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*
Department of Biology, Thomas More College, Crestview Hills, KY 41017;
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH 45267
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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than those from water-fed control animals.
These results indicate that while TGF-ß1 may play a role in
suppression, inhibitory cytokines are not the exclusive mechanism by
which low dose oral tolerance is induced. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown that oral tolerance is due primarily to the inactivation of the CD4+ cell, which plays a pivotal role in the induction of both humoral and cellular immune responses (10). However, the mechanism by which Ag-specific CD4+ cells are inactivated remains a point of considerable controversy. Experiments by others have indicated that the secretion of cytokines is responsible for mediating suppression, a phenomenon termed active or bystander suppression (3, 11). The cytokine primarily responsible for this suppression was suggested to be TGF-ß1 (12, 13). In contrast, we and others have produced evidence indicating that direct inactivation of the Ag-specific CD4+ cell occurs via a phenomenon known as anergy (the functional inactivation of a cell) rather than by active suppression (14, 15, 16, 17, 18, 19, 20).
To resolve the controversy over which mechanism was responsible for the generation of oral tolerance, it was proposed that the dose of Ag fed was the critical factor (21). Feeding high doses of Ag resulted in tolerance characterized by anergy or deletion, with little or no active suppression evident; in contrast, low doses of fed Ag resulted in tolerance induction characterized by the presence of regulatory cells secreting cytokines (21, 22). In addition to TGF-ß1, increased production of IL-4 and IL-10 has been observed in both lymph node cells and CD4+ clones derived from mice fed low doses of Ag (23, 24, 25, 26).
Given the pivotal role that TGF-ß1 appears to play in the generation
of low dose oral tolerance, it is essential to clarify its function in
this process. Three isoforms of TGF-ß exist in mammals: TGF-ß1,
TGF-ß2, and TGF-ß3. TGF-ß1 has been identified as an
imunoregulatory molecule with both immunogenic and immonsuppressive
properties depending on the cellular environment (27, 28). Systemic
administration of TGF-ß1 has been shown to suppress immune responses,
while local administration enhanced inflammatory responses (28).
Recently, knockout mice were generated using embryonic stem cells with
a disrupted TGF-ß1 gene (29, 30). TGF-ß1 null mutants exhibit
inflammation leading to tissue necrosis of numerous organs,
particularly heart and stomach. These lesions are characterized by
elevated levels of peripheral lymphocytes and immature neutrophils (29)
as well as enhanced adhesiveness of the TGF-ß1 null leukocytes (31).
Gross development of TGF-ß1 null mice appears normal until
approximately the third week after birth, at which time they exhibit an
acute wasting syndrome that quickly leads to death. As it is difficult
to induce oral tolerance in mice before 2 to 3 wk of age, it was
previously impossible to use these mice as models to assess the role of
TGF-ß1 in the generation of oral tolerance. However, due to recent
advances, the lives of these TGF-ß1 null mice can now be extended to
approximately 40 days or more by repeated injection of an Ab
(anti-CD11
) to LFA-1 (32). LFA-1 is an integrin found on
lymphocytes that allows them to bind to other cells, such as ICAM-1 on
endothelial cells, thus facilitating transendothelial migration. It has
been suggested that the anti-CD11 mAb inhibits the extravasation
and migration of lymphocytes into surrounding tissues (i.e., heart and
stomach) and thus delays the onset of organ inflammation and subsequent
death (32).
Given the above finding, it is now possible to use
anti-CD11-treated TGF-ß1 null mice as models to evaluate the
role of TGF-ß1 in the generation of oral tolerance. Therefore,
anti-CD11-treated TGF-ß1 null mice were fed both high and low
doses of OVA, and the ability of these mice to establish oral tolerance
was assessed. The results of our studies indicate that high dose
feeding did not interfere with oral tolerance induction in TGF-ß1
null mice. However, we also found that a significant number of the
TGF-ß1 null mice fed low doses of OVA were able to be tolerized; this
was an unexpected result, since TGF-ß1 is thought to play a primary
role in the induction of low dose oral tolerance. In addition, cytokine
analysis revealed that levels of other putative inhibitory cytokines
(IL-4 and IL-10) were decreased, rather than enhanced, in tolerized
TGF-ß1 null mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male and female (129 x CF1) mice
heterozygous for the TGF-ß1 null allele were housed in the animal
facility at the University of Cincinnati Medical Center under barrier
housing conditions. Neonates from heterozygote intercrosses were
identified as TGF-ß1 null mice within 48 h of birth using the
PCR techniques described below. Neonates identified as either control
mice (wild-type, +/+, or heterozygous, +/-) or TGF-ß1 null mice
(-/-) were immediately given injections of anti-CD11 (60
µg/injection) every other day from day 2 after birth until weaning
(approximately day 18 after birth). All experiments used 3- to
4-wk-old mice.
RT-PCR genotyping
RT-PCR genotyping was performed as described previously (29). Briefly, mRNA was isolated from tail clips of newborn mice; the isolated mRNA was reverse transcribed and examined by PCR analysis using an upstream primer (5'-GAGAAGAACTGCTGTGTGCG-3') and a downstream primer (5'-GTGTCCAGGCTCCAAATATAGG-3') corresponding to the exon 6 sequences flanking the neo insert in the TGF-ß1 null mutant. PCR was performed on a thermal cycler in a 20-µl volume at 95°C for 20 s, at 55°C for 50 s, and at 72°C for 1 min for 30 cycles. Amplified products were size fractionated by electrophoresis through agarose and visualized by UV illumination on ethidium bromide-stained gels. Positive controls consisted of total RNA from spleen cells cultured for 48 h with Con A.
Reagents
Chicken egg albumin (OVA; grade V) and Con A were obtained from
Sigma (St. Louis, MO). Anti-CD11 was a gift from Genentech (San
Francisco, CA).
Effect of anti-CD11 treatment on immune responsiveness
Neonatal mice (+/+) were divided into two groups and injected
with 0.02 ml of either PBS or anti-CD11 (60 µg) from day 2
until day 18 after birth. On day 31 after birth, all mice were injected
with 10 µg of OVA in IFA in the footpad and tail base. Ten days
later, mice were bled via the retro-orbital plexus and then sacrificed;
their popliteal lymph nodes
(PLN)3 were removed, and
cells were incubated in vitro with OVA (100 µg/ml, final
concentration). Serum Ab responses were assessed using ELISA, and in
vitro lymphocyte responsiveness was determined using liquid
scintillation spectrometry.
Induction of immune responsiveness in anti-CD11-treated
TGF-ß1 null mice
Anti-CD11-treated TGF-ß1 null (-/-) mice and control (+/+
or +/-) mice were injected with 10 µg of OVA in IFA in the footpad
and tail base on day 31 after birth; 10 days later, mice were
sacrificed, PLN were removed, and cells were incubated in vitro with
either Con A (2.5 µg/ml, final concentration) or OVA (1000 or 100
µg/ml, final concentration). In vitro lymphocyte responsiveness was
determined using liquid scintillation spectrometry.
Induction of oral tolerance in anti-CD11-treated TGF-ß1
null mice
Anti-CD11-treated TGF-ß1 null (-/-) mice and control (+/+
or +/-) mice were each divided into two groups; mice in one group were
orally tolerized by gavaging either 10 to 20 mg (high dose) or 1 mg
(low dose) of OVA/mouse dissolved in 0.5 ml of water three times over a
3-day period, while mice in the second group received water only. Eight
days after the last feeding, all mice were injected in the tail base
and footpad with 10 µg of OVA in IFA. Ten days after immunization,
mice were then sacrificed, and lymphocytes were obtained from the PLN
to assess the OVA-specific proliferative response.
Ab titer determination
Ab titer was determined as described previously (19). Briefly, serum samples were serially diluted on plates coated with OVA and incubated for 1 h at 37°C. Goat anti-mouse IgG coupled to alkaline phosphatase (1/1000; Boehringer Mannheim, Indianapolis, IN) was then added, and the plate was incubated for an additional 1 h at 37°C. 4-Nitrophenylphosphate (Boehringer Mannheim), at a concentration of 1 mg/ml, was added as substrate, and the color change was measured using a Bio-Rad microplate reader (Bio-Rad, Hercules, CA) at a wavelength of 405 nm. All samples were pooled, assayed in duplicate, and expressed as Ab titer ± SD. Standard curves were run each time an assay was performed using hyperimmune anti-OVA antisera, and sample titers were calculated based on dilutions that resulted in absorbance equal to 1/400 diluted standard sera. Serum samples were considered positive if their titers exhibited a fivefold or greater increase over titers of unimmunized mice (previous studies have consistently shown that unimmunized mice exhibit titers <50; data not shown).
Assessment of in vitro lymphocyte proliferative responses
Proliferative responses were assessed as described previously (19). Briefly, 10 days after footpad and tail base immunization, draining PLN were removed, and single cell suspensions were prepared in supplemented RPMI 1640 medium and 5% heat-inactivated FCS. Cells were then aliquoted at 5 x 105 cells/well in 96-well flat-bottom plates along with OVA at a concentration of 1000 or 100 µg/ml (maximal stimulation from previous experiments was seen with these doses) and incubated at 37°C in humidified 5% CO2 for 48 h. Cells were pulsed for 18 h with 1 µCi of [3H]thymidine and harvested onto glass-fiber filtermats using a Skatron multiple automated sample harvester (Skatron, Sterling, VA). Radioactivity was determined using liquid scintillation spectrometry. Addition of 100 µg/ml of an unrelated Ag, cytochrome c, was added to control wells to assess nonspecific proliferation.
Assessment of cytokine secretion in anti-CD11-treated
TGF-ß1 null mice fed low doses of Ag
Anti-CD11-treated TGF-ß1 null mice (-/-) and control (+/+
or +/-) mice were divided into two groups. Mice in one group were
orally tolerized by gavaging 1.0 mg of OVA/mouse dissolved in 0.5 ml of
water three times over a 3-day period, while mice in the second group
received water only. Eight days after the last feeding, all mice were
injected in the tail base and footpad with 10 µg of OVA in IFA. Ten
days after immunization, mice were sacrificed, lymphocytes were
obtained from the spleen and PLN, and single cell suspensions were
prepared. RBC were lysed from spleen cell suspensions using
Tris-buffered ammonium chloride. Spleen cells and PLN cells were then
aliquoted at 8 x 105 and 5 x
105 cells/well, respectively, in 24-well flat-bottom plates
along with either medium or 100 µg/ml OVA. All cells were incubated
at 37°C in humidified 5% CO2 for 48 h. Supernatants
were then harvested and stored at -70°C.
IL-4, IL-10, and IFN- production was quantified using sandwich ELISA
techniques. The concentrations of IL-4 and IFN- in cell supernatants
were quantified using murine IL-4 and IFN- ELISA kits, respectively
(Genzyme, Cambridge, MA). The concentration of IL-10 in cell
supernatants was quantified using an Ag capture ELISA with Abs (capture
and secondary biotinylated Abs) purchased from PharMingen (San Diego,
CA); murine rIL-2 was also purchased from PharMingen. All ELISAs were
conducted according to the manufacturer’s recommendation.
Statistical analysis
Due to the small number of TGF-ß1 null mice available from
each litter, experiments were repeated a minimum of three times.
Results obtained from the in vitro lymphocyte responsiveness
experiments are indicated as the counts per minute ± SD of
triplicate cultures. The statistical significance of differences among
experimental values was determined using the Student’s t
test for multiple comparisons. A value of p 
0.05 was
considered significant. Stimulation indexes were generated for each
experimental group or mouse by dividing the total counts per minute by
the background counts per minute.
For cytokine analysis experiments, cell supernatants from mice in each
group were pooled and run in triplicate; the SD was determined. The
statistical significance of differences among experimental values was
determined using Student’s t test for multiple comparisons.
A value of p 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment on immune responsiveness and
the induction of oral tolerance
Initially, it was necessary to determine whether anti-CD11
treatment affected the ability of treated mice to mount an immune
response. To assess this, wild-type mice were treated with
anti-CD11 up until day 18 after birth (the treatment time used
in all subsequent experiments), while controls were injected with PBS.
As seen from the results in Figure 1
,
treatment with anti-CD11, while having a slight inhibitory
effect, did not result in a drastic reduction in cellular
proliferation. In contrast, humoral responses of the mice in the
anti-CD11-treated group exhibited profound inhibition and were
not used for assessment purposes in future investigations.
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-treated mice
was slightly less effective than that in untreated mice (data not
shown), the results shown in Figures 2
and 3 (for wild-type mice) clearly show that significant oral tolerance
can be induced in these mice using both high and low dose feeding
regimens.
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-treated TGF-ß1 null
mice
The prolonged life of TGF-ß1 null mice treated with
anti-CD11 permitted these animals to be the subject of oral
tolerance experiments. However, since previous reports have indicated
that lymphocytes from TGF-ß1 null mutant mice (without
anti-CD11 treatment) may be maximally stimulated inherently
(33), we first determined whether lymph nodes from
anti-CD11-treated TGF-ß1 null mutant mice exhibited an
increased proliferative response to a given stimulus. To test this,
cells were incubated with either the mitogen Con A or various doses of
OVA Ag.
The results showed that the response of PLN from
anti-CD11-treated TGF-ß1 null mutant mice to Con A stimulation
was similar to that of anti-CD11-treated wild-type mice
(214,219 ± 8,046 and 170,114 ± 19,098, respectively; counts
are over background). These data suggested that lymphocytes from
anti-CD11-treated TGF-ß1 null mutant mice were not maximally
stimulated and were capable of proliferating in response to antigenic
stimulation. This was confirmed by results from experiments showing
that TGF-ß1 null mutant mice were able to respond specifically to
various doses of a given Ag (Table I);
the in vitro proliferative response to an unrelated Ag, cytochrome
c, was <10% over background (data not shown). It is
interesting to note that the proliferative responses of the PLN from
TGF-ß1 null mutant mice were almost always significantly higher than
those of PLN from the wild-type controls (Table I). However, when
stimulation indexes were generated for comparative purposes, little if
any difference between wild-type and mutant mice could be observed.
Stimulation indexes for each group were as follows. In Experiment 1,
values for lymphocytes from +/+ and -/- mice at an in vitro
concentration of 1000 µg/ml were 7.1 and 6.3, respectively, and at an
in vitro concentration of 100 µg/ml, they were 4.4 and 4.8,
respectively. In Experiment 2, values for lymphocytes from +/+ and
-/- mice at an in vitro concentration of 100 µg/ml were 4.6 and
4.0, respectively, and at an in vitro concentration of 10 µg/ml, they
were 3.1 and 3.7, respectively. In Experiment 3, values for lymphocytes
from +/+ and -/- mice at an in vitro concentration of 1000 µg/ml
were 6.7 and 5.7, respectively; at an in vitro concentration of 100
µg/ml, they were 4.6 and 5.0, respectively; and at an in vitro
concentration of 10 µg/ml, they were 3.6 and 4.0, respectively.
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-treated
TGF-ß1 null mice
To test whether TGF-ß1 played a significant role in the
generation of high dose oral tolerance, PLN from mice fed 10 to 20
mg/mouse of OVA for 3 days were stimulated in vitro with either 100 or
1000 µg/ml of OVA; the results are shown in Figure 2
. In all
experiments performed, high doses of OVA resulted in Ag-specific
tolerance in the anti-CD11-treated wild-type/heterozygote mice,
indicating that the high dose feeding regimen used was capable of
inducing tolerance. Similarly, all the TGF-ß1 null OVA-fed mice
showed highly significant levels of suppression. In addition, all
OVA-fed mice had lower stimulation indexes than the corresponding
water-fed control mice (data not shown). It should be noted that the
suppression observed in the TGF-ß1 null OVA-fed mice did not appear
to be as profound as that in the wild-type mice.
Induction of low dose oral tolerance in anti-CD11-treated
TGF-ß1 null mice
To test whether TGF-ß1 played a significant role in the
generation of low dose oral tolerance, the procedure described above
was used, except that 1 mg/mouse of OVA was fed for 3 consecutive days.
Results showed that anti-CD11-treated wild-type/heterozygote
mice were tolerized compared with water-fed controls, indicating that
the low dose feeding regimen used was capable of inducing tolerance
(Fig. 3). Of the nine TGF-ß1 null
OVA-fed mice, seven exhibited significant suppression compared with the
TGF-ß1 null H20-fed control(s) when stimulated with 100 µg/ml of
OVA in vitro. The lymph node cells from one TGF-ß1 null OVA-fed mice
mouse showed no significant change in proliferation compared with
controls (Fig. 3A), while another showed a
significant increase in proliferation (Fig. 3D). When
the PLN of TGF-ß1 null OVA-fed mice were stimulated with 1000 µg/ml
of OVA in vitro, only four of the nine mutant mice exhibited a
suppressed proliferative response; three showed no change in the
proliferative response compared with the water-fed controls, and one
showed a significant increase in the proliferative response (Fig. 3).
All OVA-fed mice that had counts per minute values significantly lower
then the corresponding water-fed control mice also had lower
stimulation indexes (data not shown). Taken together, these results
indicate that TGF-ß1 null mice fed low doses of OVA are able to be
tolerized, but less effectively than the controls.
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levels in
anti-CD11-treated TGF-ß1 null mice fed low doses of OVA
Since seven of nine anti-CD11-treated TGF-ß1 null mutant
mice fed a low dose of OVA exhibited a significantly decreased
proliferative response when stimulated with 100 µg/ml of OVA in
vitro, this indicated that factors other than TGF-ß1 must be involved
in the induction of low dose oral tolerance. Studies by others have
shown that secretion of cytokines other than TGF-ß1 may play a role
in inducing oral tolerance. Therefore, IL-4 and IL-10 levels were
quantitated in stimulated spleen cells and lymph node cells from
wild-type/heterozygote and TGF-ß1 null mutant mice fed low doses of
OVA. The results presented in Table II
show that IL-4, IL-10, and IFN- levels were reduced in spleen cells
from mice fed OVA compared with those in spleen cells from mice fed
water (with the exception of IL-4 in the wild-type control group, Expt.
2). Similarly, levels of IL-10 and IFN- were reduced in PLN from
mice fed OVA compared with those in PLN from mice fed water (Table III); levels of IL-4 could not be
assessed in PLN due to the lack of detectable levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in vivo significantly prolonged their life-span (32).
Thus, it is now possible to feed Ag to TGF-ß1 null mice and determine
the role of TGF-ß1 in the generation of oral suppression.
The results of preliminary studies showed that mice treated with
anti-CD11 were capable of mounting an immune response to
parenterally administered Ag at the level of cellular proliferation.
However, humoral responses were drastically reduced in
anti-CD11-treated mice, suggesting that the concentration of
anti-CD11 used in our experiments interfered with critical
LFA-1-dependent associations needed for Ab production. Therefore,
assessment of oral tolerance induction was restricted to cellular
proliferation assays.
Lymphocytes from TGF-ß1 null mutant mice treated with
anti-CD11 in vivo were capable of responding to Ag-specific
stimulation in a dose-dependent fashion. It is interesting to note that
in almost all cases, cells from anti-CD11-treated mutant mice
proliferated more vigorously in response to antigenic stimulation than
did anti-CD11-treated wild-type mice. However, when stimulation
indexes were calculated, a consistent pattern of enhanced proliferation
in anti-CD11-treated mutant mice was not observed.
With respect to the induction of oral tolerance, we have shown that,
compared with controls, lymph node cells from anti-CD11-treated
TGF-ß1 null mutant mice fed high doses of Ag exhibited suppressed
proliferation in response to in vitro stimulation with 1000 and 100
µg/ml of OVA. These results indicate that lymphocytes from
anti-CD11-treated TGF-ß1 null mutant animals are being
suppressed and confirm the findings of others that cytokines do not
play a primary role in the induction of high dose oral tolerance
(14, 15, 16, 17, 18, 19, 21). However, it should be noted that although lymph node
cells from all anti-CD11-treated TGF-ß1 null mice fed high
doses of OVA were significantly suppressed, this suppression appeared
to be less effective than that in similarly treated wild-type controls.
Such results may be explained by the aforementioned increase in
responsiveness of lymphocytes from TGF-ß1 null mice. A generalized
increase in the proliferative response might tend to diminish and thus
obscure any observable suppression occurring in these cells. An
alternative explanation is that TGF-ß1 may also play a role (albeit a
minor one) in high dose oral tolerance.
In contrast to results obtained with high dose feeding regimens,
anti-CD11-treated TGF-ß1 null mutant mice fed low doses of OVA
varied in their ability to generate oral tolerance. Lymph node cells
from mutant mice fed low doses of OVA were not tolerized as efficiently
as those from their wild-type controls; lymph node cells from seven of
nine and four of nine mice were significantly suppressed when incubated
in vitro with 100 and 1000 µg/ml OVA, respectively. However, the
ability of the majority of mice to exhibit a suppressed proliferative
response when incubated in vitro with 100 µg/ml of OVA would argue
that TGF-ß1 may play a partial, but not exclusive, role in the
induction of low dose oral tolerance. The possibility that suppressed
proliferative responses in TGF-ß1 null mutant mice may be obscured
due to heightened cellular responsiveness cannot be excluded.
The significant suppression observed in the majority of TGF-ß1 null
mice fed low doses of OVA suggests that factors other than the
production and secretion of TGF-ß1 are involved in the induction of
low dose oral tolerance. It has been reported that feeding low doses of
OVA stimulates the production of IL-4 and IL-10 in addition to that of
TGF-ß1 with or without in vitro stimulation (21, 23). It has been
proposed that these cytokines, in a manner similar to that of TGF-ß1,
may play a role in inhibition of the immune response via active
suppression (21, 23). Such a mechanism assumes the preferential
generation of Th2 cells (or putative Th3 cells) while Th1 cells are
down-regulated. It was possible that in our system, Th2 cells secreting
elevated levels of IL-4 and IL-10 were at least partially responsible
for the suppressed proliferative response in TGF-ß1 null mice fed low
doses of OVA. To test for this possibility, levels of IFN- (a Th1
cytokine), IL-4, and IL-10 were assessed in anti-CD11-treated
TGF-ß1 null mutant mice fed either water or low doses of OVA. Our
results show that after in vitro stimulation, levels of all three of
these cytokines decreased, rather than increased, in OVA-fed mice
compared with those in water-fed controls. In addition, although
constitutive production of IL-4, IL-10, and IFN- was observed, no
significant increase in the levels of these cytokines was found in any
of the unstimulated lymphocytes from OVA-fed mice (wild type or
mutant). These findings imply that a generalized suppression mechanism
was induced in low dose OVA-fed mice that affects both Th1 and Th2
cells. Such a mechanism is more consistent with the phenomenon of
anergy and/or deletion rather than active suppression. Repeated
attempts were made to determine whether anergy served as a mechanism
for the observed suppression by incubating PLNs from OVA-fed mutant
mice in vitro with rIL-2. However, due to extensive cell death during
the incubation period, conclusive results could not be obtained. The
inability of enough cells to survive during the experimental period
could be due in part to the fact that lymphocytes were obtained from
129 x CF1 mice; the CF1 mouse is a
partially outbred strain.
Using the TGF-ß1 null mice in our studies has the primary advantage
of eliminating a major variable that has been implicated in generating
low dose oral tolerance. Both TGF-ß1 null mice as well as wild-type
mice were used to ensure that the results observed in mutant mice were
not due to compensatory mechanisms unique to the TGF-ß1 null mice.
Our data support the findings of others, which indicate that clonal
anergy is the primary mechanism involved in inducing oral tolerance
regardless of whether high or low doses of Ag are fed (34, 35). It is
uncertain why our results differ from those of others, which suggest
that active suppression is the primary mechanism for inducing low dose
oral tolerance (36). The discrepancy could be due to the fact that
different strains of mice were used or that mice in our system were
treated with anti-CD11. In preliminary experiments it was shown
that anti-CD11 treatment had a profound, detrimental effect on
the production of humoral Ab. It is conceivable that this treatment may
have also altered the immune response at the T cell level, thus
favoring anergy over active suppression in our model. It is also
possible that the amount of Ag fed (1 mg, three times) was too high to
be considered low dose in 129 x CF1 mice. However, in
previous studies by Weiner and colleagues, 1 mg of Ag fed five times
was considered a low dose (21, 37). In addition, preliminary studies
designed to assess the concentration of a low dose in our experimental
model indicated inconsistent suppression of cellular proliferation in
wild-type mice if fed doses were lowered to 0.5 mg, three times (data
not shown). Whether additional feedings of OVA (i.e., 1 or 0.5 mg of
OVA fed five times) are needed to observe increased cytokine levels
remains to be determined.
In summary, our results indicate that both high and low dose oral tolerance can be generated in mice in the absence of TGF-ß1. Our findings suggest that mechanisms other than the secretion of inhibitory cytokines may be associated with low dose oral tolerance and that these mechanisms may be the primary ones involved in our model system.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. K. Siobhan Barone, Department of Biology, Thomas More College, 333 Thomas More Pkwy., Crestview Hills, KY 41017.
3 Abbreviation used in this paper: PLN, popliteal lymph nodes.
Received for publication December 4, 1997. Accepted for publication March 3, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 K. M. SMITH, A. D. EATON, L. M. FINLAYSON, and P. GARSIDE Oral Tolerance Am. J. Respir. Crit. Care Med., October 1, 2000; 162(4): S175 - 178. [Abstract] [Full Text] [PDF]
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D. C. Tsitoura, R. H. DeKruyff, J. R. Lamb, and D. T. Umetsu Intranasal Exposure to Protein Antigen Induces Immunological Tolerance Mediated by Functionally Disabled CD4+ T Cells J. Immunol., September 1, 1999; 163(5): 2592 - 2600. [Abstract] [Full Text] [PDF]
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J. M. Benson, S. S. Stuckman, K. L. Cox, R. M. Wardrop, I. E. Gienapp, A. H. Cross, J. L. Trotter, and C. C. Whitacre Oral Administration of Myelin Basic Protein Is Superior to Myelin in Suppressing Established Relapsing Experimental Autoimmune Encephalomyelitis J. Immunol., May 15, 1999; 162(10): 6247 - 6254. [Abstract] [Full Text] [PDF]
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M.-N. Kweon, K. Fujihashi, Y. Wakatsuki, T. Koga, M. Yamamoto, J. R. McGhee, and H. Kiyono Mucosally Induced Systemic T Cell Unresponsiveness to Ovalbumin Requires CD40 Ligand-CD40 Interactions J. Immunol., February 15, 1999; 162(4): 1904 - 1909. [Abstract] [Full Text] [PDF]
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