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Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2
either decreased or showed no change, with the exception of a small
transient increase in IL-2 at 6 h after a low dose. Increases in
IL-4 and IL-10 were found in the dome of the Peyer’s patch, and
increases in TGF-ß were observed in the interfollicular region and
the villi. IL-10 was also substantially increased in the villi. IL-4
and IL-10 were produced predominately by CD4+ T
cells. TGF-ß was found predominately in macrophages and
CD4+ T cells. Peyer’s patches had a marked up-regulation
of TGF-ß mRNA as measured by RT-PCR. These results demonstrate the
differential activation of cytokine production in discrete regions of
gut-associated lymphoid tissue. The induction of cytokines known to
inhibit autoimmune disease at the site of Ag absorption indicates an
important role for the mucosal immune system in the establishment of
oral tolerance. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the consequences of oral Ag administration is the induction of oral tolerance. Oral tolerance is evidenced by the suppression of humoral and cell-mediated responses to immunization with Ag following oral administration of the same Ag (5, 6). Depending upon the dose of Ag that was fed, oral tolerance may be mediated by anergy, deletion, or the induction of regulatory T cells (7, 8, 20). Regulatory T cells generated during the course of oral tolerance function by producing suppressive cytokines such as IL-4, IL-10, and TGF-ß (9, 10). TGF-ß is an important gut-associated cytokine, as it provides help for IgA production, and regulatory T cells induced by oral Ag preferentially secrete TGF-ß. In the rat, TGF-ß-secreting regulatory cells can be found in Peyer’s patches that are removed 24 to 48 h after one feeding of myelin basic protein (MBP) and can adoptively transfer protection in vivo (11). In murine models, CD4+ regulatory cells from mesenteric lymph nodes (MLNs) which secrete TGF-ß, IL-4, and IL-10 have been cloned (9). Oral tolerance has been used to successfully treat cell-mediated autoimmune diseases in animals and is currently being tested in humans (12). In the present study, we used OVA TCR-transgenic mice to examine the cytokine milieu in the Peyer’s patch and lamina propria in response to orally administered OVA. Low-dose and high-dose feeding was compared. The transgenic model enabled us to characterize the T cell response to feeding in vivo; such a response could not be defined in conventional animals without immunization due to the low frequency of Ag-specific T cells. In addition, the transgenic model allowed us to examine immunologic events associated with the generation of immune responses in GALT.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs were obtained as follows: rat anti-mouse IL-2 (clone
S4B6) and rat anti-mouse IL-10 (clone JESJ-2A5) were purchased from
PharMingen (San Diego, CA); rabbit anti-porcine TGF-ß was
obtained from R&D Systems (Minneapolis, MN); rat anti-mouse IL-4
(BVD6.24G) was provided by Dr. A. Lichtman (Harvard Medical School,
Boston, MA); and hamster anti-mouse IFN- was purchased from
Genzyme (Cambridge, MA). The specificities of these Abs were
established by bioassays and by blocking staining with recombinant
cytokines.
Ab concentrations were as follows: IL-10, IL-2 and IFN- (5 µg/ml),
IL-4 (hybridoma supernatant); TGF-ß (2 µg/ml).
The secondary Abs used for immunofluorescence staining of IL-4 and
TGF-ß were R-phycoerythrin (R-PE)-conjugated goat anti-rat IgG
(Caltag, Burlingame, CA) and FITC-conjugated goat anti rabbit IgG
(Caltag), respectively. IFN- was labeled with FITC-conjugated goat
anti-hamster IgG (Caltag). For double staining, FITC- or
R-PE-labeled anti-murine CD4 or CD8 were obtained from PharMingen.
Macrophages (M
) were labeled with rat anti-mouse F4/80 (Caltag)
followed by R-PE-labeled anti-rat IgG (Caltag). Abs for
immunofluorescence were used at a concentration of 5 µg/ml.
Mice
OVA-specific TCR transgenic mice (BALB/c) expressing the
V
1.3/Vß8.2 TCR on 97% of peripheral T cells (20) were fed 0.5 mg
or 500 mg of OVA either one or five times on alternate days. Tissues
were taken from the jejunum of the small bowel. A section of bowel
(8–12 cm) that was localized 
2 to 3 cm beyond the pyloric sphincter
was examined. Peyer’s patches were harvested at 1, 2, 4, 6, 24, and
48 h after a single low-dose feeding and at 48 h after five
low-dose feedings. Tissues from animals given a single high-dose
feeding or five high-dose feedings were harvested 48 h after the
last feeding. Control mice that had been designated unfed were given
PBS instead of OVA. Some controls were fed hen egg lysozyme (HEL). In
an initial series of experiments, we fed animals PBS or equivalent
amounts of HEL or OVA and found no difference between HEL- and PBS-fed
animals in terms of immunohistochemistry or the release of cytokines
after secondary in vitro stimulation.
Preparation of cryosections
Peyer’s patches and villus regions from OVA TCR-transgenic mice were excised, rinsed in PBS, immersed in OCT freezing medium (Miles, Elkhart, IL), and snap frozen in liquid nitrogen-cooled isopentane. Tissues were sectioned at -20°C, fixed for 5 to 10 s in chilled acetone, and stored at -70°C.
Immunohistochemistry
Frozen sections were thawed and fixed in acetone for 2 min. Endogenous peroxidase activity was quenched by incubation in periodic acid (0.005 M in water, pH 2.45) for 10 min followed by washing in PBS and immersion in 0.003 M sodium borohydride for 30 min (13). After washing, tissues were incubated for 20 min in diluted normal blocking serum that was prepared from the species from which the secondary Ab is made. Endogenous biotin was blocked by 1-h incubations in avidin D followed by biotin (vector blocking kit, Vector Laboratories, Burlingame, CA). Sections were rinsed in PBS and immersed in primary antiserum that had been diluted in blocking serum for incubation overnight. Next, sections were washed for 10 min in PBS and incubated in diluted, biotinylated, secondary Ab for 1 h. After washing, sections were incubated in Vectastain Elite avidin-biotin complex reagent, washed again, and incubated in peroxidase substrate solution containing 3,3'-diaminobenzidine (DAB Substrate Kit, Vector Laboratories) for 5 to 7 min. Sections were washed for 5 min in tap water, counterstained in hematoxylin, cleared, and mounted.
Frozen sections were prepared for immunofluorescence microscopy by
fixation in acetone for 2 min followed by incubation for 20 min in
diluted normal blocking serum as described above. To block nonspecific
staining by fluorochrome-conjugated Abs that were mediated by FcRs,
sections were incubated with 2.4G2 Ab (PharMingen, 10 µg/ml) that was
directed against FcII/IIIRs. For cytokine labeling, sections were
incubated overnight in primary antiserum. After washing for 10 min in
PBS, sections were incubated in R-PE- or FITC-labeled secondary Ab for
1 to 2 h. For double staining, sections were incubated in FITC- or
R-PE-labeled anti-CD4 (PharMingen) or CD8 (PharMingen) for 1
h. M were incubated with rat anti-mouse F4-80 for 1 to 2 h
followed by R-PE-labeled anti-rat IgG (Caltag) for 1 h.
Sections were washed in PBS and mounted in Vectashield mounting medium
(Vector Laboratories). The specificity of fluorescence staining was
demonstrated by preincubation of the Ab with its target Ag or by
incubation of tissue with unlabeled anti-cytokine Ab before
staining with the fluorochrome-conjugated form of the same
anti-cytokine Ab. Nine sections from three animals (three per
animal) were examined both for animals fed 0.5 mg once (or 500 mg) or
five times. No staining was observed following preincubation of the Ab
with its target Ag (100-fold excess) or following incubation of tissue
with unlabeled anti-cytokine Ab (100-fold excess) before incubation
with the fluorochrome-conjugated form.
Preparation of Peyer’s patch cells and extraction of RNA
Proximal Peyer’s patches were surgically removed from the gut and placed in DMEM. Isolated patches were incubated in 0.02% EDTA in HBSS and placed on a rotating wheel (20 revolutions per minute (rpm)) for 30 min at 37°C. Next, patches were incubated in 0.05% collagenase (90 min, 37°C, 20 rpm) with fresh collagenase at 30-min intervals. Cells were centrifuged at 1200 rpm for 5 min at 4°C and washed twice in HBSS. The cells were subsequently lysed with trizol solution (Life Technologies, Gaithersburg, MD). Total RNA was extracted with isopropanol according to the manufacturer’s instructions.
RT-PCR for detection of cytokine mRNAs
Total RNA (10 µg) was quantitated by spectrophotometer and
reverse transcribed using oligo(dt)12–18 (Life
Technologies) and RNaseH-reverse transcriptase (Superscript II, Life
Technologies) at 42°C for 2 h. The reverse transcriptase was
inactivated at 95°C. To ensure that each sample had the same amount
of cDNA, the hypoxanthine phosphoribosyltransferase (HPRT) cDNA
concentration of each sample was first determined using HPRT-specific
primers. Then cDNA samples were amplified with IFN-- and
TGF-ß1-specific primers, and products were separated on agarose gels
and stained with ethidium bromide. PCR reactions were incubated for 36
cycles (denaturation for 30 s at 94°C, annealing for 1 min at
55°C, and extension for 1 min at 72°C). The sequences of primers
for the cytokine genes are as follows: IFN- forward,
5'CACACTGCATCTTGGCTT-3'; IFN- reverse, 5'-ACTCCTTTTCCGCTTCCT-3';
TGF-ß1 forward, 5'-CTTTAGGAAGGACCTGGGTT-3'; TGF-ß1
reverse, 5'-CAGGAGCGCACAATCATGTT-3'; HPRT forward,
5'-CTCGAAGTGTTGGATACAGG-3'; HPRT reverse, 5'-TGGCCTATAGGCTCAT
AGTG-3'.
Quantification
Tissues from two experiments were examined. Two animals were fed per dose in one experiment, and one animal was fed per dose in a second experiment. Nine sections (three per animal) were randomly selected and quantitated for each feeding regimen. The number of stained cells in one centimeter field per section was counted under x400 magnification. Data are expressed as the average number of cells per nine high-power fields ± SD and were obtained by the Student t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dome.
The dome of the Peyer’s patch is the primary site of Ag entry after
being taken up by M cells. Within 6 h, OVA TCR-transgenic mice
that were given a single low-dose feeding of 0.5 mg of OVA had a large
increase in both IL-10- and IL-4-stained cells in the dome of the
Peyer’s patch (Fig. 1
). IL-10 was
significantly increased by 1 h (from 1.7 ± 4.0 to 16.3
± 10.1, p = 0.001) and increased further by 6 h
(61.8 ± 5.7, p < 0.001). By 24 h, the
number of IL-10-secreting cells had returned to control levels. IL-4
also increased in the dome after a single low-dose feeding, although
not as rapidly as IL-10. Significant increases were observed at 6
h (from 9.7 ± 3.1 to 26.1 ± 1.1, p =
0.002). Unlike IL-10, which returned to control levels by 24 h,
the number of IL-4-positive cells increased at 24 h (28.9 ±
10.1, p = 0.014) and returned to control levels by
48 h. Figure 2 shows the
immunohistochemical localization of IL-4 in the Peyer’s patch dome of
animals that were fed OVA vs those that were fed HEL. No increase in
IL-4 or IL-10 was observed in animals that were fed single or multiple
high doses (500 mg) of OVA or a control protein (HEL). There was no
expression of TGF-ß at 6 h in the dome of the Peyer’s patch in
animals that were fed a single dose of OVA (Fig. 3). In fact, there was little expression
of TGF-ß at any of the time points examined (1–48 h), regardless of
whether animals had five feedings of 0.5 mg of OVA or high-dose (500
mg) feedings of OVA one or five times.
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and IL-2 were expressed in the dome of control animals; IFN-
decreased at 6 h after a single low-dose feeding, whereas there
was no change in IL-2 (Fig. 3
). The expression of IFN- or IL-2 in
the dome did not significantly change at other time points or as a
result of animals receiving multiple low- or high-dose feedings of OVA
(Fig. 4). Thus, the primary change in the
dome area involved the marked increase in expression of IL-10 and IL-4
after low-dose feeding.
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The expression of cytokine in the corona, the area in the Peyer’s
patch just underneath the dome, was similar to that seen in the dome
itself, with the exception that a large increase in IL-4 was observed
after multiple low-dose feedings. As in the dome, there was a rapid
increase in the expression of IL-10 after a single low-dose feeding
(within 1 h) that decreased by 24 h. There was virtually no
expression of TGF-ß at any time point or with any feeding regimen.
IFN- decreased and IL-2 increased slightly after a single low-dose
feeding, and both were similar to controls after other feeding
regimens. The levels of IL-10 in unfed mice (0.1 ± 0.3) increased
in the corona by 1 h (10.8 ± 9.3) after a low-dose feeding,
and increases were also evident at 4 (14.4 ± 8.3) and 6
(24.4 ± 18.5) h. By 24 h after feeding, IL-10 had returned
to the levels found in unfed animals. IL-10 levels remained similar to
the levels seen in unfed animals at 48 h after other feeding
protocols.
In the corona, levels of IL-4 showed an tendency to increase, with a large SD by 6 h (30.4 ± 6.7), but returned to unfed levels (19.4 ± 6.8) at 24 and 48 h after a single low-dose feeding. However, a large increase (2.5-fold) in IL-4 was found following a five low-dose feedings (45 ± 3.3). Amounts similar to those seen in unfed mice were found with other feeding regimens. No significant changes for TGF-ß were detected in the corona (data not shown).
For IFN-, a decrease from unfed levels (16.6 ± 8.1) was seen
at 48 h after a single low-dose feeding (5.1 ± 3.9). The
results from all other feeding regimens were similar to those found in
unfed mice. IL-2 showed a small increase from unfed mice (5.2 ±
2.3 to 12.9 ± 2.9) at 6 h after a low-dose feeding and then
returned to unfed levels at 24 and 48 h. All other feeding
regimens had levels that were similar to unfed levels.
Germinal centers
Germinal centers are B cell-rich areas in the Peyer’s patch. The
primary change observed in germinal centers was an increase in the
expression of IL-4 (Fig. 5). This
increase was seen at 24 h after a single-dose feeding but
subsequently returned to control levels. No changes in IL-4 were seen
at high doses. There was no increase in TGF-ß or IL-10 staining.
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staining showed no change compared with
control levels, with the exception an increase with a large SD (from
48.4 ± 12.9 to 70.7 ± 12.4) that was seen at 48 h
after a low-dose feeding. Interfollicular region
The interfollicular area was notable for a marked expression of
TGF-ß. This peaked at 6 h and was seen at 24 and 48 h as
well (Figs. 6A and
7). An increase in IL-2 was also observed
at 6 h after a single low-dose feeding (Fig. 6B). No changes were observed in the expression of
IL-4, IL-10, or IFN- in the interfollicular region.
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A marked increase in TGF-ß was observed in the lamina propria of
fed mice. This was observed at 6 and 48 h after a single low-dose
feeding and was even more pronounced after five low-dose feedings
(Figs. 8A and
9). A single high dose did not induce
TGF-ß expression, whereas five high-dose feedings resulted in
increased TGF-ß expression.
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B). In contrast, no significant change was
detected after a single high-dose feeding, while increased IL-10 was
detected after five feedings. Of particular note, constitutive staining
of the epithelium for IL-10 was routinely observed (Fig. 10).
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). IL-2-stained cells were localized predominately underlying the
epithelium and increased transiently at 6 h after a single
low-dose feeding (Fig. 11). No change
was seen following a single high-dose feeding. IFN--stained cells
were scattered throughout the lamina propria, and no change in IFN-
was found after either low- or high-dose feedings.
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A), while other
cytokines were similar to controls. Similarly, IL-10 and TGF-ß
increased at 48 h after five low-dose feedings, with TGF-ß
showing a greater increase than that seen at 6 h after a single
feeding (Fig. 12B). Thus, increased expression of
IL-10 and TGF-ß are dominant responses to oral Ag in the lamina
propria of the villi.
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At 6 h after a low dose of OVA for IL-4 and IL-10 and after
five feedings for TGF-ß, CD4+ and CD8+ T
cells and M were examined for the production of cytokines by
double-label immunofluorescence microscopy. A 6-h period was chosen for
IL-4 and IL-10, since up-regulation of these cytokines in the dome of
the Peyer’s patch (Fig. 1) occurs at 6 h after feeding. As shown
in Figure 13, IL-4-producing cells in
the dome of the Peyer’s patch and in the lamina propria of the villi
were predominately CD4+ T cells at 6 h. IL-10 staining
in the dome, corona, and villi was also found predominately in
CD4+ T cells (data not shown). Only occasional staining of
CD8+ T cells for IL-4 and IL-10 was found in the Peyer’s
patch and villi. As shown in Figure 14,
TGF-ß up-regulation in the interfollicular region and the lamina
propria after five low-dose feedings was predominately due to M and
CD4+ T cells. An occasional staining of CD8+ T
cells was also observed (Fig. 14). At 6 h after one feeding of 0.5
mg, it was found that staining of TGF-ß occurred predominately in
M and CD4+ T cells. Some IFN- and IL-2 staining of
CD4+ T cells was detected in the Peyer’s patch and in the
lamina propria (data not shown).
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Isolated Peyer’s patch cells were examined for response to
oral Ag by RT-PCR. Levels of TGF-ß and IFN- mRNA were examined in
Peyer’s patch cells from OVA- and PBS-fed OVA TCR-transgenic
mice at 6 h after a single feeding of 0.5 mg. As shown in Figure 15, a marked increase in TGF-ß mRNA
was found in OVA-fed animals when compared with PBS-fed animals. A
slight increase in IFN- expression was also observed in OVA-fed
animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TGF-ß is an important gut-associated cytokine, as it serves as switch factor for IgA production and may also be involved in the homing mechanism of cells to high endothelial venules. Little expression of TGF-ß was observed in the dome, corona, or germinal center of the Peyer’s patch, but there was a marked increase seen in the interfollicular region at 6 h after a single low-dose feeding. The interfollicular region is a T cell migration area. TGF-ß-secreting cells can be found in the Peyer’s patch at 24 to 48 h after feeding of MBP (11). In addition, there was a marked increase in the expression of TGF-ß in the lamina propria at 6 h after a single feeding of 0.5 mg.
Although it is well established that absorption of Ag, especially particulate Ag, occurs via the M cells overlying the Peyer’s patch, soluble Ag may also be absorbed into the lamina propria via the villous epithelium. In addition to an increase of TGF-ß in the lamina propria, we also observed increased expression of IL-4 and IL-10 in the lamina propria after a single feeding of 0.5 mg of OVA. IL-4 increased at 2 and 4 h after feeding but returned to control levels by 48 h, whereas IL-10 increased after 1 h and remained elevated at 48 h. The area of the Peyer’s patch that most resembles the cytokine increases found in the lamina propria is the interfollicular region, a T cell migration region.
T cells in lymphoid organs drained by mucosal sites secrete IL-4 as a
primary T cell growth factor, whereas those drained by nonmucosal sites
secrete IL-2 (14). IL-4 is an important growth factor for the
generation of Th2-type and TGF-ß-secreting CD4+ T
cells (15, 16, 34, 35). As described above, we found marked increases
of IL-4 in several anatomic regions of GALT. There were minimal changes
in terms of IL-2 expression, with a slight increase in the expression
of IL-2 in the corona, interfollicular region, and lamina propria at
6 h after a single low dose. Notably, we did not observe an
increased expression of IFN- by immunohistochemistry in any of the
regions examined, apart from a slight increase in the germinal center.
In fact, there was a decrease in IFN- expression in the dome of the
Peyer’s patch after a single feeding, which is consistent with the
increased expression of IL-4 and IL-10 that occurs in this region.
These observations are consistent with studies of Peyer’s patches from
wild-type mice. Activated T cells from the Peyer’s patches produced
IL-4 as the major T cell growth factor (14). Peyer’s patch
CD8+ T cells purified by flow cytometry have been shown to
contain transcripts for IL-4, IL-5, and IL-10 (17). Also, oral
immunization with sheep RBCs has been shown to result in predominately
Th2-type cells in the Peyer’s patch (18). Nonetheless, other
investigators have observed increased IFN- secretion in Peyer’s
patch cells after secondary stimulation in vitro (19). In these
studies, patches were taken from the entire small bowel of OVA
TCR-transgenic animals after three feedings of 250 mg each. A similar
phenomenon has been observed after multiple low-dose feedings when
patches taken from the entire small bowel are used (J. Inobe and
H. L. Weiner, unpublished observations). However, when we examined
patches from the proximal bowel of OVA TCR-transgenic mice given three
250-mg feedings, we did not observe the up-regulation of IFN- by
immunohistochemical analysis. These results suggest that the observed
in vitro IFN- secretion occurs only upon secondary stimulation or
when distal patches are included.
RT-PCR analysis of Peyer’s patch cells taken 6 h after feeding
with 0.5 mg of OVA was undertaken to determine the response to feeding
at the level of mRNA production. TGF-ß mRNA showed a marked increase
at 6 h that was also evident at 6 h and after five low-dose
feedings according to immunohistochemistry. There was not a marked
change in IFN- mRNA, although there was a suggestion that a slight
increase occurred. This finding is consistent with the increase in
IFN- seen by immunohistochemistry in the germinal center at
48 h.
The mechanism of immune hyporesponsiveness following the oral
administration of Ag is dose dependent, with higher doses inducing
anergy and deletion. We previously reported that five high-dose (500
mg) feedings in OVA TCR-transgenic animals led to the deletion of cells
in the dome region of the Peyer’s patch and to a decrease in IFN-
and IL-4/IL-10 production in the spleen following in vitro stimulation
with Ag. It should be noted that there was no decrease in TGF-ß
secretion (20). Our immunohistochemical studies of GALT are consistent
in that there was increased TGF-ß expression in both the villi and
the Peyer’s patch interfollicular region even with multiple high-dose
feedings. IL-4 and IL-2 expression decreased. Significantly, we
observed increased IL-10 secretion in the villi with high-dose
feeding.
We found constitutive expression of IL-4 and IL-10 in the lamina propria before the administration of Ag. There was little expression of TGF-ß in any of the areas of the GALT before feeding, suggesting the importance of Ag in inducing TGF-ß. Animals were not fasted before the oral administration of OVA. IL-10 staining of the epithelium as well as in underlying lymphoid tissue before feeding was a prominent finding. Whether IL-10 is produced by intestinal absorptive cells or absorbed from underlying lymphoid tissue is not known. The results of Pania et al. (21) suggest that the intestinal epithelium may produce IL-10. They used density gradient centrifugation and RT-PCR to show that IL-10 production segregated with intestinal epithelial cells and not with contaminating T cells. The importance of IL-10 for the intestine is indicated by studies indicating that IL-10-deficient mice develop chronic enterocolitis (22), and that tolerance to resident intestinal flora is abrogated in experimental colitis and restored by treatment with IL-10 (23). In other studies, we have found that IL-10 can prime naive dendritic cells to induce IL-4 secretion, and that IL-10 down-regulates IL-12 production (36). Thus, IL-10 may be an important cytokine in GALT that serves to prime for Th2 responses, inhibiting IL-12 and, consequently, Th1 responses.
The predominance of cytokine changes in the gut that involve the
increased expression of IL-4, IL-10, and TGF-ß is consistent with the
host preventing potentially harmful Th1-type responses. Indeed, oral
tolerance may broadly be defined as the inhibition of Th1 responses in
the periphery. Factors that affect Th1-type responses would be expected
to affect oral tolerance, and the administration of IFN- abrogated
oral tolerance (24), whereas anti-IL-12 enhanced it (19).
Consistent with this observation, we observed a decrease in IFN- and
an increase in IL-4 and IL-10 in the dome of the Peyer’s patch at
6 h after feeding.
It has been shown that orally administered LPS enhances protection by orally administered MBP; this protection is not seen when LPS is given systemically (25). The LPS synergistic effect was found to be associated with the expression of IL-4 in the brain of orally tolerized animals (26). It is possible that LPS in the gut enhances the Th2 response to orally administered OVA. The effect of LPS may be mediated by the polysaccharide o-side chain, which has been shown to be transported across the intestinal epithelium (27). In support of this hypothesis, it has been shown that oligosaccharide from schistosome-infected mice is a potent inducer of IL-10 production by B220 cells (28). Studies of IL-4-deficient mice concluded that IL-4 and probably Th2 cells are required for the induction of gut mucosal Ab responses, since the administration of Ag to IL-4-targeted mice failed to stimulate Ag-specific T cell responses even though switch differentiation from IgM to IgA was not impaired (29).
LPS has also been found to mediate immunization via the gut as well as oral tolerance. The expression of dietary protein in Escherichia coli renders it strongly antigenic to GALT as measured by biliary IgA Abs (30, 31, 32). Whether LPS is involved in tolerization or immunization may depend upon how the protein is presented to GALT. Presentation that involves the polysaccharide portion may induce Th2 responses, while presentation involving the inflammatory lipid A portion of the molecule may favor Th1 responses.
We found that the cells producing IL-4 and IL-10 in the dome of the
Peyer’s patch at 6 h after low-dose feeding were predominately
CD4+ T cells. TGF-ß seen in the interfollicular region
and villi at 6 h after a low dose and after five low-dose feedings
was predominately present in M and CD4+ T cells. Only an
occasional staining of CD8+ T cells for TGF-ß, IL-4, and
IL-10 was observed. It should be noted that the majority of T cells in
these animals are CD4+ TCR-transgenic T cells. However,
since they are not on a recombinant-activating gene-1-deficient
background, the transgene expression is not absolute, and some
CD8+ T cells may be present.
Whether cells secreting TGF-ß and Th2 cytokines are initially
generated in the Peyer’s patch, lamina propria, or MLNs is not
definitively known. Previous studies of the MLNs of orally tolerized
(SJLxPLJ)F1 mice found that both CD8+ T cells
which secrete TGF-ß and CD4+ cells which secrete IL-4 and
IL-10 in addition to TGF-ß are induced by oral tolerization (9).
MBP-specific CD4+ T cell clones generated from the MLNs of
MBP-fed SJL mice secrete TGF-ß, IL-4, and IL-10 but not IFN-.
Other investigators have shown by immunohistochemical studies of murine
Peyer’s patches that both CD4+ and CD8+ cells
are found in the interfollicular region of the patch (33). Whether
TGF-ß-secreting cells of the Peyer’s patch also secrete Th2
cytokines was not examined. Other cell types may also contribute to the
cytokine milieu of GALT.
In summary, we have found that oral Ag induces Th2-type and TGF-ß responses in the Peyer’s patches and villi of OVA TCR-transgenic mice. The mechanisms by which a predominately Th2 environment is induced in the subepithelial dome and TGF-ß is induced preferentially in the interfollicular region and in the villi are not well understood. However, our finding of differential cytokine induction in specific anatomic locations in GALT provides a basis for understanding the mechanisms of mucosal immune responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Patricia A. Gonnella, Center for Neurologic Diseases, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address:
3 Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; M, macrophage; MBP, myelin basic protein; R-PE, R-phycoerythrin; HEL, hen egg lysozyme; MLN, mesenteric lymph node; HPRT, hypoxanthine phosphoribosyltransferase; rpm, revolutions per minute.
Received for publication March 27, 1997. Accepted for publication January 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment. J. Immunol. 144:4163.[Abstract]
have opposing effects while TGF-ß
positively regulates its own production. J. Immunol. In
press.
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 K. Heiss, N. Janner, B. Mahnss, V. Schumacher, F. Koch-Nolte, F. Haag, and H.-W. Mittrucker High Sensitivity of Intestinal CD8+ T Cells to Nucleotides Indicates P2X7 as a Regulator for Intestinal T Cell Responses J. Immunol., September 15, 2008; 181(6): 3861 - 3869. [Abstract] [Full Text] [PDF]
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X. Wu, K. Roelofs-Haarhuis, J. Zhang, M. Nowak, L. Layland, E. Jermann, and E. Gleichmann Dose dependence of oral tolerance to nickel Int. Immunol., August 13, 2007; (2007) dxm066v1. [Abstract] [Full Text] [PDF]
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K. Wing, Z. Fehervari, and S. Sakaguchi Emerging possibilities in the development and function of regulatory T cells Int. Immunol., July 1, 2006; 18(7): 991 - 1000. [Abstract] [Full Text] [PDF]
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J. Klein, W. Ju, J. Heyer, B. Wittek, T. Haneke, P. Knaus, R. Kucherlapati, E. P. Bottinger, L. Nitschke, and B. Kneitz B Cell-Specific Deficiency for Smad2 In Vivo Leads to Defects in TGF-beta-Directed IgA Switching and Changes in B Cell Fate J. Immunol., February 15, 2006; 176(4): 2389 - 2396. [Abstract] [Full Text] [PDF]
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D. Alvarez, F. K. Swirski, T.-C. Yang, R. Fattouh, K. Croitoru, J. L. Bramson, M. R. Stampfli, and M. Jordana Inhalation Tolerance Is Induced Selectively in Thoracic Lymph Nodes but Executed Pervasively at Distant Mucosal and Nonmucosal Tissues J. Immunol., February 15, 2006; 176(4): 2568 - 2580. [Abstract] [Full Text] [PDF]
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K. Kalies, M. Blessenohl, J. Nietsch, and J. Westermann T Cell Zones of Lymphoid Organs Constitutively Express Th1 Cytokine mRNA: Specific Changes during the Early Phase of an Immune Response J. Immunol., January 15, 2006; 176(2): 741 - 749. [Abstract] [Full Text] [PDF]
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 Y. Chung, S.-H. Lee, D.-H. Kim, and C.-Y. Kang Complementary role of CD4+CD25+ regulatory T cells and TGF-{beta} in oral tolerance J. Leukoc. Biol., June 1, 2005; 77(6): 906 - 913. [Abstract] [Full Text] [PDF]
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S. Borsutzky, B. B. Cazac, J. Roes, and C. A. Guzman TGF-{beta} Receptor Signaling Is Critical for Mucosal IgA Responses J. Immunol., September 1, 2004; 173(5): 3305 - 3309. [Abstract] [Full Text] [PDF]
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J. Rey, N. Garin, F. Spertini, and B. Corthesy Targeting of Secretory IgA to Peyer's Patch Dendritic and T Cells after Transport by Intestinal M Cells J. Immunol., March 1, 2004; 172(5): 3026 - 3033. [Abstract] [Full Text] [PDF]
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I. T. Tobagus, W. R. Thomas, and P. G. Holt Adjuvant Costimulation during Secondary Antigen Challenge Directs Qualitative Aspects of Oral Tolerance Induction, Particularly during the Neonatal Period J. Immunol., February 15, 2004; 172(4): 2274 - 2285. [Abstract] [Full Text] [PDF]
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R. W. DePaolo, B. J. Rollins, W. Kuziel, and W. J. Karpus CC Chemokine Ligand 2 and Its Receptor Regulate Mucosal Production of IL-12 and TGF-{beta} in High Dose Oral Tolerance J. Immunol., October 1, 2003; 171(7): 3560 - 3567. [Abstract] [Full Text] [PDF]
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 K. Becker, D. Kindrick, R. McCarron, J. Hallenbeck, and R. Winn Adoptive Transfer of Myelin Basic Protein-Tolerized Splenocytes to Naive Animals Reduces Infarct Size: A Role for Lymphocytes in Ischemic Brain Injury? Stroke, July 1, 2003; 34(7): 1809 - 1815. [Abstract] [Full Text] [PDF]
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 A. Finamore, M. Roselli, N. Merendino, F. Nobili, F. Vignolini, and E. Mengheri Zinc Deficiency Suppresses the Development of Oral Tolerance in Rats J. Nutr., January 1, 2003; 133(1): 191 - 198. [Abstract] [Full Text] [PDF]
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 P. L. Ogra, H. Faden, and R. C. Welliver Vaccination Strategies for Mucosal Immune Responses Clin. Microbiol. Rev., April 1, 2001; 14(2): 430 - 445. [Abstract] [Full Text] [PDF]
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P. A. Gonnella, H. P. Waldner, and H. L. Weiner B Cell-Deficient ({micro}MT) Mice Have Alterations in the Cytokine Microenvironment of the Gut-Associated Lymphoid Tissue (GALT) and a Defect in the Low Dose Mechanism of Oral Tolerance J. Immunol., April 1, 2001; 166(7): 4456 - 4464. [Abstract] [Full Text] [PDF]
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R. D. Newberry, J. S. McDonough, W. F. Stenson, and R. G. Lorenz Spontaneous and Continuous Cyclooxygenase-2-Dependent Prostaglandin E2 Production by Stromal Cells in the Murine Small Intestine Lamina Propria: Directing the Tone of the Intestinal Immune Response J. Immunol., April 1, 2001; 166(7): 4465 - 4472. [Abstract] [Full Text] [PDF]
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 K. Fujihashi, T. Dohi, P. D. Rennert, M. Yamamoto, T. Koga, H. Kiyono, and J. R. McGhee Peyer's patches are required for oral tolerance to proteins PNAS, March 13, 2001; 98(6): 3310 - 3315. [Abstract] [Full Text] [PDF]
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D. A. Kahn, D. C. Archer, and C. J. Kelly Absence of Functional Inducible NO Synthase Enhances the Efficacy of Tolerance Induced by High Dose Antigen Feeding J. Immunol., December 1, 2000; 165(11): 6116 - 6122. [Abstract] [Full Text] [PDF]
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V. Julia, S. S. McSorley, L. Malherbe, J.-P. Breittmayer, F. Girard-Pipau, A. Beck, and N. Glaichenhaus Priming by Microbial Antigens from the Intestinal Flora Determines the Ability of CD4+ T Cells to Rapidly Secrete IL-4 in BALB/c Mice Infected with Leishmania major J. Immunol., November 15, 2000; 165(10): 5637 - 5645. [Abstract] [Full Text] [PDF]
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S. Nagata, C. McKenzie, S. L. F. Pender, M. Bajaj-Elliott, P. D. Fairclough, J. A. Walker-Smith, G. Monteleone, and T. T. MacDonald Human Peyer's Patch T Cells Are Sensitized to Dietary Antigen and Display a Th Cell Type 1 Cytokine Profile J. Immunol., November 1, 2000; 165(9): 5315 - 5321. [Abstract] [Full Text] [PDF]
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 C. ASSEMAN, S. FOWLER, and F. POWRIE Control of Experimental Inflammatory Bowel Disease by Regulatory T cells Am. J. Respir. Crit. Care Med., October 1, 2000; 162(4): S185 - 189. [Abstract] [Full Text] [PDF]
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A. McI. Mowat, M. Steel, A. J. Leishman, and P. Garside Normal Induction of Oral Tolerance in the Absence of a Functional IL-12-Dependent IFN-{gamma} Signaling Pathway J. Immunol., November 1, 1999; 163(9): 4728 - 4736. [Abstract] [Full Text] [PDF]
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F. W. v. Ginkel, S. M. Wahl, J. F. Kearney, M.-N. Kweon, K. Fujihashi, P. D. Burrows, H. Kiyono, and J. R. McGhee Partial IgA-Deficiency with Increased Th2-Type Cytokines in TGF-{beta}1 Knockout Mice J. Immunol., August 15, 1999; 163(4): 1951 - 1957. [Abstract] [Full Text] [PDF]
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J. Sun, B. Dirden-Kramer, K. Ito, P. B. Ernst, and N. Van Houten Antigen-Specific T Cell Activation and Proliferation During Oral Tolerance Induction J. Immunol., May 15, 1999; 162(10): 5868 - 5875. [Abstract] [Full Text] [PDF]
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H. L. Weiner Oral tolerance with Copolymer 1 for the treatment of multiple sclerosis PNAS, March 30, 1999; 96(7): 3333 - 3335. [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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