HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chung, Y. Articles by Kang, C.-Y. Search for Related Content PubMed PubMed Citation Articles by Chung, Y. Articles by Kang, C.-Y.

Kinetic Analysis of Oral Tolerance: Memory Lymphocytes Are Refractory to Oral Tolerance¹

Laboratory of Immunology, College of Pharmacy, Seoul National University, Shillimdong, Kwanakgu, Seoul, Korea

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Oral administration of soluble Ag before immunization induces peripheral tolerance and is effective in suppressing animal models of autoimmune diseases. Although tolerance induction in primed animals is more clinically relevant, it is not well studied. Therefore, this study was designed to examine the feeding effects on different phases of the immune response. We observed that feeding a single high dose (250 mg) of OVA to OVA-primed BALB/c mice could induce OVA-specific suppression in the Ab production and T cell proliferation only at the naive and the activation phases of the immune response, whereas multiple high doses (100 mg/feed for 10 days) were effective at the effector phase. OVA-specific IL-4 production in culture supernatant was also suppressed in the tolerized groups. However, when the mice had resting memory lymphocytes, even multiple feeding regimens were not effective in tolerance induction, although multiple low doses (1 mg/feed for 10 days) partially suppressed Ab production. This phenomenon was confirmed by adoptive transfer study. Nevertheless, the reactivated memory response was suppressed partially by multiple high doses. Our findings have an important implication for understanding the mechanism of oral tolerance and for the therapeutic applications of oral tolerance to autoimmune diseases.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immunologic tolerance is a state of unresponsiveness specific for a particular Ag. Oral administration of Ags is a classical method of inducing Ag-specific systemic immune tolerance (1, 2). It is well documented that a single high dose of Ag induced a state of tolerance characterized by clonal anergy or deletion, whereas multiple low doses induced a state of tolerance characterized by active suppression (3, 4, 5, 6). Many studies have shown that orally fed relevant Ags before systemic immunization suppressed immune response and inflammatory diseases, such as experimental autoimmune encephalitis (7, 8, 9, 10), experimental autoimmune uveoretinitis (11, 12) and collagen-induced arthritis (13, 14, 15). Furthermore, clinical trials based on the concept of oral tolerance are being applied to humans for multiple sclerosis and rheumatoid arthritis (16, 17, 18). These autoimmune diseases occur when a specific adaptive immune response is mounted against self-Ag (19).

The adaptive immune response can be divided into four phases, which are naive, activating, effector, and memory phase (20, 21). When the memory lymphocytes meet the same Ag, they lead the secondary immune response characterized by more rapid kinetics, isotype switching, and higher amount of Ag-specific Ab, thus effectively eliminating the Ag (22, 23). Most studies of oral tolerance are the case that oral administration of Ag is followed by systemic immunization (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). In this case, naive lymphocytes met orally administered Ag. However, in the disease state the immune system has already met the causing Ag and established the immune response to the Ag. Hence, tolerance induction in the primed animals is more clinically relevant. A few studies have reported the effect of Ag feeding after immunization (24, 25, 26, 27, 28); however, the results were controversial. For example, it was reported that feeding after immunization with low doses of Ag led to worsening of experimental autoimmune uveoretinitis (26). Similarly, it was also reported that orally Ag was not effective in decreasing ongoing clinical experimental autoimmune encephalitis (27). But, another study reported that feeding Ag 7 days after immunization produced dose-dependent suppression of T cell response, although serum IgG levels were unaffected (28). Despite mixed results, most groups showed that proximal feeding after immunization was more effective in tolerance induction. Although there are so many studies investigating oral tolerance, no study has shown clearly the feeding effects on different phases of the immune response.

The present study was designed to show whether single or multiple feeding regimens could induce Ag-specific unresponsiveness in primed animals. The results of this study indicate that the early phases of the immune response could be tolerized by orally administered Ag. However, we also found that once the immune system established memory to a certain Ag, feeding Ag is not effective in inducing tolerance to the Ag.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Female BALB/c mice (5–8 wk of age) were obtained from Charles River (Tokyo, Japan). The mice housed at Seoul National University until use and kept in a specific pathogen-free condition in a germ-free isolator (Techniplast, Buguggiate, Italy) during the experiments.

Induction and assessment of oral tolerance

For feeding a single high dose of Ag, mice were immunized i.p. with 20 µg of OVA (grade V; Sigma, St. Louis, MO) emulsified in CFA (Sigma). Mice were given a single feed of 250 mg of OVA or PBS as a control at 2 days before or 0, 1, 2, 3, 5, 7, 14, 20, 30, and 50 days after immunization, respectively. Three weeks after feeding, the mice were boosted i.p. with 20 µg of OVA in incomplete Freund’s adjuvant (IFA;³ Sigma). Abs in serum were measured 7 days after boosting. Eight days after boosting, spleens were removed for assessment of proliferation and for cytokine assays. In some experiments, groups of mice were immunized i.p. with 20 µg of type II collagen (Sigma) and fed 250 mg of OVA or PBS 1 day after the immunization.

For feeding multiple doses of Ag, mice were immunized i.p. at day 0 and fed daily 1 mg or 100 mg of OVA or PBS during days 5–14 or days 30–39, respectively. In memory reactivation experiment, mice were immunized i.p. twice at day 0 and day 21, and fed daily 1 mg or 100 mg of OVA or PBS during days 21–30. Three days after the last feeding, the mice were boosted i.p. and tested as described above.

ELISA for Ag-specific IgG quantification

Microtiter plates (Maxisorp F96; Nunc, Roskilde, Denmark) were coated overnight at 4°C with 100 µl/well OVA (5 µg/ml) in PBS (pH 7.4) or type II collagen in 0.05 M acetate buffer. After washing with PBS, the plates were blocked with 150 µl/well with 1% BSA (Sigma) for 30 min at room temperature. Serially diluted serum samples were added to the wells and incubated for 3 h at room temperature. Alkaline phosphatase-conjugated goat anti-mouse IgG (Pierce, Rockford, IL) diluted in PBS/0.1% BSA/Tween 20 (1:2000) was added and incubated for 2 h at room temperature. The plates were washed, and then 100 µl of phosphatase substrate (p-nitrophenyl-phosphate in a carbonate buffer, pH 9.6) was added to each well. The absorbance was read at 405 nm using a Emax microplate reader (Molecular Devices, Menlo Park, CA). Ab concentrations in tested serum were determined from standard curves constructed using immuno-affinity purified anti-OVA IgG.

Splenocyte proliferation assay

Single cell suspensions were prepared from spleens placed in RPMI 1640 (BioWhittaker, Walkersville, MD) and erythrocytes were depleted. After three washes, the cells were resuspended in RPMI 1640 containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD). Cells were plated at 5 x 10⁵ spleen cells per well in 96-well round-bottom microtiter plates (Nunc). Cells were cultured for 4 days with 20 µg/ml OVA (grade V) or alone in 200 µl of medium. After 96 h of incubation, including a final 22 h pulse with [³H]thymidine (0.5 µCi per well), the cultured splenocytes were harvested using an automatic harvester (Skatron, Sterling, VA), and incorporation of label was measured using a liquid scintillation counter (Wallac, Turku, Finland).

Cytokine assay

Single cell suspensions were prepared as described above. Cells were plated at 8 x 10⁶ cells per well in a 1 ml aliquot in a 24-well tissue culture plate (Nunc). Cells were cultured for 72 h with 20 µg/ml OVA or alone. Supernatants were harvested and frozen at -20°C until assayed.

Culture supernatants were tested for the presence of IFN- and IL-4 using sandwich ELISA. Purified rat anti-mouse IFN- mAb (clone R4-6A2), biotinylated rat anti-mouse IFN- mAb (clone XMG1.2), and recombinant mouse IFN- were purchased from PharMingen (San Diego, CA). For determination of IFN- production, 96-well microtiter plates were coated with capture Ab (2 µg/ml in sodium phosphate buffer) at 4°C overnight. After washing, the plates were blocked with 1% BSA for 30 min at room temperature. Serial dilution of recombinant mouse IFN- and sample were added to wells and incubated overnight at 4°C. Biotinylated detection Ab was diluted in 1% BSA/Tween 20 to 1 µg/ml and added to wells for 1 h at room temperature. The plates were washed, and 100 µl/well streptavidin-HRP (Pierce), diluted to 1:1000, was added. After incubation for 30 min, 3,3',5,5'-tetramethylbenzidine peroxidase substrate (Sigma) was added and incubated until color development. The color reaction was stopped by adding 100 µl of 0.2 M H₂SO₄, and the plates were read at 450 nm using a Emax microplate reader (Molecular Devices).

For the IL-4 assay, the mouse IL-4 Minikit was used (Endogen, Cambridge, MA). IL-4 concentration was assayed according to manufacturer’s recommendations. Cytokine concentrations in test supernatants were determined from standard curves constructed using serial dilutions of appropriate recombinant cytokines.

Adoptive lymphocyte transfer

BALB/c mice were immunized i.p. with 20 µg of OVA in CFA. Thirty days after immunization, the mice were sacrificed and their spleens harvested. After depletion of erythrocytes, single-cell suspensions of whole splenocytes from OVA-immunized mice were transferred i.v. via tail vein to groups of five or six naive recipient mice at doses of 10⁷ splenocytes per transfer (200 µl). The recipient mice were fed 100 mg, 1 mg of OVA, or PBS for 10 days and then boosted i.p. with 20 µg of OVA in IFA 4 days after the last feeding to determine whether immunologic memory response was transferred and whether multiple feeding regimens were effective to tolerance induction in this system. For negative control, groups of mice of the same age as recipient mice were immunized with OVA in IFA in a similar fashion to the last step of the cell transfer study described above. Sera were obtained 5, 10, and 15 days after immunization, and OVA-specific Ab was quantified using ELISA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
A single high dose feeding effects different stages of the immune response

We first investigated whether a single feeding of high dose of Ag at different stages of the immune response could induce immunologic tolerance. Groups of mice were immunized i.p. with OVA in CFA. The mice were then fed 250 mg of OVA or PBS as a control in a single dose at different days and boosted i.p. with OVA in IFA (Fig. 1B). To compare the naive state with the postimmunized state in our system, a day -2 group was added (Fig. 1A). In the day -2 group, mice were fed 2 days before primary immunization. One week after boosting, these mice were bled and the sera from each group of mice were tested for anti-OVA IgG by ELISA. The effect of a single high dose feeding of Ag was also investigated in cellular responses. Splenocytes were obtained from the same mice and tested by an in vitro proliferation assay.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Experimental schedules. Groups of mice were treated as indicated above. Mice were immunized i.p. with 20 µg OVA in CFA and boosted i.p. with the same amount of OVA in IFA. In A and B, mice were fed 250 mg of OVA or PBS as a control. In C and D, mice were fed 100 mg, 1 mg of OVA, or PBS as a control, respectively.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Feeding effects of single high dose of OVA on Ab production and T cell proliferation in primed mice. All experiments were performed as described in Fig. 1, A and B. The described days indicated the feeding day after primary immunization. The obtained sera were quantified for OVA-specific IgG by ELISA. The 5 x 105 splenocytes per well were cultured with OVA for 96 h, and [3H]thymidine incorporations were measured. Results for individual mice are indicated, and the crossbars show the average of each group. The data were analyzed by Student’s t test and shown as p values. The formula used was: % suppression = (PBS - OVA) x 100/PBS.

We also examined the production of IFN- and IL-4, which were used as the indicator of Th1 and Th2 response, respectively. Culture supernatants of splenocytes were obtained after 72-h incubation, as described above. As showed in Fig. 3, IFN- production was not affected in day -2 group. However, IFN- production was increased significantly in all the other groups fed OVA regardless of suppression of humoral and cellular response. In contrast, a different pattern of IL-4 production was observed. There was almost complete suppression of IL-4 in days -2, 0, 1, and 2 groups OVA-fed compared with PBS-fed groups. From the day 3 group, however, the suppression was broken gradually. Finally, in the day 14 group, the amount of IL-4 was near the OVA-fed group and PBS-fed control group. Collectively, the results indicated that in those groups that IL-4 production was inhibited, and the in vitro proliferation of splenocytes was also suppressed.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Feeding effects of single high dose of OVA on cytokine production. All experiments were performed as described in Fig. 1, A and B. The 8 x 106/ml splenocytes were cultured with 20 µg/ml OVA, and the supernatants were harvested after 72 h. Each cytokine was quantified using sandwich ELISA. These values were averaged and are presented as the mean ± SE.

A similar protocol was used to examine the effects of multiple feeding of high or low dose OVA at the time in which a single dose feeding could not induce tolerance (Fig. 1C).

OVA-specific IgG response was suppressed profoundly by multiple high dose feeds from day 5 to 14 (Fig. 4A, p < 0.001). However, multiple low dose feeds at this period did not suppress IgG response. The cellular response was suppressed profoundly at high dose feeds (p < 0.01), whereas low dose feeds induced moderate suppression (p < 0.1). These data indicated that systemic Ag-specific unresponsiveness could be induced at the effector stage of immune response by a multiple high dose feeding regimen.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Multiple feeding effects on Ab production and T cell proliferation in the effector or memory phase of the immune response. All experiments were performed as described in Fig. 1C. The data of mice fed OVA daily from days 5 to 14 were shown in A, and those of mice fed OVA from days 30 to 39 were shown in B. Results for individual mice are indicated, and the crossbars show the average of each group. The data were analyzed by Student’s t test and shown as p value. The formula used was: % suppression = (control - fed)/control.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. Multiple feeding effects on cytokine production in the primed mice. All experiments were performed as described in Fig. 1, C and D. The splenocyte culture supernatants were harvested after 72-h culture. Each cytokine was quantified using sandwich ELISA. These values were averaged and are presented as mean ± SE.

We also examined the effect of multiple feeds on reactivated memory response. Groups of mice were immunized on days 0 and 21, and then fed 10 times with 1 mg, 100 mg OVA, or PBS as a control (Fig. 1D). Three days after the last feeding, these mice were boosted with the same Ag, and ELISA and proliferation assays were performed as described above.

The OVA-specific Ab amount was decreased moderately (37%) in the 100 mg fed group (p < 0.05). Ag-specific proliferation was also decreased (48%) in the 100 mg fed group. In the 1 mg fed group, Ag-specific immune response was suppressed slightly in both humoral and cellular level (Fig. 5, 22% and 33%, respectively). IL-4 production was almost completely suppressed by multiple high doses of OVA while suppressed partially by multiple low doses. On the other hand, IFN- production was increased only in high doses fed group (Fig. 6). Thus we concluded that the reactivated memory response could be suppressed partially by multiple feeds.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Multiple feeding effects on the Ab production and T cell proliferation in the reactivated memory immune response. All experiments were performed as described in Fig. 1D. Results for individual mice are indicated, and the crossbars show the average of each group. The data were analyzed by Student’s t test and shown as a p value. The formula used was: % suppression = (control - fed)/control.

Because single or multiple feeding regimens could not induce Ag-specific unresponsiveness after establishment of memory, we next examined whether these phenomena were dependent on the number of OVA-specific memory lymphocytes using an adoptive transfer model. In preliminary experiments, we determined the minimal cell number to evoke memory response in the recipient mice. When various number of total splenocytes, obtained 30 days after immunization, were transferred i.v. into naive recipient mice, 10⁷ cells per transfer appeared to be the minimal cell number to evoke memory Ab response to OVA while 10⁶ cells did not (data not shown). Therefore we used 10⁷ cells per transfer in the following experiment. Donor mice were immunized with OVA, and 30 days after immunization a single splenocyte suspension was prepared from donor mice. A total of 10⁷ cells were transferred i.v. into each recipient mouse, and 4 days after the transfer recipient mice were fed 100 mg, 1 mg, or PBS as control for 10 days, and then these mice were boosted and bled. Again, 10⁷ cells per mouse were sufficient to evoke memory response in recipient mice (Fig. 7). We could observe the characteristics of memory response most significantly in sera obtained at day 10. Compared with the results of the primary response, the kinetics was more rapid, the isotype was switched from IgM to IgG (data not shown), and the amount of OVA-specific IgG was 26 times higher (at day 10). As shown in Fig. 7, however, these memory responses were not inhibited by multiple feeds. On the contrary, the Ab response was somewhat enhanced by high dose feeds. At 5 days after immunization, compared with other groups, there was a remarkable increase in the amount of Ab in the high dose fed group, indicating that these feeds activated the immune system. At day 10, no apparent difference in the level of Ab production was observed among those three recipient groups. Taken together, these data confirmed that once the immune system established memory to a certain Ag, tolerance to the Ag could not be induced by oral administration of the Ag, even when the number of memory cells were reduced to the least measurable level.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. Adoptive transfer of immunologic memory for OVA and multiple feeding effect on Ab production in the recipient mice. BALB/c mice were immunized i.p. with OVA, and 30 days later 107 splenocytes were transferred i.v. into groups of naive recipient mice. The recipient mice were immunized i.p. 4 days later with OVA. In control group, naive mice in the same age with the recipient mice were immunized i.p. without adoptive transfer. The OVA-specific IgG in serum was measured at 5, 10, and 15 days after immunization using ELISA. These values were averaged and are presented as mean ± SE.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Tolerance induction in naive animals after oral administration of an Ag has been well documented by many groups (3, 4, 5, 6). Our previous study has shown that the state of tolerance established by prior feeding of high dose of Ag was maintained after 26 wk (29). Furthermore, several studies have demonstrated the effectiveness of oral tolerance in animal models of autoimmune disease (7, 8, 9, 10, 11, 12, 13, 14, 15). However, attempts to induce tolerance by oral Ag after immunization have yield mixed results. In this regard, we have investigated the effect of oral Ag administration on different stages of the immune response. Initially we performed experiments to examine whether single feeding of high dose of Ag could induce tolerance at each stage of the immune response in BALB/c mice. Feeding a single high dose of OVA suppressed production of anti-OVA IgG and in vitro proliferation of splenocytes completely when OVA was fed at 0, 1, or 2 days after immunization, respectively. It seemed that the remaining fed OVA in circulation did not affect our assays because the half-life of the fed OVA was less than 12 h in BALB/c mice (data not shown) and there was no apparent difference in proliferation in the absence of Ag between OVA-fed groups and PBS-fed groups. The observed OVA-specific suppression was caused by quenching the ongoing primary response. The degree of suppression was similar to that of group fed before immunization. Thus it was concluded that at activation stage of immune response, a single high dose of Ag could induce profound Ag-specific tolerance. However, such a tolerance was not induced in mice fed OVA 3 days after immunization. Furthermore, T cell proliferation was not affected by feeding in this group. Feeding Ag to the mice at 7 days or 14 days after immunization (day 7 and day 14 group, respectively) induced slightly stronger suppression in Ab production than day 3 and day 5 group. We have repeated the same experiment and obtained similar results. At 7 days after immunization, Abs were produced vigorously in primed animals. Thus it seems likely that Ab produced by immunization and Ag administered formed immune complexes, which bound to FcR on B cells and regulated Ab production in T cell-independent manner (30, 31). These observations indicated that feeding single high dose of Ag could partially suppress the B cell response at the effector phase.

It is now widely accepted that multiple feeding is more effective in tolerance induction than single feeding (24, 32). Actually, feeding multiple high doses of OVA suppressed the effector phase of the immune response. The degree of suppression was profound for both Ab production and T cell proliferation. However, once an immune system establishes memory to the Ag, neither single nor multiple feeding regimens induced Ag-specific unresponsiveness. Interestingly, multiple feeding of a low dose of Ag to the memory stage of mice showed partial suppression of Ab production. On the other hand, at the memory reactivation stage of the immune response, multiple feeding induced partial suppression. Because the immune response in this condition could be induced by both newly generated naive cells and previously formed memory cells, we interpret the partial suppression of the OVA-fed mice was mainly due to the suppression of newly generated OVA-specific naive cells. We finally asked if the number of Ag-specific memory cells were decreased to the least measurable level, tolerance could be induced by multiple feeding. A total of 10⁷ cells per transfer (10% of donor splenocytes) were the minimal cell numbers to evoke memory response in our system. Neither high doses nor low doses were effective in recipient mice. Thus, we conclude that the refractory phenomenon of memory cell to orally administered Ag is due to qualitative characteristics rather than quantitative. We have another piece of evidence compatible with this idea. When primed mice were treated by other methods for tolerance induction, such as i.v. or i.p. tolerization, these mice failed to induce tolerance to the Ag, moreover they produced about twice more large amount of Ag-specific Ab than control mice (our unpublished data). It will be interesting to dissect the underlying cellular and molecular mechanism for this phenomenon.

Based on our observations, the immune response could be divided into two steps: one is the feeding-susceptible step that includes the naive, activating, and effector phases, and the other is the feeding-resistant step that includes the memory phase. Thus at the feeding-susceptible step, the feeding regimen by multiple high dose could induce complete tolerance to the Ag, and at the feeding-resistant step the feeding regimen by multiple low dose could suppress partially only the production of Abs. We speculate that multiple low doses might induce suppressor T cells to OVA and suppress Ab production by means of active suppression as reported by several groups (5, 7). However, this mechanism may not be applicable when there were so many Ag-specific effector lymphocytes in blood circulation at the effector phase of the immune response.

It was also notable that feeding OVA to OVA-primed mice elevated the production of IFN- in splenocytes culture. There is good evidence that IFN- can mediate immune suppression (33). It has been reported that IFN- led lymphocytes to anergy state and induced tolerance (34, 35). In parallel to this report, IFN- null mice failed to induce tolerance at a high dose of Ag, thus indicating that IFN--producing T cells are essential for the suppression of the systemic immune response (36). Furthermore, this cytokine led to the reduction of IL-4 in the tolerized mice in our study. However, IFN- production does not seem to be the unique condition to lead anergy. It is likely that the phase of the immune response, the nature of administered Ag, and the property of Ag-presenting cell are also important (2, 37, 38).

Understanding the mechanisms that underlie these overall observations is important because manipulation of immune responses at disease states through oral Ag administration may provide a novel therapeutic strategy for the control of autoimmunity and allergy. Many studies have shown that oral administration of Ag was effective in suppressing immune response and inflammatory disease. Weiner et al. (17) have reported that a trial of oral tolerization with myelin Ags to individuals with early relapsing multiple sclerosis showed some therapeutic effect. In contrast, some studies have reported that oral administration of Ag was able to activate immune response to the Ag. For example, Blanas et al. (39) have reported oral administration of autoantigen in mice induced a cytotoxic T lymphocyte response that could lead to the onset of autoimmune diabetes. Furthermore, several studies reported that feeding after disease induction was not effective, and low doses of Ag led to worsening of mild disease. These mixed results of the oral tolerance in primed animals might be dependent on the time, the dose, and the frequency of Ag administration. To our knowledge the results in this study are the first to show that once the immune system established memory to the Ag, complete tolerance was not induced by Ag feeding. Our findings have an important implication not only for understanding the mechanisms of oral tolerance, but also for the therapeutic applications of oral tolerance. Considering our results, multiple or continuous feeding of the causing Ag would ameliorate the autoimmune diseases by tolerizing newly generated naive cells. However, our most surprising observation was that the resting memory lymphocytes could enhance the immune response to the Ag rather than induce tolerance by oral administration of Ag. Thus feeding Ag can cause stronger immune response at a certain condition, which suggests that feeding regimens should be cautious when applying this approach to the treatment of human autoimmune diseases.

	Acknowledgments

 
We thank Dr. R. Ward at the U.S. Environmental Protection Agency for review of the manuscript.

	Footnotes

 
¹ This work was supported by Grant HMP-96-D-1-1023 from the Ministry of Health and Welfare of Korea and the Korea Science and Engineering Foundation (KOSEF) through the Research Center for New Drug Development at Seoul National University.

² Address correspondence and reprint request to Dr. Chang-Yuil Kang, Laboratory of Immunology, College of Pharmacy, Seoul National University, Shillimdong, Kwanakgu, Seoul 151-742, Korea. E-mail address:

³ Abbreviation used in this paper: IFA, incomplete Freund’s adjuvant.

Received for publication April 13, 1999. Accepted for publication July 20, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Mowat, A. M.. 1987. The regulation of immune responses to dietary protein antigens. Immunol. Today 8:93.
2. Strobel, S., A. M. Mowat. 1998. Immune responses to dietary antigens: oral tolerance. Immunol. Today 19:173.[Medline]
3. Weiner, H. L.. 1997. Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol. Today 18:335.[Medline]
4. Weiner, H. L., A. Friedman, A. Miller, S. J. Khoury, A. Al-Sabbagh, L. Santos, M. Sayegh, R. B. Nussenblatt, D. E. Trentham, D. A. Hafler. 1994. Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu. Rev. Immunol. 12:809.[Medline]
5. Friedman, A., H. L. Weiner. 1994. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc. Natl. Acad. Sci. USA 91:6688.[Abstract/Free Full Text]
6. Chen, Y., J. Inobe, R. Marks, P. Gonnella, V. K. Kuchroo, H. L. Weiner. 1995. Peripheral deletion of antigen-reactive T cells in oral tolerance. Nature 376:177.[Medline]
7. Chen, Y., V. K. Kuchroo, J. Inobe, D. A. Hafler, H. L. Weiner. 1994. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265:1237.[Abstract/Free Full Text]
8. Higgins, P. J., H. L. Weiner. 1988. Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein and its fragments. J. Immunol. 140:440.[Abstract]
9. Javed, N. H., I. E. Gienapp, K. L. Cox, C.C. Whitacre. 1995. Exquisite peptide specificity of oral tolerance in experimental autoimmune encephalomyelitis. J. Immunol. 155:1599.[Abstract]
10. Meyer, A. L., J. M. Benson, I. E. Gienapp, K. L. Cox., C. C. Whitacre. 1996. Suppression of murine chronic relapsing experimental autoimmune encephalomyelitis by the oral administration of myelin basic protein. J. Immunol. 157:4230.[Abstract]
11. Gregerson, D. S., W. F. Obritsch, L. A. Donoso. 1993. Oral tolerance in experimental autoimmune uveoretinitis. J. Immunol. 151:5751.[Abstract]
12. Nussenblatt, R. B., R. R. Caspi, R. Mahid, C. C. Chan, F. Roberge, O. Lider, H. L. Weiner. 1990. Inhibition of S-antigen induced experimental autoimmune uveoretinitis by oral induction of tolerance with S-antigen. J. Immunol. 144:1689.[Abstract]
13. Nagler-Anderson, C., L. A. Bober, M. E. Robinson, G. W. Siskind, G. J. Thorbecke. 1986. Suppression of type II collagen-induced arthritis by gastric administration of soluble type II collagen. Proc. Natl. Acad. Sci. USA 83:7443.[Abstract/Free Full Text]
14. Zhang, Z. J., C. S. Y. Lee, O. Lider, H. L. Weiner. 1990. Suppression of adjuvant arthritis in Lewis rats by oral administration of type II collagen. J. Immunol. 145:2489.[Abstract]
15. Khare, S. D., C. J. Krco, M. M. Griffiths, H. S. Luthra, C. S. David. 1995. Oral administration of an immunodominant human collagen peptide modulates collagen-induced arthritis. J. Immunol. 155:3653.[Abstract]
16. Husby, S., J. Mestecky, Z. Moldoveanu, S. Holland, C. O. Elson. 1994. Oral tolerance in humans. J. Immunol. 152:4663.[Abstract]
17. Weiner, H. L., G. A. Mackin, M. Matsui, E. J. Khoury, D. M. Dawson, D. A. Hafler. 1993. Double-blind pilot trial of oral tolerization with myelin antigens in multiple sclerosis. Science 259:1321.[Abstract/Free Full Text]
18. Trentham, D. E., R. A. Dyneisius-Trentham, E. J. Orav, D. Combitchi, C. Lorenzo, K. L. Swell, D. A. Hafler, H. L. Weiner. 1993. Effects of oral administration of type II collagen on rheumatoid arthritis. Science 261:1727.[Abstract/Free Full Text]
19. Theofilopoulos, A. N.. 1995. The basis of autoimmunity. I. Mechanisms of aberrant self-recognition. Immunol. Today 16:90.[Medline]
20. Tough, D. F., J. Sprent. 1995. Lifespan of lymphocytes. Immunol. Res. 14:1.[Medline]
21. Croft, M.. 1994. Activation of naïve, memory and effector T cells. Curr. Opin. Immunol. 6:431.[Medline]
22. Sprent, J.. 1994. T and B memory cells. Cell 76:315.[Medline]
23. Guttormsen, H.-K., L. M. Wetzler, R. W. Finberg, D. L. Kasper. 1998. Immunologic memory induced by a glycoconjugate vaccine in a murine adoptive lymphocyte transfer model. Infect. Immun. 66:2026.[Abstract/Free Full Text]
24. Peng, H.-J., M. W. Turner, S. Strobel. 1989. The kinetics of oral hyposensitization to a protein antigen are determined by immune status and the timing, dose and frequency of antigen administration. Immunology 67:425.[Medline]
25. Leishman, A. J., P. Garside, A. M. Mowat. 1998. Immunological consequences of intervention in established responses by feeding protein antigens. Cell. Immunol. 183:137.[Medline]
26. Torseth, J. W., D. S. Gregerson. 1998. Oral tolerance in experimental autoimmune uveoretinitis: feeding after disease induction is less protective than prefeeding. Clin. Immunol. Immunopathol. 88:297.[Medline]
27. Kennedy, K. J., W. S. Smith, S. D. Miller, W. J. Karpus. 1997. Induction of antigen-specific tolerance for the treatment of ongoing, relapsing autoimmune encephalomyelitis. J. Immunol. 159:1036.[Abstract]
28. Yoshino, S.. 1995. Antigen-induced arthritis in rats is suppressed by the inducing antigen administered orally before, but not after immunization. Cell. Immunol. 163:55.[Medline]
29. Kang, B., K.-M. Kim, C.-Y. Kang. 1999. Oral tolerance by high dose OVA in BALB/c mice is more pronounced and persistent in the Th2 mediated immune responses than in the Th1 responses. Immunobiology 200:264.[Medline]
30. Miyama-Inaba, M., T. Suzuki, Y.-H. Paku, T. Masuda. 1982. Feedback regulation of immune responses by immune complexes; possible involvement of a suppressive lymphokine by FcR-bearing B cell. J. Immunol. 128:882.[Abstract]
31. Dai, S., G. Moller. 1988. Antigen-antibody complex-induced immunosuppression: effect of F(ab')₂ antibodies and protein A. Scan. J. Immunol. 27:413.[Medline]
32. Friedman, A.. 1997. Induction of anergy in Th1 Lymphocytes by oral tolerance. Ann. NY Acad. Sci. 778:103.[Medline]
33. Koblish, H. K., C. A. Hunter, M. Wysocka, G. Trinchieri, W. M. F. Lee. 1998. Immune suppression by recombinant interleukin (rIL)-12 involves interferon induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect. J. Exp. Med. 188:1603.[Abstract/Free Full Text]
34. Marth, T., W. Strober, B. L. Kelsall. 1996. High dose oral tolerance in ovalbumin TCR-transgenic mice. J. Immunol. 157:2348.[Abstract]
35. Kelsall, B. L., R. O. Ehrhardt, W. Strober. 1994. Induction of a primary IFN- response by Peyer’s patch dendritic cells is mediated by IL-12 and inhibited by IL-4. FASEB J. 8:A207.
36. Kweon, M.-N., K. Fujihashi, J. L. VanCott, K. Higuchi, M. Yamamoto, J. R. McGhee, and H. Kiyono. Lack of orally induced systemic unresponsiveness in IFN- knockout mice. J. Immunol. 160:1687.
37. Finkelman, F. D., A. Lees, R. Birnbaum, W. C. Gause, S. C. Morris. 1996. Dendritic cells can present antigen in vivo in a tolerogenic or immunogenic fashion. J. Immunol. 157:1406.[Abstract]
38. Viney, J. L., A. M. Mowat, J. M. O’Malley, E. Williamson, N. A. Fanger. 1998. Expanding dendritic cells in vivo enhances the induction of oral tolerance. J. Immunol. 160:5815.[Abstract/Free Full Text]
39. Blanas, E., F. R. Carbone, J. Allison, J. F. A. P. Miller, W. R. Heath. 1996. Induction of autoimmune diabetes by oral administration of autoantigen. Science 274:1707.[Abstract/Free Full Text]

This article has been cited by other articles:

				F. Blumenthal-Barby, K. Eulenburg, A. Schrage, M. Zeitz, A. Hamann, and K. Klugewitz In vivo modulation of antigen-experienced cells in response to high-dose oral antigen: deletion but no evidence for alterations in the cytokine phenotype Int. Immunol., July 1, 2008; 20(7): 893 - 900. [Abstract] [Full Text] [PDF]

				T. J. Kenna, R. Thomas, and R. J. Steptoe Steady-state dendritic cells expressing cognate antigen terminate memory CD8+ T-cell responses Blood, February 15, 2008; 111(4): 2091 - 2100. [Abstract] [Full Text] [PDF]

				N. Parameswaran, D. J. Samuvel, R. Kumar, S. Thatai, V. Bal, S. Rath, and A. George Oral Tolerance in T Cells Is Accompanied by Induction of Effector Function in Lymphoid Organs after Systemic Immunization Infect. Immun., July 1, 2004; 72(7): 3803 - 3811. [Abstract] [Full Text] [PDF]

				P. M. Cobelens, A. Kavelaars, A. Vroon, M. Ringeling, R. van der Zee, W. van Eden, and C. J. Heijnen The {beta}2-Adrenergic Agonist Salbutamol Potentiates Oral Induction of Tolerance, Suppressing Adjuvant Arthritis and Antigen-Specific Immunity J. Immunol., November 1, 2002; 169(9): 5028 - 5035. [Abstract] [Full Text] [PDF]

				H.-o. Lee, C. J. Cooper, J.-h. Choi, Z. Alnadjim, and T. A. Barrett The State of CD4+ T Cell Activation Is a Major Factor for Determining the Kinetics and Location of T Cell Responses to Oral Antigen J. Immunol., April 15, 2002; 168(8): 3833 - 3838. [Abstract] [Full Text] [PDF]

				J. Todt, J. Sonstein, T. Polak, G. D. Seitzman, B. Hu, and J. L. Curtis Repeated Intratracheal Challenge with Particulate Antigen Modulates Murine Lung Cytokines ,2 J. Immunol., April 15, 2000; 164(8): 4037 - 4047. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chung, Y. Articles by Kang, C.-Y. Search for Related Content PubMed PubMed Citation Articles by Chung, Y. Articles by Kang, C.-Y.