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Mucosal Immunology Lab, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Systemic nonresponsiveness has been observed after feeding of a wide variety of Ags, including soluble proteins, contact sensitizing agents, and heterologous RBC (reviewed in 2 . The demonstration that oral administration of type II collagen suppresses the induction of collagen-induced arthritis (3, 4) and myelin basic protein suppresses the induction of experimental autoimmune encephalitis (5), and the subsequent generalization of these findings to many other experimental models of autoimmune disease (reviewed in 6 have sparked renewed interest in understanding the mechanism(s) by which oral Ag leads to peripheral nonresponsiveness. The suppression of both humoral and cell-mediated immune responses in the peripheral lymphoid tissue is often accompanied by a local mucosal secretory IgA response. All of the known mechanisms for the induction of peripheral tolerance, including clonal deletion, clonal anergy, and regulation by cytokines (as well as TGF-ß-mediated active suppression) have been postulated to play a role in oral tolerance, the relative contributions of each varying with the dose of Ag fed (6). However, whether or not the microenvironment of the gut-associated lymphoid tissue (GALT)3 plays a unique role in the induction of oral tolerance has not been clear. It has been suggested that oral tolerance to high doses of soluble Ag results when small amounts of Ag gain access to the systemic circulation (2) and are then presented by nonprofessional APCs in the absence of appropriate costimulation (6, 7), implying little role for the GALT.
CD4+ T cells can be divided into two functional subsets,
Th1 and Th2, which are defined by their pattern of cytokine secretion.
Differentiation of Th cells into Th1 or Th2 subsets is determined by
the presence of the priming cytokines IL-12 or IL-4, respectively,
during a primary antigenic stimulus. Indeed, recent evidence has
suggested that the immune response to a variety of infectious agents is
accompanied by preferential expansion of one Th subset with a
concomitant down-regulation of the other (8). To explore the role of
the GALT, we examined oral tolerance to the model Ag OVA in the context
of a mucosal infection. Heligmosomoides polygyrus is a
natural murine parasite with a strictly enteric life cycle that induces
a vigorous, polarized Th2-type immune response characterized by
peripheral blood eosinophilia and marked increases in serum IgG1 and
IgE, and accompanied by a marked induction of the costimulatory
molecules B7.1 and B7.2 (9). To induce tolerance to OVA, mice were fed
a single high dose of OVA before immunization with OVA in
CFA. Draining popliteal lymph node (PLN) cells from OVA-fed,
noninfected mice were impaired in their ability to produce both Th1
(IL-2 and IFN-
) and Th2 (IL-5 and IL-10) cytokines upon
restimulation with OVA in vitro, but responded as well as PBS-fed
controls to stimulation with plate-bound anti-CD3. Both
Th1-dependent (IgG2a) and Th2-dependent (IgG1) OVA-specific serum Ab
responses were reduced in OVA-fed mice compared with controls.
Intragastric administration of OVA to mice at the peak of the mucosal
immune response to H. polygyrus at 8 days postinfection
(p.i.) resulted in maintenance of Th1 cytokine tolerance upon
stimulation with OVA in vitro, while Th2 responses were similar to or
higher than those of PBS-fed controls. In keeping with these findings,
serum OVA-specific IgG2a, but not IgG1, was reduced compared with
PBS-fed controls. Treatment of OVA-fed, H.
polygyrus-infected mice with rIL-12 during the first 8 days p.i.
restored and potentiated tolerance of serum OVA-specific Ab responses
of both Th1- and Th2-dependent isotypes as well as Th1 and Th2 cytokine
responses to OVA in vitro. We favor the interpretation that these
results point to a unique role for the cytokine microenvironment of the
GALT in the induction and maintenance of oral tolerance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/cByJ mice (8–10 wk of age) were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained in a specific viral pathogen-free facility. Infective, ensheathed, third-stage larvae (L3) of H. polygyrus (generously provided by Dr. Marilyn Scott, Institute of Parasitology, McGill University, Montreal, Canada) were propagated as previously described and stored at 4°C until use (10). Mice were inoculated orally with 200 L3. Adult worms in the intestinal contents were determined at sacrifice (4 wk p.i.). Parasite-induced peripheral blood eosinophilia was measured using the Unopette test (Becton Dickinson, Rutherford, NJ).
Induction of tolerance to OVA
Eight days after infection with H. polygyrus, either 0.5 ml of PBS or OVA (25 mg in PBS; Sigma Chemical Co., St. Louis, MO) was administered intragastrically using a ball-tipped feeding needle to groups of three or four mice. Control mice received PBS or OVA without concomitant H. polygyrus infection. One group of OVA-fed, H. polygyrus-infected mice was treated with murine rIL-12 (R&D Systems, Minneapolis, MN) during the first 8 days p.i. The IL-12 was diluted in PBS containing 1% BALB/c serum and was injected i.p. in 0.1 ml. For the first IL-12 treatment experiment, the mice were injected seven times with 950 ng/mouse/injection. After seven doses, the mice showed some signs of toxicity and the dose was reduced to six injections of 833 ng/mouse in subsequent experiments. Seven days after feeding, mice were immunized in the hind footpads with 100 µg of OVA in CFA. Two weeks after immunization, cells from the draining PLN were harvested for restimulation in vitro and serum was collected for determination of OVA-specific Ab responses.
In vitro restimulation
Pooled PLN cells from each group of three or four mice (2 x 106/ml) were cultured in triplicate in flat-bottom microtiter plates (Costar, Cambridge, MA) in complete DMEM (Life Technologies, Grand Island, NY; complete DMEM contains 10% FCS (HyClone Laboratories, Logan, UT), 10 mM HEPES, 2 mM L-glutamine, 100 U penicillin/ml, 100 µg streptomycin/ml, 50 µM ß-mercaptoethanol, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate) in 200 µl with or without varying concentrations of OVA. Some wells were coated with purified anti-CD3 (PharMingen, San Diego, CA; 10 µg/ml) as a positive control. To measure cellular proliferation, plates were pulsed at 48, 72, and 96 h with 1 µCi/well of [3H]TdR (DuPont NEN, Boston, MA) and harvested 16 h later. [3H]Thymidine incorporation was determined by liquid scintillation counting (Beckman LS1801, Fullerton, CA).
Measurement of cytokine production
At 24 h after the initiation of the cultures, 50 µl of supernatant was harvested for assessment of IL-2 production using the indicator cell line HT-2 (American Type Culture Collection (ATCC), Rockville, MD) in the presence of neutralizing concentrations of the IL-4-specific Ab 11B11 (also obtained from ATCC). HT-2 cells (5 x 103) were incubated with culture supernatants, in triplicate, for 24 h; 1 µCi/well of [3H]TdR was added for the last 6 h of culture. Murine rIL-2 (Genzyme Corp., Boston, MA) was used as the standard.
Additional cultures in complete DMEM were set up in 24-well plates at 4
to 5 x 106 cells/ml in 1 ml for the assessment of
IL-4, IFN-, IL-5, and IL-10 production in 48- and 72-h culture
supernatants by ELISA. Cultures were stimulated with 1, 10, or 100
µg/ml OVA, 5 µg/ml Con A, or plate-bound anti-CD3 (10 µg/ml).
ELISA capture (BVD4-1D11, IL-4; R4-6A2, IFN-, TRFK-5, IL-5, and
JESS-2A5, IL-10) and biotinylated second Abs (BVD6-24G2, IL-4; XMG1.2,
IFN-; TRFK4, IL-5, and SXC-1, IL-10) were purchased from PharMingen.
Immulon 2 ELISA plates (Dynatech Labs., Chantilly, VA) were coated with
capture Abs in PBS overnight at 4°C before blocking in PBS/3% FCS
for 2 h at room temperature. After washing with PBS/0.05% Tween,
culture supernatants or standards were incubated, in triplicate, on
coated plates overnight at 4°C. The plates were then washed and
incubated with the biotinylated second Abs for 1 h at 37°C. The
wells were washed with PBS/Tween and incubated with
peroxidase-conjugated streptavidin (0.5 µg/ml; Zymed Labs., San
Francisco, CA), developed with O-phenylenediamine (OPD;
Zymed Labs.), stopped with 2 N H2SO4, and read at 492 nm.
Standard curves were obtained using recombinant murine IFN-, IL-4
(Genzyme Corp.), IL-10 (R&D Systems), and IL-5 (PharMingen), and are
expressed in pg/ml ± SEM. OD values were converted to pg/ml for
each cytokine by linear regression with Delta Soft II (Biometallics,
Princeton, NJ). The limits of detection of the ELISA assays are 5 pg/ml
for IFN- and IL-4, 40 pg/ml for IL-10, and 8 pg/ml for
IL-5.
Ab assays
Each mouse was bled at sacrifice, and individual sera were assayed for OVA-specific IgG1 and IgG2a by ELISA on OVA-coated Immulon 2 plates. Plates were blocked with PBS/3% BSA for 1 h at RT and washed in PBS/Tween-20 before addition of diluted serum samples, in triplicate. After 2.5 h at RT, isotype-specific Ab responses were detected using horseradish peroxidase-conjugated goat anti-mouse IgG1 and IgG2a (Southern Biotechnology, Birmingham, AL). The reaction was developed with OPD and read at 492 nm. OD values were converted to µg/ml of OVA-specific IgG1 or IgG2a by comparison with a standard curve of purified OVA-specific Ig (developed with either anti-mouse IgG1 or anti-mouse IgG2a) with Delta Soft linear regression analysis, and are expressed as the mean concentration for each group of mice ± SEM. OVA-specific Ig was purified from the pooled serum of OVA-immunized mice by affinity chromatography on an OVA-Sepharose 4B column (Pharmacia, Piscataway, NJ) prepared according to the manufacturer’s instructions.
Mice were also bled weekly to monitor the course of the parasitic infection. Total IgG1 and total IgE were detected by ELISA on plates coated with goat anti-mouse Ab to IgG1 (Southern Biotechnology) or rat anti-mouse Ab to IgE (PharMingen). Blocked, washed plates were incubated with diluted serum samples in triplicate for 1.5 h at RT. After washing with PBS/Tween, IgG1 was detected with HRP-conjugated goat anti-mouse IgG1 (Southern Biotechnology); IgE was detected with biotin-conjugated rat anti-mouse IgE (PharMingen) and peroxidase-conjugated streptavidin (Zymed Labs.). Both reactions were developed with OPD and read at 492 nm. OD values were converted to µg/ml of IgG1 and IgE by comparison with standard curves of purified IgG1 (Southern Biotechnology) or IgE (PharMingen) by linear regression analysis, and are expressed as the mean concentration for each group of mice ± SEM.
Statistical differences in serum Ab levels were determined using a two-tailed Student’s t test with StatView software (Abacus Concepts, Berkeley, CA). A p value < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that the mucosal immune response to
H. polygyrus peaks at 8 days p.i. Gause and colleagues
demonstrated that B220+ B cells in the mesenteric lymph
node (MLN) of infected mice exhibit a maximal increase in cell size,
MHC class II, and B7-2 expression at this time point (11). Moreover,
reverse-transcriptase PCR analysis showed that cells in the MLN and
Peyer’s patch already exhibited a polarized Th2 cytokine response by
day 8 (12). We were interested in determining whether a polarized,
parasite-induced, Th2 mucosal cytokine response influenced subsequent
systemic responses to orally administered Ag. We therefore used the
oral tolerance protocol shown in Figure 1
. Eight days after H.
polygyrus infection, mice were fed a single high (25 mg) dose of
OVA or PBS and immunized in the footpads 1 wk later with OVA/CFA. One
group of infected mice was treated with rIL-12 six times during the
first 8 days p.i., a protocol previously shown to inhibit Th2 cytokine
production and enhance Th1 responses to primary nematode infection
(13). Two weeks after immunization, cells from the draining PLN were
restimulated in vitro with varying doses of OVA or plate-bound
anti-CD3. Proliferative responses were determined by
[3H]thymidine incorporation at various time points, and
cytokines secreted into the culture supernatant were analyzed by ELISA.
Three independent experiments produced comparable results. The results
shown in Figures 2 through 4 are from the
same representative experiment. Figure 2A shows that T cells
from both H. polygyrus-infected (closed symbols) and
noninfected (open symbols) PBS-fed mice proliferate in response to
stimulation with OVA in vitro. The response increased markedly over the
time period examined. As expected from previous reports (14, 15), oral
administration of OVA virtually abrogated the proliferative response to
OVA in vitro. The proliferation of T cells from OVA-fed, infected mice
is partially reduced. Treatment of OVA-fed infected mice with rIL-12 in
vivo to block the parasite-induced Th2 response restored
nonresponsiveness to OVA restimulation in vitro.
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B shows that all five cell
populations responded similarly to anti-CD3 after 66 h of
culture, indicating that all of the cell populations were viable and
contained similar numbers of potentially responsive T cells, and
emphasizing the specificity of the nonresponsiveness to OVA seen in
Figure 2A. T cells from noninfected mice decline or plateau
after 66 h in culture. The anti-CD3 response from OVA-fed mice
declined more rapidly than that of PBS-fed mice, perhaps reflecting an
enhanced susceptibility of orally tolerized lymphocytes to cell death
in vitro (16). The response of T cells from both PBS- and OVA-fed
infected mice to plate-bound anti-CD3 continued to climb throughout
the culture period examined, presumably driven by parasite-specific
priming in vivo. The response to the parasite is diminished in
IL-12-treated infected mice (see Fig. 4), and T cells from these mice
proliferate less vigorously to anti-CD3 stimulation in vitro, but
at levels at least comparable with those seen in noninfected mice.
However, whereas the response to anti-CD3 declined over time in
noninfected mice, the response of T cells from OVA-fed, IL-12-treated
infected mice continued to increase from 66 to 114 h in culture in
a manner similar to that observed for the other groups of
parasite-infected mice.
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We next asked whether oral administration of OVA in the polarized,
Th2-biased mucosal environment present in H.
polygyrus-infected mice influenced the cytokine profile seen after
Ag restimulation in vitro. Culture supernatants were analyzed for a
panel of Th1 (IFN-) and Th2 (IL-4, IL-5, IL-10) cytokines at both 48
and 72 h after the initiation of the culture by ELISA. IL-2 was
measured in 24-h supernatants by bioassay (see Materials and
Methods). The data shown in Figure 3 represent the peak of the Ag-specific
cytokine response. The Ag specificity of the cytokine responses is
demonstrated by their dose dependence; in vitro restimulation with
increasing concentrations of OVA elicited secretion of higher levels of
cytokines for each cytokine assayed. We confirmed that secretion of
both Th1 and Th2 cytokines was reduced markedly by feeding of OVA to
noninfected mice (15). Figure 3 shows that production of the Th1
cytokines IL-2 (Fig. 3A) and IFN- (Fig. 3B) was tolerized by OVA feeding in both infected and
noninfected mice. PLN from each group responded similarly to
restimulation with anti-CD3 in vitro. Markedly different results
were observed when the same culture supernatants were analyzed for the
Th2 cytokines IL-5 and IL-10. Th2 cytokine production was tolerized by
OVA feeding in noninfected mice. However, OVA-fed infected mice
produced levels of IL-10 comparable with that seen in PBS-fed infected
and noninfected controls (Fig. 3C). Tolerance to OVA
was maintained in PLN cells from OVA-fed, infected mice treated with
rIL-12 in vivo. OVA-fed, infected mice produced more IL-10 in response
to anti-CD3 stimulation than that observed for PBS controls; PLN
cells from OVA-fed noninfected mice secreted less IL-10 in response to
anti-CD3. Although observed repeatedly, the mechanism by which OVA
feeding suppresses the Th2 cytokine response to stimulation with
anti-CD3 in noninfected mice is not clear. Enhanced responsiveness
to restimulation with anti-CD3 by cells from infected mice may
reflect parasite-induced activation in vivo. In the experiment shown in
Figure 3D, OVA feeding markedly enhanced IL-5 production by
PLN cells from infected mice in response to OVA restimulation in vitro;
in other experiments, OVA- and PBS-fed infected mice produced similar
amounts of IL-5 (data not shown). IL-12 treatment of OVA-fed infected
mice restored tolerance of IL-5 responses to OVA. As noted for IL-10,
feeding primed for enhanced production of IL-5 in response to
stimulation with anti-CD3 in infected mice, while noninfected,
OVA-fed mice produced less IL-5 in response to cross-linking with
anti-CD3. H. polygyrus infection is known to induce an
elevation of IL-4 mRNA in the gut-associated lymphoid tissue (12), and
IL-4 is clearly involved in the protective immune response to the
parasite (17). Although IL-4 was readily detectable in cultures of
anti-CD3-stimulated splenic or PLN lymphocytes from infected mice,
we could not reproducibly detect an OVA-specific IL-4 response in the
culture supernatants of PLN lymphocytes restimulated with OVA in vitro.
Accordingly, IL-4 is not included in the Th2 cytokine panel shown in
Figure 3. Therefore, noninfected mice exhibit tolerance for Ag-specific
Th1 and Th2 responses in vitro. Mice fed Ag during a Th2-biased mucosal
immune response cannot be tolerized for Th2 cytokine restimulation in
vitro.
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shows
that both OVA-specific IgG1 and IgG2a were reduced by OVA feeding in
noninfected mice. However, the Th2-dependent (IgG1) Ab response was
tolerized by OVA feeding in noninfected mice, but not in mice infected
with H. polygyrus. A statistically significant reduction in
the OVA-specific IgG1 response was restored by treatment of infected
mice with rIL-12. Thus, oral administration of OVA during a Th2-biased
mucosal immune response prevents induction of tolerance for a
Th2-dependent Ab response.
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Infection with H. polygyrus induces a marked increase
in serum IgG1 and IgE (17). To monitor the effects of OVA feeding and
rIL-12 treatment on the response to the parasite, we determined
eosinophil counts and serum IgE and IgG1 levels weekly during the
course of the oral tolerance experiments. rIL-12 treatment has been
shown to inhibit IgE and eosinophil responses during a primary
infection with the intestinal nematode parasite Nippostrongylus
brasiliensis (13). Figure 4A shows that the IgE response
to H. polygyrus infection is also significantly inhibited by
rIL-12 treatment during the first 8 days p.i. The parasite-induced IgE
response is reduced by about 50% at day 8 p.i.
(p < 0.05), the time point at which OVA is
fed, and remains at about 75% of PBS control levels even at 30 days
p.i. The eosinophil response to H. polygyrus infection at
day 8 p.i. is abrogated completely by rIL-12 treatment (Fig. 4B) and remains below levels seen in infected,
untreated mice throughout the course of the experiment. The
parasite-induced IgG1 response is not affected by rIL-12 treatment
during the first 8 days of infection (Fig. 4C). This
was expected, since previous work has shown that while the polyclonal
IgE response to H. polygyrus is inhibited by Abs to IL-4 and
eosinophilia is inhibited by Abs to IL-5, neither Ab treatment
inhibited total IgG1 (18). In addition, IgE responses are generally
more readily inhibited by rIL-12 treatment than IgG responses (19).
Finally, we determined the impact of rIL-12 treatment and OVA feeding
on parasite survival. Figure 4D shows that the number of
parasites recovered from mice fed with OVA at 29 days p.i. did not
differ from that in the control, PBS-fed mice. However, the number of
parasites in rIL-12-treated mice was elevated, indicating that IL-12
treatment resulted in prolonged survival. Taken together, these results
suggest that IL-12 treatment of infected mice resulted in a marked
diminution in the parasite-induced Th2 cytokine response.
As further confirmation of the effectiveness of IL-12 treatment in
reversing the cytokine profile of H. polygyrus-infected
mice, we stimulated MLN and spleen cells at 8 days p.i. from
noninfected, infected, and infected plus IL-12-treated mice in vitro
with Con A or anti-CD3 and examined the secretion of the Th1
cytokine IFN- and the Th2 cytokines IL-4, IL-5, and IL-10 into 48-h
culture supernatants by ELISA. Table II
shows a clear Th2 bias in the response to Con A or anti-CD3 in
cells derived from the MLN of infected mice. The MLN shifts from 30%
B220+ and 70% TCR-
ß+ in uninfected mice
to 70% B220+ and 30% TCR-ß+ in infected
mice at 8 days p.i. (data not shown). The polarized Th2 response in the
MLN is clear even without normalizing the data for this marked downward
shift in the proportion of T cells in infected mice. IL-12 treatment
greatly reduces, but does not completely remove, the Th2 response, as
would be expected from the data on the response to the parasite in
Figure 4. Th2 cytokine production is enhanced (and Th1 cytokine
production reduced) in Con A- or anti-CD3-stimulated spleen cells
from infected mice. The splenic T cell response is not polarized to a
Th2-type response as it is in the MLN, and we did not obtain evidence
for any alteration in the proportion of T and B cells in the spleens of
infected mice. The increased Th2 cytokine response in infected mice was
eliminated by treatment with rIL-12 in vivo. These results are
consistent with previous analyses of cytokine mRNA induced in response
to nematode infection. When examined in the MLN and Peyer’s patch 8
days after primary infection with another nematode parasite, N.
brasiliensis, IL-12 treatment inhibited the induction of mRNA for
the Th2 cytokines IL-4 and IL-5, but enhanced the production of the Th1
cytokine IFN- as well as the Th2 cytokine IL-10 (13). We did not see
any enhancement of IL-10 responses in infected mice by examining
secretion of cytokines by T cells restimulated with Con A or
anti-CD3. The up-regulated production of IL-10 mRNA noted in the
previous study might be derived from non-T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and a Th2-dependent isotype of IgG
(Table I) was broken in H. polygyrus-infected mice.
Differentiation of Th cells into Th1 or Th2 subsets is determined by
the presence of the priming cytokines IL-12 or IL-4, respectively,
during a primary antigenic stimulus (8). One interpretation of our
results (and the one that we favor) is that the induction of large
quantities of IL-4 in the GALT by parasitic infection primes for a Th2
response to orally administered OVA and prevents the development of
tolerance. Work in other systems has documented the ability of a
polarized Th2 response to one Ag to alter or deviate the cytokine
profile of the response to another Ag (20, 21) and its reversal by
treatment with rIL-12 (22, 23). Tolerance of Th2 responses to orally
administered OVA is restored by treatment of infected mice with rIL-12,
providing further support for our contention that parasite-induced Th2
cytokines are responsible for the break in Th2 tolerance to OVA. In
another model system, an established T cell tolerance to i.v. injected
staphylococcal enterotoxin B was broken by infection with the nematode
parasite N. brasiliensis (24). Anergic, staphylococcal
enterotoxin B-tolerized, CD4+ T cells proliferated and
produced IL-4 following infection with N. brasiliensis,
suggesting activation, by infection, of previously tolerant cells. We
show in this study that tolerance for Th1-mediated responses to OVA can
still be induced during an ongoing infection, and that it is the
polarized Th2 response induced by the parasite that dictates whether
the response to a normally tolerizing form of orally administered Ag
will be immunity or tolerance. However, although H.
polygyrus maintains a strictly enteric life cycle, it does lead to
some Ag nonspecific stimulation in peripheral sites, as can be seen in
the responses to in vitro restimulation of spleen and PLN cells from
infected mice with anti-CD3 in Figure 2 and Table II. This suggests
that either activated T cells or APC bearing H. polygyrus
Ags can migrate out of the GALT to peripheral sites. Although this
response does not exhibit the marked Th2 polarity seen in the mucosal
(MLN) response to H. polygyrus, it suggests the alternative
possibility that the induction of cytokine release in the periphery in
response to this mucosal infection or cellular traffic to the PLN may
play a role in influencing peripheral tolerance induction to orally
administered Ag.
It has been suggested that oral administration of high doses of Ag
leads to tolerance because small amounts of Ag gain access to the
peripheral circulation and are then presented, in the periphery, in the
absence of the costimulatory signals required to induce immunity.
Although not addressed directly in this study, use of this mucosal
infection model should provide insight into the role of the GALT in the
processing and presentation of mucosal Ag. During H.
polygyrus infection, the costimulatory molecules B7.1 and B7.2 are
markedly up-regulated on B220+ cells (with concomitant
down-regulation on TCR-ß+ cells) in the MLN by 8 days
p.i. (Ref. 9 and data not shown). Moreover, the induction of a
protective immune response to H. polygyrus has been shown
recently to be B7 dependent, demonstrating that the levels of
costimulation induced are sufficient for the development of immunity to
the parasite (25). H. polygyrus infection is restricted to
the intestine, but some parasite-induced activation of B cells and
macrophages occurs in the spleen (data not shown and Table II). These
initial observations have led us to speculate on the role of
costimulation in the development of tolerance to orally administered
Ag. If Ag presentation in the absence of costimulation is responsible
for the Th1 nonresponsiveness observed in our model, one would have to
assume no role for presentation of orally administered Ag in the MLN
and preferential Ag presentation in the spleen. Yet the marked Th2
cytokine bias present only in the MLN in response to this enteric
parasite (Ref. 12 and Table II) clearly alters Th2 nonresponsiveness to
OVA. The ability to suppress both Th1 and Th2 responses in
IL-12-treated, infected mice in the absence of parasite-induced Th2
cytokines shows that both types of Th responses can be tolerized in the
presence of parasite-induced up-regulation of costimulation, which is
not altered substantially by IL-12 treatment (data not shown). We favor
the interpretation that the induction of peripheral tolerance is
preceded by transient T cell activation (26) and requires a
B7-dependent signal (27, 28). The ability of an Ag to induce tolerance
or immunity may be decided by whether it is recognized in the context
of CD28 (which seems to induce immunity) or CTLA-4 (which induces
tolerance). It will be of great interest to examine the role of these
ligands in orally induced nonresponsiveness in H.
polygyrus-infected mice.
Taken together, we think that our results favor the interpretation that the cytokine environment in the GALT plays a key role in regulating peripheral nonresponsiveness to orally administered Ag. Whether this involves a unique role for the GALT in Ag presentation, recruitment of cells from the periphery into the GALT, or the generation and/or migration of cells that secrete regulatory cytokines, will be the subject of future experiments. Since helminthic infections with polarized cytokine responses are endemic in many developing countries (incidence can reach 100% by age 10 in some areas (9)), our observations are clearly of clinical relevance. Mucosal infection is likely to have an impact on both the therapeutic use of orally administered Ag in the treatment of autoimmune disease (6) and the disregulation of mucosal cytokine responses that appears to predispose for intestinal inflammation and inflammatory bowel disease (1).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cathryn Nagler-Anderson, Mucosal Immunology Laboratory, Room 3308, Massachusetts General Hospital East, 3 West, Building 149, 13th Street, Charlestown, MA 02129. E-mail address:
3 Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; MLN, mesenteric lymph node; OPD, O-phenylenediamine; p.i., postinfection; PLN, popliteal lymph node; RT, room temperature.
Received for publication August 13, 1997. Accepted for publication November 17, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T. Matysiak-Budnik, G. van Niel, F. Megraud, K. Mayo, C. Bevilacqua, V. Gaboriau-Routhiau, M.-C. Moreau, and M. Heyman Gastric Helicobacter Infection Inhibits Development of Oral Tolerance to Food Antigens in Mice Infect. Immun., September 1, 2003; 71(9): 5219 - 5224. [Abstract] [Full Text] [PDF]
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J. C. M. Paterson, P. Garside, M. W. Kennedy, and C. E. Lawrence Modulation of a Heterologous Immune Response by the Products of Ascaris suum Infect. Immun., November 1, 2002; 70(11): 6058 - 6067. [Abstract] [Full Text] [PDF]
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M. E. H. Bashir, P. Andersen, I. J. Fuss, H. N. Shi, and C. Nagler-Anderson An Enteric Helminth Infection Protects Against an Allergic Response to Dietary Antigen J. Immunol., September 15, 2002; 169(6): 3284 - 3292. [Abstract] [Full Text] [PDF]
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S. D. Hurst, B. W. P. Seymour, T. Muchamuel, V. P. Kurup, and R. L. Coffman Modulation of Inhaled Antigen-Induced IgE Tolerance by Ongoing Th2 Responses in the Lung J. Immunol., April 15, 2001; 166(8): 4922 - 4930. [Abstract] [Full Text] [PDF]
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H. N. Shi, H. Y. Liu, and C. Nagler-Anderson Enteric Infection Acts as an Adjuvant for the Response to a Model Food Antigen J. Immunol., December 1, 2000; 165(11): 6174 - 6182. [Abstract] [Full Text] [PDF]
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 M. E. Scott and K. G. Koski Zinc Deficiency Impairs Immune Responses against Parasitic Nematode Infections at Intestinal and Systemic Sites J. Nutr., May 1, 2000; 130(5): 1412S - 1420. [Abstract] [Full Text]
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H. N. Shi, M. J. Grusby, and C. Nagler-Anderson Orally Induced Peripheral Nonresponsiveness Is Maintained in the Absence of Functional Th1 or Th2 Cells J. Immunol., May 1, 1999; 162(9): 5143 - 5148. [Abstract] [Full Text] [PDF]
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,
, PBS fed; 
,•, OVA fed; 
, OVA-fed
mice treated with rIL-12. The results are displayed as the mean ±
SEM of triplicate wells. Three independent experiments produced
comparable results. The data in Figures 2 through 4
