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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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in the GALT of wild-type but not µMT
mice. Upon stimulation of popliteal node cells, in vitro induction of
regulatory cytokines TGF- and IL-10 was observed in wild-type but
not µMT mice. Greater protection against experimental autoimmune
encephalomyelitis was found in wild-type mice. Oral tolerance in µMT
and wild-type mice was found to proceed by different mechanisms. Anergy
was observed from 0.5 mg to 250 ng in µMT mice but not in wild-type
mice. Increased Ag was detected in the lymph of µMT mice. No
cytokine-mediated suppression was found following lower doses from 100
ng to 500 pg in either group. These results demonstrate the importance
of the B cell for the induction of cytokine-mediated suppression
associated with low doses of Ag. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production was observed after both a low
(5) and high (4) dose of Ag. These results
indicated that B cells were not the only tolerogenic APC in vivo.
Indeed, Ag targeted to dendritic cells has been shown to induce a
tolerogenic T cell response (7). Also, peripheral T cells
have been shown to be tolerized by Ag presented by nontraditional APCs
in the absence of appropriate costimulation (8, 9, 10).
Several factors have been shown to affect the generation of tolerance.
Differences in the route of Ag administration can determine the nature
of the immune response (11). Intravenous administration
induced tolerance in an Ag-specific manner as evidenced by suppression
of disease, and no increase in Th2 cells was found (4, 5, 6, 11). Low doses of orally administered Ag resulted in induction
of tolerance characterized by an up-regulation in Th2 cells and TGF-
(12). These results indicated that tolerance by i.v.
administration of Ag and low dose oral tolerance may proceed by
different mechanisms (12, 13).
Dose of Ag can determine the nature of the immune response. Intravenous tolerance and high dose oral tolerance were found to proceed by the mechanism of anergy, which was characterized by a suppression of proliferation that was reversible following incubation of cells in IL-2 (14, 15). In contrast, low dose oral tolerance was found to proceed by the mechanism of active suppression and was characterized by the production of anti-inflammatory cytokines and suppression of proliferation, which was not reversible following incubation in IL-2.
Another factor that has been shown to determine the nature of the
immune response is the cytokine microenvironment. The critical role of
cytokines in the induction of tolerance was evidenced by abrogation of
oral tolerance following injection of IFN- (16).
Peyer’s patches are a major source of IgA producing B cells and have also been shown to be a site where regulatory cells that mediate the active suppression component of oral tolerance are generated (13, 17, 18). Thus it is possible that B cells may play an important role in the generation of oral tolerance. To evaluate the role of B cells in the induction of oral tolerization, we studied µMT mice, which were generated by the introduction of a nonsense mutation into the transmembrane exon of the IgM heavy chain resulting in a total deletion of B cells (19). T cell tolerance was measured by T cell proliferation and cytokine responses. Anergy was measured by reversal of suppression of proliferation by IL-2, and the cytokine microenvironment of the gut-associated lymphoid tissue (GALT) was determined by immunohistochemical analysis. Oral tolerance in µMT mice was found to proceed by the mechanism of anergy. µMT mice had profound differences in the GALT cytokine microenvironment and a defect in the induction of cytokine-mediated suppression with low doses of Ag.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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µMT and wild-type C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All animals were housed at the Harvard Institutes of Medicine under virus-free conditions.
Abs, Ags, and recombinant cytokines
Abs used for immunohistochemical analysis were: rat
anti-mouse IL-2 clone S4B6 and rat anti-mouse IL-10 clone
JESJ-2A5 (PharMingen, San Diego, CA); rabbit anti-porcine TGF-
(R&D Systems, Minneapolis, MN); rat anti-mouse IL-4, BVD6.24G
(provided by A. Lichtman, Harvard Medical School, Boston, MA); and
hamster anti-mouse IFN- clone H22.1 and rat anti-mouse IL-6
clone M-10 (Genzyme, Cambridge, MA). Abs were used at the following
concentrations: IL-10, IL-6, IL-2, and IFN- (5 µg/ml), IL-4
(hybridoma supernatant), and TGF- (2 µg/ml).
Abs used for ELISA were purified rat anti-mouse IL-2 (clone
JES6-5H4), IL-4 (clone BVD4-1D11), IL-10 (clone JES5-2A5), and IFN-
(clone R4-6A2) mAb; biotinylated rat anti-mouse IL-2 (clone
JES6-5H4), IL-4 (clone BVD4-24G2), IL-10 (clone SXC-1), and IFN-
(clone XMG1.2) mAb (PharMingen); and polyclonal chicken
anti-TGF-1 (R&D Systems) and monoclonal
mouse anti-TGF- (clone 1D11.16; Celtrix Pharmaceuticals, Santa
Clara, CA). Recombinant cytokines were mouse IL-2, IL-4, IL-10, IFN-
(PharMingen), and purified bovine TGF-1
(Celtrix Pharmaceuticals).
Oral administration of Ag
Mice were fed various doses (25 mg, 0.5 mg, 250 µg, 250 ng, 100 ng, 10 ng, 1 ng, and 500 pg) of OVA dissolved in PBS by gastric intubation with an 18-gauge stainless steel feeding needle (Thomas Scientific, Swedesboro, NJ). In some experiments mice were fed 250 µg myelin oligodendrocyte glycoprotein (MOG35–55). Ag was administered every other day for a total of five times. All fed mice were immunized and compared with unfed immunized mice.
Immunization of OVA-fed mice
Mice fed OVA were immunized in the foot pad with 200 µg OVA in 0.1 ml PBS emulsified in an equal volume of CFA containing 2 mg/ml of mycobacterium tuberculosis H37 RA (Difco, Detroit, MI).
Induction and clinical evaluation of experimental autoimmune encephalomyelitis (EAE)
Mice were immunized with a s.c. injection in the flank of 50 µg MOG in 0.1 ml of dH2O:PBS (1:9) emulsified in an equal volume of CFA containing 4 mg/ml of mycobacterium tuberculosis H37 RA (Difco) and an i.v. injection of 150 ng pertussis toxin (List Biological Laboratories, Campbell, CA) in 0.1 ml of PBS. Then, animals received a second injection of pertussis toxin 48 h later. Animals were scored for EAE as follows: 0, no disease; 1, limp tail; 2, hindlimb weakness; 3, hindlimb paralysis; 4, hindlimb plus forelimb paralysis; 5 moribund.
Cell culture and proliferation assay
Cells were cultured at 0.5 x 106 or
1 x 106 per well in 0.2 ml of serum-free
medium X-Vivo 20 (BioWhittaker, Walkersville, MD) supplemented with 0.1
mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 U/ml of
streptomycin and containing various concentrations of Ag. For cytokine
assays, culture supernatants were collected at 48 h for IL-2,
IL-4, IL-10, and IFN- and at 72 h for TGF-. For
proliferation assays, 1 µCi of [3H]thymidine
(1 Ci = 376) was added to each culture at 72 h. Cells were
harvested and radioactivity incorporated was counted 16 h later
using a flatbed beta counter (Wallac, Gaithersburg, MD).
Detection of anergy
Anergy was demonstrated according to the method of Gaur et al. (19) as follows: cells were cultured in DMEM supplemented with10% FCS in a 96-well plate at 0.5 x 106 cells/well. Pooled cells from each group of mice were cultured in the absence of Ag (medium alone) and in the presence of varying doses of Ag. In addition, cells from fed immunized mice were cultured in the presence or absence of recombinant IL-2 (rIL-2, 5 U/ml) and compared with cells from unfed immunized mice that were cultured in the absence of rIL-2. IL-2 was added at the time lymph node or spleen cells were placed in culture. Cells were cultured for 4 days, and [3H]thymidine (1 µCi) was added for the last 18 h. Results are presented as the mean ± SD of triplicate cultures after subtraction of background radioactivity. Background counts were obtained by culturing cells in the absence of Ag. Experiments were repeated three times. Results of individual experiments are shown.
Collection of lymph
Lymph was collected according to the method of Korngold and Bennink (20) with the following modifications: mice were fed 0.5 mg OVA and 30 min later fed 0.3 cc corn oil for visualization of the thoracic duct. Because it was not necessary to collect lymph over a prolonged period of time, the animal was not suspended over a wheel. Lymph was collected by cannulation from 1 to 2 and from 2 to 3 h after feeding.
ELISA for cytokines
Quantitative ELISAs for IL-2, IL-4, IL-10, and IFN- were
performed using paired mAbs specific for corresponding cytokines per
manufacturer’s recommendations (PharMingen). TGF- ELISA was
performed as previously described (21). Results are
expressed as mean values from triplicate cultures. Data presented are
representative of two or three experiments.
ELISA for OVA
Quantitative ELISA for OVA was performed as follows: 96-well microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated overnight at 4°C with 1 µg/ml sheep anti-OVA polyclonal Abs (Cortex, San Leandro, CA) in 100 µl carbonate buffer, ph 8.2. The plates were then washed three times with PBS containing 0.5% Tween 20, blocked with 1% BSA in PBS, washed, and incubated with lymph or OVA standard overnight at 4°C. The plates were washed again and incubated with 1 µg/ml biotinylated rabbit anti-OVA (Rockland, Gilbertsville, PA) for 1 h at room temperature. Avidin-peroxidase was added, and color was developed with a one-component tetramethylbenzidine reagent (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
Immunostaining of light microscopic sections
Tissues were excised from anesthetized animals embedded in OCT Tissue-TeK (Miles, Elkhart, IL) snap frozen in liquid nitrogen-cooled isopentane. Cryosections were fixed in acetone and stored at -20°C. 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) for 10 min followed by washing in PBS and immersion in 0.003 M sodium borohydride for 30 min. After washing, tissues were incubated for 20 min in diluted normal blocking serum that was prepared from the species from which the secondary Ab was made. Endogenous biotin was blocked by incubation in avidin D for 1 h followed by incubation in biotin for 1 h. After washing in PBS, sections were incubated overnight in primary Ab diluted in blocking serum. Sections were rinsed in PBS and incubated in diluted biotinylated secondary Ab for 1 h. After washing, sections were incubated in Vectastain Elite ABC reagent, washed again, and incubated in peroxidase substrate solutions containing 3,3'-diaminobenzidine (DAB Substrate Kit; Vector Laboratories) for 5–7 min. Sections were washed for 5 min in tap water, counterstained in hematoxylin, cleared, and mounted.
Up-regulation of cytokine staining was determined by comparing fed immunized with unfed immunized mice. Quantification of data was performed by computerized image analysis (IP Labs Systems; Scanalytics, Fairfax, VA). Three mice per group were studied, and experiments were repeated three times. Nine sections (three per animal) were randomly selected and quantitated for each group. The region of interest (ROI) represents a defined area (116 µm x 44 µm) at x100 magnification. The percent staining within the ROI was measured in 20 randomly selected regions. This analysis was conducted for the dome, corona, interfollicular, and germinal center of the Peyer’s patch and for the lamina propria. Data are expressed as the mean percent staining per ROI ± SD and were analyzed by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We examined wild-type and µMT C57BL/6 mice for induction of oral
tolerance following feeding of 0.5 mg OVA five times followed by
immunization in the foot pad. Popliteal node cells were harvested 7
days after immunization. Upon stimulation with Ag in vitro both
wild-type and µMT mice showed induction of oral tolerance evidenced
by suppression of proliferation and IFN- production (Fig. 1
). The increased level of proliferation
seen in µMT mice compared with littermate controls may be due to a
larger number of T cells in µMT cultures due to the absence of B
cells. Although both wild-type and µMT mice showed induction of oral
tolerance, wild-type mice showed up-regulation of IL-10 and TGF-
that was not seen in µMT mice. Small amounts of IL-2 (50 pg) were
detected in wild-type mice that were suppressed with feeding to 10 pg.
No IL-2 was detected in µMT, and no IL-4 was detected in either
group. Similar results were found in spleen cells at 30 days after
immunization (data not shown).
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As shown in Figs. 2 and 3, there were dramatic differences in
cytokine expression both in the Peyer’s patch and lamina propria of
fed immunized wild-type and µMT mice fed 0.5 mg OVA. In Fig. 2, IL-4
was present throughout the GALT of wild-type mice (Fig. 2A)
but was absent in µMT animals (Fig. 2B). Although IL-10
was present in the epithelium of both groups it was only up-regulated
in the dome (Fig. 2, C and D) and lamina propria
of wild-type mice. As shown in Fig. 3, there was up-regulation of
TGF- in wild-type mice (Fig. 3a), whereas TGF- was not
detected in µMT mice (Fig. 3b). IFN- was expressed in
the lamina propria of wild-type (Fig. 3c) mice but not in
µMT mice (Fig. 3d). IL-2 was expressed in wild-type mice
(Fig. 3e) and only sparsely distributed in the lamina
propria of µMT mice (Fig. 3f). IL-6 was expressed in both
wild-type and µMT mice (Fig. 3, g and h). In
animals fed 25 mg five times, up-regulation of TGF- and an
increasing trend for IL-10 were observed in the GALT of wild-type but
not in µMT mice (data not shown). No detectable changes were found
with feeding alone.
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, and IFN-
were present in the GALT of wild-type mice but not in µMT mice. Small
amounts of IL-10 were seen in the lamina propria of wild-type mice
(0.28% ± 0.27) with a decreasing trend (0.02% ± 0.04) seen in µMT
mice. In addition, similar amounts of IL-2 were detected in the corona
of the Peyer’s patch of wild-type and µMT mice (0.28 ± 0.44
and 0.29 ± 0.3%, respectively). Thus, the cells of the GALT of
µMT mice lack the Th2 cytokines and TGF- found in wild-type mice
indicating a different microenvironment for the presentation of
Ag.
When MOG was fed as Ag, similar results to those seen with OVA were
found in the GALT of wild-type and µMT mice. Mice were examined at 30
days after immunization. In wild-type mice IL-4 was increased in the
dome, corona, and interfollicular region of the Peyer’s patch (Fig. 4A). IL-10 was increased in
the dome of the patch and in the lamina propria of the villi (Fig. 4B). TGF- was increased in the lamina propria (3.5
± 2.7 fed vs 0.0 ± 0.0% unfed). In contrast, the GALT of both
fed and unfed µMT mice was characterized by the absence of IL-4,
TGF-, and IFN-. Although IL-10 was present in the epithelium,
only small amounts were found in the lamina propria, and no
up-regulation with feeding was seen (data not shown).
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Given the results in the GALT we examined in situ cytokine
expression in the popliteal node. The cortex of the popliteal node of
fed immunized wild-type mice fed 0.5 mg OVA showed decreased staining
for IFN- and increased TGF- with no significant increase in IL-4
or IL-10 when compared with unfed immunized mice (Fig. 5). In contrast, fed immunized µMT mice
showed no detectable IFN- and no increase in TGF-. Unfed
immunized µMT mice expressed much more IL-10 than fed immunized mice
with a large SD observed. µMT nodes were also characterized by the
absence of IL-4. Thus, up-regulation of TGF-, IL-4, or IL-10 was not
seen in the popliteal node of fed immunized µMT mice. After feeding a
high dose of 25 mg five times no up-regulation of TGF-, IL-4, and
IL-10 was seen in either group (data not shown).
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Because up-regulation of IL-4, IL-10, and TGF- associated with
cytokine-mediated active suppression in wild-type mice was not seen in
µMT mice, we examined these mice for the induction of anergy.
Popliteal node cells from OVA-fed mice (0.5 mg) were incubated in 5 U
recombinant IL-2 (rIL-2) and compared with cells from fed mice not
incubated in rIL-2 and cells from unfed immunized animals. Results
shown in Fig. 6 indicate that cell
proliferation remained suppressed in fed wild-type mice after
incubation in rIL-2. In contrast, µMT mice showed reversal of
suppression following rIL-2 treatment indicating anergy. Next we tested
for anergy in spleen cells from µMT and wild-type mice at 30 days
after immunization. Similar to the popliteal node, reversal of
suppression after incubation in rIL-2 was observed in µMT but not in
wild-type animals (Fig. 7A).
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. Analogous to what was seen with OVA,
incubation in rIL-2 reversed the suppression in µMT mice but not in
wild-type animals (Fig. 7
B). Furthermore, there was less
suppression of EAE in µMT vs wild type (Fig. 8).
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Because we observed anergy in the popliteal node and spleen of
µMT mice, we examined thoracic duct lymphatic fluid to determine
whether there were differences in absorption of Ag in the two groups of
mice. Lymphatic fluid from wild-type and µMT mice fed 0.5 mg OVA was
collected by cannulation of the thoracic duct according to the method
of Korngold and Bennink and assayed for detection of OVA by ELISA.
Lymph was collected from 1 to 2 and from 2 to 3 h after feeding.
As shown in Fig. 9 increased
concentration of OVA was found in lymph of µMT mice compared with
wild-type mice.
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We then asked whether reversal of suppression with IL-2 was also
present at a lower dose of Ag administration or whether we could
identify a low dose in µMT mice that induced cytokine-mediated
suppression. Popliteal node cells from mice fed a 250 ng dose of OVA
five times showed suppression of proliferation and IFN- production
in both µMT and wild-type mice; however, suppression of proliferation
was reversed in µMT mice but not in wild-type mice following
incubation in rIL-2 (Fig. 10).
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) or up-regulation of IL-4, IL-10, or
TGF- was observed in either wild-type or µMT mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. This was not only seen in animals
orally administered Ag, but similar patterns were observed in the GALT
of naive µMT mice. The different cytokine microenvironment appears to
have the following effect. First, we did not observe the up-regulation
of IL-4, IL-10, and TGF- following orally administered Ags in µMT
mice. These cytokines are associated with the induction of
cytokine-mediated active suppression when low doses of Ag are
administered orally. However, we found that the oral tolerance induced
in µMT mice related to anergy as measured by reversal of suppression
by rIL-2 at all doses that suppression was observed. Consistent with
this we found increased amount of Ag entered the lymph in µMT animals
than wild type, and we observed no induction of cytokines even at
extremely low doses. The absence of B cells reduces the Th2/Th3 microenvironment and enhances Ag absorption. The actual mechanisms underlying these observations are not known and could be mediated through other local cell populations. The reason that more Ag appears in the lymph in µMT mice could relate to either an altered structure of the GALT allowing greater epithelial permeability (absorptive or paracellular) and removal of Ag into the lymph, greater availability of Ag due to absence of luminal IgA or the binding of Ag by low-affinity Ab that is normally picked up by B cells but does not occur in µMT mice. This would alter the amount and form of Ag presented to macrophages and dendritic cells both in the GALT, and the draining lymphatic structures. Furthermore, if there is paracellular entry of Ag, our findings may represent access of Ag to conventional APCs in the lamina propria. The nature of the OVA in the thoracic duct was not examined. Previous studies of macromolecular uptake in the rat have found that some OVA absorbed from the gastrointestinal tract can be detected intact in the plasma and lymph (22). Both intact or processed OVA that retains antigenicity would be detected by our assay.
The mechanism of anergy is normally associated with high doses of oral Ag in wild-type mice or with Ag administered by the i.v. route in wild-type or µMT mice. The ability of µMT mice to tolerize following i.v. Ag is well established (4, 5, 6), and high dose (25 mg) oral tolerance has been reported (23). This study demonstrates that oral tolerance following low doses of Ag proceeds by the mechanism of anergy in µMT mice, whereas a nonanergic, cytokine-mediated mechanism was found in wild-type mice.
In this study we performed a titration of doses to determine whether a
low dose could be identified in which oral Ag induced tolerance due to
a cytokine-mediated suppression mechanism in the µMT mice. However,
doses as low as 250 ng resulted in suppression of cellular
proliferation in µMT, which was reversible following incubation in
rIL-2 indicating anergy with no IL-4, IL-10, and TGF-. In contrast,
suppression in wild-type mice was not reversed by IL-2. Oral doses of
100 ng to 500 pg resulted in no suppression of cellular proliferation
in either wild-type or µMT mice. The reason that titration to a low
dose that induces active suppression in µMT mice is not achievable is
not known but may be due to vast differences in the cytokine
microenvironment in the GALT of wild-type and µMT mice. Our
immunohistochemical analysis shows that unfed wild-type mice have IL-4,
IL10, TGF-, and IFN- that are reduced or absent in the GALT of
µMT mice. Both wild-type and µMT mice have similar amounts of IL-2
in the corona of the Peyer’s patch, and IL-6 was found in the Peyer’s
patch and lamina propria of both groups. We found small amounts of
IL-12 in wild-type animals but none in µMT (data not shown). Thus the
data suggest that Ag presentation takes place in a different
environment in µMT mice. Previous studies have shown that injection
of Th1 cytokines can circumvent the induction of oral tolerance
(16). Taken together the data indicate that the B cell is
necessary for the generation of the Th2 environment in the GALT and is
essential for the induction of a low dose active suppression mechanism
of tolerance.
In µMT mice Peyer’s patches are not visible to the eye as pronounced elevations along the intestinal wall as seen in wild-type mice, rather they appear as small opaque areas. Also the patches in µMT mice are reduced in size due to absence of B cells and are fewer in number than in wild-type mice. Thus some investigators may not have detected patches, whereas other investigators have called them lymphoid aggregates due to their reduced size. Peyer’s patches are characterized by the presence of follicle-associated epithelium (FAE), which contain M cells scattered among the columnar epithelial cells. The FAE covers the dome region of the patch, which is free of intestinal villi. Golovkina et al. (24) have published pictures of M cells in the FAE of Peyer’s patches of µMT mice and have reported that 1.2–6.6 M cells are present in µMT mice per every 1000 M cells in normal intestine.
Our immunohistochemical analysis of the GALT shows that in
response to feeding low doses of OVA or MOG the up-regulation of IL-4,
IL-10, and TGF- occurs in wild-type but not µMT mice. In the
popliteal node in situ we found that low dose feeding resulted in the
up-regulation of TGF- in wild-type but not µMT mice. Thus similar
to results in vitro we observed cytokine up-regulation only in fed
immunized wild-type mice. In addition, we observed that in the
popliteal node of µMT mice much more IL-10 was present in unfed
immunized than fed immunized mice. The reason for this is not known but
may be due to apoptosis of Th2 cells following oral administration of
an anergy-inducing dose of Ag. Previously we have shown that oral Ag
can delete Ag-reactive T cells by apoptosis. Deletion was dependent on
dosage and frequency of feeding (25). At lower doses
apoptosis was not observed; however, at higher doses both Th1 and Th2
cells were deleted. In the case of µMT mice we have shown increased
Ag in the lymph and the demonstration of anergy in the popliteal
node.
Our titration experiments showed that in addition to the absence
of tolerance, the feeding of a 500-pg dose resulted in a priming
response in both wild-type and µMT mice. Previous studies have
reported the phenomenon of priming in response to varying low doses of
Ag (26, 27). Lamont et al. found the priming of systemic
and local delayed-type hypersensitivity responses by feeding low doses
of 5 µg of OVA to BALB/c mice (26). Furthermore, Stokes
et al. reported that sensitization of systemic DTH following oral Ag
occurred only in one of two mouse strains examined (27).
Taken together, the data suggest that under certain circumstances in
specific mouse strains priming can occur in response to very low doses
of Ag. The reason for this is unknown, but we hypothesize that this may
relate to differential Ag presentation and strength of the signal to
the T cell that occurs at very low doses; we are presently
investigating this. Furthermore, Lamont et al. have also speculated
that it reflects different effects of fed Ag on distinct subsets of Th1
and Th2 cells (26). Our studies suggest that the cytokine
microenvironment also may play a role because we observed evidence of
priming in µMT mice fed nanogram doses of OVA after cells were
challenged with 1000 µg in vitro (Fig. 11). Thus the priming at very
low doses appears to be a real biological event whose mechanism remains
unexplained at this time.
Evidence from several studies suggests that B cells acting as APC
skew the differentiation of helper T cells toward a Th2 response
(28, 29, 30, 31). The generation of Th2- and TGF--producing
cells characterizes the active suppression mechanism of oral tolerance
(12). Because T cells producing IL-4 and IL-10, and
TGF- have been shown to regulate Th1 cells in EAE (32),
the absence of active suppression in µMT mice may explain the reduced
protection against MOG-induced EAE in our µMT mice compared with wild
type. The importance of the B cell and its possible role in the
generation of a Th2 response was also suggested by studies of EAE that
have shown that in the absence of the B cell there is a failure of
spontaneous recovery in B10.PL µMT mice compared with B10.PL mice
(33).
In conclusion, the data demonstrate that the B cell is essential for maintenance of a predominately Th2 cytokine environment in the GALT and the induction of the low dose cytokine-mediated suppression mechanism of oral tolerance in C57BL/6 mice. Furthermore, the data suggest that the cytokine microenvironment in the GALT and lymph nodes is important for the generation of regulatory cells indicative of this mechanism.
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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, Harvard Institutes of Medicine, 77 Louis Pasteur Avenue, Boston, MA 02115.
3 Abbreviations used in this paper: µMT, B cell-deficient; GALT, gut-associated lymphoid tissue; MOG, myelin oligodendrocyte glycoprotein; EAE, experimental autoimmune encephalomyelitis; ROI, region of interest.
Received for publication October 18, 2000. Accepted for publication January 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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V. V. Parekh, D. V. R. Prasad, P. P. Banerjee, B. N. Joshi, A. Kumar, and G. C. Mishra B Cells Activated by Lipopolysaccharide, But Not By Anti-Ig and Anti-CD40 Antibody, Induce Anergy in CD8+ T Cells: Role of TGF-{beta}1 J. Immunol., June 15, 2003; 170(12): 5897 - 5911. [Abstract] [Full Text] [PDF]
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A. J. Buendia, L. Del Rio, N. Ortega, J. Sanchez, M. C. Gallego, M. R. Caro, J. A. Navarro, F. Cuello, and J. Salinas B-Cell-Deficient Mice Show an Exacerbated Inflammatory Response in a Model of Chlamydophila abortus Infection Infect. Immun., December 1, 2002; 70(12): 6911 - 6918. [Abstract] [Full Text] [PDF]
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 D. C. Tsitoura, V. P. Yeung, R. H. DeKruyff, and D. T. Umetsu Critical role of B cells in the development of T cell tolerance to aeroallergens Int. Immunol., June 1, 2002; 14(6): 659 - 667. [Abstract] [Full Text] [PDF]
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