-treated
whole splenocytes or splenocytes depleted of CD25⫹ cells. Splenocytes from mice (n ⫽ 12) given four 20-␮g intranasal doses of EtxB
were harvested 25 days after immunization with type II collagen/
Freund’s complete adjuvant (CII/CFA). Groups of naive mice were
inoculated intravenously with 1 ⫻ 107 splenocytes (■; n ⫽ 10), 1 ⫻ 107
CD25⫹ depleted splenocytes (Œ; n ⫽ 14), or vehicle (Hanks’ balanced
salt solution) (E; n ⫽ 9). Three days after adoptive transfer, mice were
challenged with CII/CFA to induce CIA (day 0). Values are the
mean ⫾ SEM clinical score of arthritis, the incidence, and the severity
of disease in affected animals. ⴱ ⫽ P ⬍ 0.05, ⴱⴱ ⫽ P ⬍ 0.001 versus
controls. See Figure 1 for other definitions.
the target of the T cell response. Mice were treated
intranasally with PBS or EtxB before challenge with
KLH/CFA. On day 25, CD4⫹CD25⫹ cells were purified
and added to cultures of ILN cells from untreated,
KLH/CFA-primed animals (Figure 6B). The results
showed that CD4⫹CD25⫹ cells from EtxB-treated mice
were capable of reducing IFN␥ production in response
to KLH. Once again, this effect was not associated with
altered levels of proliferation or with IL-4 and IL-10
production. A marginal decrease in KLH-associated
IFN ␥ production was seen in cocultures of
CD4⫹CD25⫹ cells from PBS-treated mice. Similar results were obtained in 2 identical experiments using
In a second approach to investigate the role of
CD25⫹ cells in the modulation of CIA by EtxB, splenic
cells from CII/CFA-challenged mice treated with intranasal EtxB were isolated and left unselected or were
depleted of the CD25⫹ population for adoptive transfer
into naive recipients. Three days after transfer, recipients were challenged with CII/CFA and subsequently
monitored for signs of arthritis (Figure 7). CIA developed normally in mice that had received vehicle, and, as
expected, transfer of unselected spleen cells from EtxBtreated mice inhibited development of CIA. Importantly, depletion of CD25⫹ cells from the transferred
population significantly reduced their ability to suppress
arthritis (P ⬍ 0.001 for mean clinical scores between
recipients of unselected and depleted cells from day 25
to day 46). This difference was also manifested when
separate data regarding disease incidence and severity
were analyzed.
Interestingly, although depletion of the CD25⫹
population markedly diminished the capacity of the
transferred cells to prevent arthritis, some protective
effect was still observed in comparison with vehicle-only
controls. The mean clinical scores in mice receiving cells
depleted of the CD25⫹ population were lower than
those in controls (P ⬍ 0.05 on days 21, 28, and 32), an
effect that resulted primarily from a decreased severity
of disease in this group.
The results of these experiments show that mucosal administration of EtxB can block the establishment
and progression of CIA. Intranasal or intragastric treatment with EtxB at the time of disease induction reduced
clinical signs of joint swelling. However, the intranasal
route appeared to be more effective, because mice were
protected from CIA when 1 ␮g of EtxB was given
intranasally but not when it was administered intragastrically. EtxB is highly pH- and protease-resistant and
would not be expected to undergo degradation during
transit into the intestine (33). Therefore, this difference
in effective dose probably results from dilution of EtxB
over the large surface area of the gut. The ability of EtxB
to prevent clinical arthritis correlated with reductions in
inflammatory changes in the stifle joints, indicating that
EtxB is capable of reducing pathologic changes within
the joints.
Further analysis of the effects of intranasal EtxB
treatment revealed that clinical disease was prevented in
some mice (as revealed by the lowered incidence of
CIA), and mean disease severity was reduced in those
animals that did develop CIA. The incidence and severity of CIA were also reduced when intranasal EtxB
treatment was delayed until the anti-CII response had
been established. The observation that the incidence of
clinical joint swelling decreased following EtxB treatment indicates that the B subunit can ameliorate established disease in some animals. An analysis of disease
severity suggested that disease progression was attenuated in the remaining arthritic animals. Collectively,
these findings indicate that EtxB can modify the nature
of the immune response following CII challenge, blocking either the establishment or progression of joint
Investigations into the mechanisms underlying
mucosal treatment of CIA by EtxB revealed a critical
role played by B subunit–receptor interaction. The non–
receptor-binding mutant, EtxB(G33D), failed to prevent
CIA. Interestingly, as previously reported (19), intranasal administration of CtxB also failed to block CIA. The
inability of CtxB, when used alone, to modulate CIA
reveals critical differences between EtxB and CtxB,
which probably relate to their disparate stabilities (i.e.,
CtxB is unstable as a pentamer below pH 3.9, and EtxB
is stable at pH 2.0) or the slightly wider receptor
specificity of EtxB. Although CtxB was unable to prevent CIA in the absence of conjugation to CII (19), it has
been shown to prevent diabetes in NOD mice following
parenteral delivery (20) and can block trinitrobenzene
sulfonic acid–induced colitis when given orally in the
presence of a pH buffer (34).
Attenuation of CIA following mucosal delivery of
EtxB was associated with a marked reduction in the
proinflammatory anti-CII immune response. Intranasal
and intragastric EtxB treatment decreased levels of
CII-specific IgG2a antibodies, and intranasal delivery of
EtxB decreased CII-dependent splenic T cell production
of IFN␥. The reduction of the anti-CII IgG2a antibody
response was analogous to that observed using an established intranasal CII tolerization protocol (35). Suppression of the anti-CII Th1 response following subcutaneous injection of EtxB in IFA was associated with
enhanced IL-4 production, suggesting deviation toward
Th2 (21). However, enhanced Th2 responsiveness was
not the mechanism underlying EtxB-mediated protection against CIA following mucosal administration. Although intranasal delivery of 100 ␮g of EtxB did increase levels of IgG1 anti-CII antibodies, this effect was
not seen following treatment with 1 ␮g of EtxB intranasally or 100 ␮g of EtxB intragastrically, even though both
of these doses effectively prevented clinical arthritis.
Furthermore, no enhancement of IL-4 or IL-10 production was observed in cultures of splenic cells from mice
treated with EtxB intranasally.
Because suppression of the anti-CII Th1 response did not correlate with heightened Th2 reactivity,
we hypothesized that EtxB may enhance the activity of a
regulatory CD4⫹ T cell population. Such cells capable
of suppressing immune reactivity by the production of
soluble factors and/or via cell–cell contact have been
described both in vitro and in vivo (36). Although
characterization of these cell types is incomplete, they
are thought to be critical for the maintenance of immune
homeostasis, because their in vivo depletion or reconstitution, respectively, can induce or protect against Th1mediated inflammatory disease. Conclusive evidence
that mucosal administration of EtxB enhanced the activity of CD4⫹ T regulatory cells came from our finding
that adoptively transferred splenic CD4⫹ cells inhibited
clinical arthritis, provided that they were derived from B
subunit–treated mice. Moreover, the finding that depleting CD4⫹ cells ablated the capacity of spleen cells to
transfer disease protection demonstrates the critical role
played by this subset in the B subunit–mediated prevention of CIA. Our further observation that depletion of
CD25⫹ cells from spleen cells before transfer abrogated
their ability to prevent CIA suggests a role for a
CD4⫹CD25⫹ subset in mediating EtxB-induced protection against CIA. Nevertheless, differences in the pattern of disease between whole spleen cell–protected
mice and mice that had received splenocytes depleted of
CD25⫹ cells indicate that other cell populations may
contribute to immune regulation following EtxB treatment.
Experiments directly testing the ability of sorted
CD4⫹CD25⫹ cells from the spleens of EtxB-treated
mice to prevent CIA may provide further insight into the
role of this population. However, such studies would
need to consider that this population may also contain
activated CII-responsive effector cells. Additional evidence to associate EtxB treatment with enhanced regulatory activity of CD4⫹CD25⫹ T cells came from in
vitro experiments in which purified populations of these
cells were added to ILN cell cultures from CII/CFAprimed mice. These studies revealed that CD4⫹CD25⫹
cells from EtxB-treated mice are enhanced in their
capacity to block CII-specific IFN␥ production. This
reduction in IFN␥ was not associated with increased
IL-4 or IL-10 production, which suggests that the regulatory CD4⫹CD25⫹ population was not mediating its
effects due to the presence of Th2 cells (which might
share this surface phenotype). Interestingly, the addition
of CD4⫹CD25⫹ cells from EtxB-treated mice did not
alter levels of proliferation in ILN cell cultures responding to CII.
This finding differs from observations using naturally occurring CD4⫹CD25⫹ cells, in which both proliferation and cytokine production are suppressed (30).
There are several possible reasons for this difference.
First, because of technical constraints of cell isolation,
we used a relatively low ratio of CD4⫹CD25⫹ cells to
responder ILN cells. Although regulatory CD4⫹CD25⫹
cells suppressed anti-CD3–stimulated responses of
CD4⫹CD25⫺ cells at this ratio, the extent of inhibition
was suboptimal (30). Second, the CD4⫹CD25⫹ regulatory T cell population modulated by EtxB may have
functional activity different from that in naive animals.
This difference could result from altered activation of
naturally occurring CD4⫹CD25⫹ cells or may be a
consequence of EtxB-mediated induction of a novel cell
population that shares this phenotype. Finally, in our
culture system, CD4⫹CD25⫹ cells were used to modulate a Th1-dominated antigen-specific response. This
contrasts with other systems in which CD4⫹CD25⫹
cells are assessed for their ability to modulate naive T
cell responses or the responses of CD4⫹CD25⫺ cells to
polyclonal stimulation (30,37).
The reduction in IFN␥ release observed following EtxB treatment, and in cocultures of ILN cells and
CD4⫹CD25⫹ cells from EtxB-treated mice, is consistent with the attenuation of arthritis. IFN␥ is a primary
switch factor for production of IgG2a antibodies, which
are critical for the establishment of joint inflammation in
CIA. Nevertheless, although anti-CII IgG2a antibodies
were reduced following EtxB treatment, the observed
reduction was similar between mice given diseaseprotecting intranasal or high-dose intragastric treatment
and mice receiving a non–disease-protecting low intragastric dose. Taken together with the observation that
IgG2a anti-CII antibody levels were not reduced in
recipients of CD4⫹ cells from EtxB-treated mice (data
not shown), these findings highlight the importance of
modulatory effects of EtxB that are independent of
antibody production. Such effects may include a reduction in IFN␥-dependent macrophage and neutrophil
activation/recruitment into joint tissue. However, alterations to the anti-CII response, additional to suppression of IFN␥ production, may also be critical for EtxBmediated protection against CIA.
How might mucosal administration of EtxB enhance the activity of T cells that can regulate the
response to CII? As an autoantigen, CII is probably
involved in driving normal processes that maintain peripheral tolerance. Indeed, T cells from DBA/1 mice fail
to proliferate in response to autologous CII challenge
but secrete some IFN␥ (38). The enhanced regulatory
activity of CD4⫹CD25⫹ cells following EtxB treatment
was not unique to the response to CII. Similar regulatory
activity was evident when CD4⫹CD25⫹ cells from
EtxB-treated mice were added to ILN cell cultures
responding to KLH. Although the extent of IFN␥ suppression was lower than that seen in cultures with CII,
the higher magnitude of the ILN response to KLH may
mean that increased numbers of CD4⫹CD25⫹ cells
would be required to achieve a similar level of suppression. Although these findings suggest that EtxB does not
protect against CIA by activating existing CII-specific
regulatory T cells, they do not preclude the possibility
that EtxB influences differentiation of recently activated
T cells, causing them to acquire regulatory function.
Interestingly, we found that CD4⫹CD25⫹ cells from
EtxB-treated mice not given CII/CFA were unable to
suppress IFN␥ by CII-responsive ILN cells (data not
Altered T cell activation by EtxB could result
from its trafficking to lymphoid tissues that drain sites of
antigen/CFA challenge. This is unlikely, however, because relatively low doses of EtxB are protective, and
high-affinity receptors for the B subunit are found
throughout the body. Alternatively, recently activated
CII-primed cells may enter sites draining EtxB and
encounter a modified microenvironment during a critical period in their differentiation. Uncommitted activated T cells recirculate throughout peripheral lymphoid
tissues (39), and EtxB can modulate the activation of
several cell types (16). For example, EtxB triggers IL-10
secretion by monocytes and inhibits their release of
IL-12 (40). If IL-10 secretion was stimulated in the
lymphoid tissues draining mucosal EtxB, it might favor
generation of regulatory T cells, as described in other
systems (36). Such an effect would influence the immune
response to any immunizing antigen.
It is conceivable that the regulatory T cells identified in our experiments are triggered following recognition of EtxB itself. The activation and differentiation
of EtxB-specific T cells would also occur within the
modified local environment that is likely established
following B subunit–receptor interaction. As a result,
EtxB-specific regulatory T cells may be generated and
enter distant sites of inflammation, where they suppress
the ongoing immune response in an antigen-nonspecific
manner. In support of this premise, endogenous
CD4⫹CD25⫹ regulatory cells require activation
through T cell receptor engagement to become suppressive, but once stimulated, their subsequent regulatory
activity is antigen nonspecific (41). A final possible
explanation of why enhanced regulatory activity is found
within the CD4⫹CD25⫹ spleen cell population after
EtxB administration and CII/CFA challenge is that
either soluble factors (e.g., IL-10, transforming growth
factor ␤, prostaglandins) or non–antigen-specific cells
(e.g., natural killer T cells) are released at sites local to
mucosal B subunit delivery. These may then influence
the nature of T cell differentiation at distant inflammatory tissues. Further investigations will be required to
determine the specificity of the regulatory T cell population triggered by EtxB and to define the exact processes by which EtxB enhances their activity.
In conclusion, our study highlights the potential
of EtxB as a therapeutic agent in RA. Furthermore, the
finding that disease prevention by EtxB is linked to
enhanced activity of a population of regulatory T cells
indicates that EtxB may have wider applicability as a
means of interfering in the processes driving related
inflammatory disorders. Elucidation of the precise
mechanisms by which EtxB–receptor interaction enhances regulatory cell activity will be required in order
to maximize the chances of its successful application in
human disease.
We thank C. J. Elson and S. Parry for their helpful
advice, S. Sreckovic for his technical expertise in cell sorting, J.
Baker and A. J. Coad for assistance with histology, and M.
Kenny for toxin preparation and purification.
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