Recent studies have suggested that IL-12 and IFN-γ may impair the ability of fed Ag to induce systemic tolerance. Because both of these cytokines can function to directly or indirectly induce inducible NO synthase (iNOS) expression, we have investigated whether the functional expression of iNOS regulates oral tolerance. C57BL/6J wild-type or C57BL/6J NOS2−/− mice were gavaged with a single dose of 20 mg of keyhole limpet hemocyanin (KLH), followed by s.c. immunization with KLH/CFA. In the absence of feeding Ag, several parameters of the immune response were more robust in C57BL/6J NOS2−/− mice following KLH/CFA immunization, including the magnitude of the delayed-type hypersensitivity response, the proliferative response, and the production of IFN-γ and IL-2 by Ag-activated draining lymph node cells. These heightened responses in the C57BL/6J NOS2−/− mice are still effectively inhibited by feeding KLH. Feeding KLH to the C57BL/6J NOS2−/− mice elicited heightened TGF-β1 production by Ag-activated lymphocytes, as well as augmented total IgG, IgG1, and IgG2a responses to KLH/CFA compared with that seen in Ag-fed wild-type mice. Feeding Ag to the NOS2−/− mice suppressed proliferative responses and IFN-γ production, while increasing IL-4 production and the IgG1/IgG2a ratio even following a booster immunization of KLH/CFA. Administrating l-N6-(1-iminoethyl)-lysine · 2HCl to wild-type mice during the period of Ag feeding reproduced the high TGF-β1 production seen in Ag-activated lymphocytes from Ag-fed NOS2−/− mice. Feeding KLH is followed by transient up-regulation of NOS2 mRNA expression in the Peyer’s patches of wild-type mice. Selective inhibition of NOS2 may be a simple way to augment tolerogenic mucosal immune responses.

Initial exposure to Ags at mucosal surfaces can lead to systemic tolerance. Mucosal exposure to certain doses of Ag characteristically elicits T cell expression of cytokines such as IL-4, IL-10, and TGF-β1 (extensively reviewed in Ref. 1). These cytokines can be “suppressive” to cellular immune reactions and antagonize the expression of Th1-like cytokines (2, 3). At other doses of fed Ag, systemic tolerance is the result of clonal deletion and/or anergy (4, 5). The gut immune response to ingested Ag is centered in the Peyer’s patch, where activated T cells produce predominately IL-4, IL-10, and TGF-β1 (6). Following Ag feeding, such T cells migrate from Peyer’s patches through mesenteric lymph nodes to peripheral sites (1). Because feeding Ag can modulate autoimmune disease processes (7, 8, 9, 10), regulatory mechanisms that influence immune responses in the Peyer’s patch may determine the success of oral tolerance treatment strategies.

The role played by IFN-γ in the development of oral tolerance is controversial. Peyer’s patches from wild-type mice fed a large dose (25–250 mg) of OVA produce large amounts of IFN-γ (11, 12). Mice lacking IFN-γ fail to be systemically tolerized to OVA following feeding. These studies suggest that IFN-γ plays an important role in the regulation of the gut immune reaction responsible for the development of systemic tolerance. Other studies support the opposite conclusion. Targeted mutant mice lacking the IFN-γ receptor can be orally tolerized (13). Treatment of mice with IFN-γ i.p. before the feeding of Ag blocks the induction of systemic tolerance (14). Treatment of mice fed large doses of OVA with anti-IL-12 blocks the production of IFN-γ in the Peyer’s patch and augments induction of systemic tolerance as evidenced by increased production of TGF-β1 and IL-10 in the periphery (12). In the aggregate, these studies suggest a negative regulatory role for IFN-γ on the immune reactions in the gut that lead to systemic tolerance.

One possible mechanism by which IFN-γ may act as a negative regulator is through the induction of inducible NO synthase (iNOS).3 IFN-γ, among other proinflammatory cytokines, is a potent positive regulator of iNOS gene transcription (15). NO, in turn, can play an important role in the regulation of immune responses (16, 17, 18, 19). In these studies, we have investigated whether functional expression of iNOS modulates oral tolerance. Our results show that mice genetically lacking iNOS or mice treated with a selective iNOS inhibitor have augmented induction of systemic tolerance to fed Ags, reminiscent of the effects of anti-IL-12 treatment. Furthermore, mice fed a single large dose of Ag have significantly increased levels of iNOS mRNA expression in the Peyer’s patch. The ability to augment tolerance by inhibiting expression of iNOS may be beneficial to mucosal tolerance strategies for the treatment of autoimmune diseases.

Male C57BL/6 and C57BL/6NOS2−/− mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The mice were used between 4 and 6 wk of age. Mice were housed and handled in accordance with Department of Veterans Affairs and National Institutes of Health guidelines under Institutional Animal Care and Use Committee approved protocols.

Keyhole limpet hemocyanin (KLH) was purchased from Calbiochem (San Diego, CA). Freund’s adjuvant and Mycobacterium tuberculosis were obtained from Difco (Detriot, MI). CFA was prepared as 4 mg/ml M. tuberculosis in a 1:1 (v/v) emulsion of Freund’s adjuvant and PBS. l-N6-(1-iminoethyl)-lysine · 2HCl (l-NIL) was obtained from Alexis Biochemicals (San Diego, CA). Mice treated with l-NIL were given 50 μg/ml in the drinking water for a total of 6 days or ∼167 μg/day/mouse.

For feeding and immunization, mice were anesthetized with methoxyflurane obtained from Mallinckrodt (Mundelein, IL). The mice were allowed to recover on room air. Mice were gavaged with either 20 mg KLH in 250 μl PBS or 250 μl PBS with a 20-gauge feeding needle. In some experiments, mice were given 50 μg/ml l-NIL in the drinking water, starting the day before gavage and continuing until day 5 after gavage. Mice were immunized s.c. at the base of the tail with 100 μg KLH in 200 μl CFA. Some mice were given a second s.c. flank injection of 100 μg KLH in 200 μl CFA in a site distinct from the initial injection. To assess DTH, all mice involved in the studies were given 50 μg KLH in 50 μl PBS intradermally in the left foot pad and 50 μl PBS in the right foot pad 5 days after the last immunization. Foot pad swelling was measured with a micrometer (Mitutoyo, Japan) by an observer blinded to the experimental design and was recorded as the difference between the left and right foot pad.

Draining flank lymph nodes were harvested and prepared into a single cell suspension with a metal screen. Cells were plated in 96-well tissue culture plates (Falcon/Becton Dickinson, Franklin Lakes, NJ) at 1 × 106 cells/ml in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% (v/v) heat-inactivated FCS (Gemini Bio-Products, Calabasas, CA), 5.5 × 10−5 M 2-ME (Life Technologies) 130 U/ml penicillin, 130 μg/ml streptomycin, and 2.5 mM l-glutamine (Omega Scientific, Tarzana, CA). Cells were cultured at 37°C with 5% CO2 in a humidified incubator. Cells assayed for proliferation were pulsed with 1 μCi/well [3H]thymidine (Amersham, Arlington Heights, IL) on day 4 and harvested 24 h later.

Culture supernatant concentrations of IL-2, IL-4, and IFN-γ were determined by sandwich ELISA with Abs purchased from PharMingen (San Diego, CA). Levels of IL-2 were determined from culture supernatant 24 h following Ag stimulation. IL-4 and IFN-γ concentrations were determined from culture supernatants on day 5. Ab dilutions, which maximized signal to noise, were determined for each Ab pair. Briefly, 96-well Maxisorp microtiter plates (Nunc-Nalgene, Naperville, IL) were coated with the appropriate capture Ab (0.1 M carbonate buffer, pH 9.5) overnight at 4°C. Plates were blocked for 1 h at room temperature (RT) with PBS containing 10% FCS and 0.05% Tween 20 (Sigma, St. Louis, MO). Plates were incubated with supernatant samples and conjugated detection Ab (IL-2, HRP; IL-4 and IFN-γ, biotin) for 2 h at RT. When appropriate, samples were incubated with avidin-HRP at RT for 2 h. Following extensive washes, plates were developed with TMB (3,3′, 5,5′ tetramethylbenzidine) substrate reagent set purchased from PharMingen and quenched with 2 N H2SO4. Color development was evaluated in a spectophotometric microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm. Levels of TGF-β1 were determined in nonacid-activated day-5 culture supernatants with a kit from Promega (Madison, WI) as per the manufacturer’s instruction.

Serum was collected from mice by terminal cardiac puncture. Several samples of serum were analyzed at various dilutions by ELISA to determine a dilution from which concentrations of KLH-specific Igs were linear with respect to OD. Briefly, 96-well Maxisorp microtiter plates were coated with KLH (2 μg/ml in PBS) overnight at 4°C. Plates were blocked for 1 h at RT with PBS containing 4% BSA (Sigma) and 0.05% Tween 20. Plates were incubated with serum samples for 2.5 h at 37°C. Plates were developed with anti-IgG (Calbiochem, San Diego, CA), anti-IgG1 (Caltag, Burlingame, CA), or anti-IgG2a (PharMingen) alkaline phosphatase conjugates at 37°C for 2.5 h. Color was developed by incubating plates with p-nitrophenylphosphate disodium purchased from Sigma (1 mg/ml in 1 M carbonate buffer, pH 9.6) at RT for equal amounts of time. Color development was evaluated in a microplate reader at 605 nm.

Total RNA was prepared from Peyer’s Patches and non-Peyer’s Patch with the RNeasy Mini kit (Qiagen, Valencia, CA) and stored at −70°C with 40 U RNase-OUT inhibitor purchased from Life Technologies. cDNA was prepared from 2 μg of each sample using a kit from Life Technologies (Superscript II preamp) according to the manufacturer’s instructions. The cDNA was used in a PCR with serial dilutions of a known molar amount of a competitive template for iNOS (Clontech, Palo Alto, CA) and β-actin. The resulting levels of iNOS were normalized to the levels of β-actin. The primers for the murine iNOS were 5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′ and 3′-GGTTTCGGTGCTCCGAGACTGTCGG-5′. The primers for the murine β-actin were 5′-AATGTGGCTGAGGACTTTG-3′ and 3′-ACTGTCGTAACGAAGACACA-5′. The competitive template for β-actin was a generous gift from Daniel P. Gold (Sidney Kimmel Cancer Institute, San Diego, CA). The PCR buffer for both the iNOS and β-actin amplifications contained a final concentration of 2 mM MgCl2. The PCR conditions used for the iNOS and β-actin amplification were 38 cycles of 45 s at 94°C, 45 s at 55°C, and 2 min at 72°C followed by a 7-min 72°C step. PCR products were resolved on a 1.4% agarose Tris-boric acid-EDTA gel. Band intensity was evaluated on a digital imaging system (IL-1000 v2.02; Alpha Innotech, San Leandro, CA).

Differences were statistically analyzed using a one-way ANOVA with a Bonferroni/Dunn post-test. Analysis was accomplished with Statview v4.5 (Abacus Concepts, Berkeley, CA).

Because the role of NO in mucosal tolerance had not been examined, we studied the effect of NO generated through iNOS on high dose (20 mg of KLH) oral tolerance. As shown in Fig. 1 A, this protocol of feeding KLH had no effect on the magnitude of the Ag-specific DTH in wild-type C57BL/6J mice. The iNOS−/− mice had a more robust DTH to KLH than wild-type mice, consistent with previous observations regarding their heightened T cell responses both in vivo and in vitro (3, 18, 19). Despite the magnitude of the DTH response in the iNOS−/− mice, feeding KLH to these mice reduced the DTH response by almost 50%.

FIGURE 1.

A, The effect of oral tolerance on DTH. C57BL/6J NOS2+/+ (n = 5) and C57BL/6J NOS2−/− (n = 5) mice were fed 20 mg KLH (filled columns) or PBS vehicle control (open columns). On day 7, mice were immunized s.c. in the base of the tail with 100 μg KLH in CFA. On day 12, mice were challenged with 50 μg KLH in 50 μl PBS in the left foot pad and with 50 μl PBS in the right foot pad. On day 14, foot pad swelling was assessed by an observer blinded to the experimental design. ANOVA with Bonferroni/Dunn post-test; ∗, p < 0.0005. B, The effect of Ag feeding on draining lymph node proliferative response to Ag. Draining lymph nodes were taken from the mice described in A on day 14. Cellular proliferation assay was conducted at 1 × 106 cells/ml with the indicated concentrations of KLH. Cultures were pulsed with 1 μCi/well [3H]TdR on day 4 and incorporation assayed on day 5. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0013; ∗∗, p = 0.0919. C, Feeding induced alterations of the humoral immune response. Sera from the mice described in A were collected on day 14. KLH-specific Ig response was determined by ELISA. The OD values shown here are from a serum dilution determined to be on the linear part of the OD vs dilution curve. The same dilution was used for all samples. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0147; ∗∗, p = 0.0004; ∗∗∗, p = 0.0003; †, p = 0.0048.

FIGURE 1.

A, The effect of oral tolerance on DTH. C57BL/6J NOS2+/+ (n = 5) and C57BL/6J NOS2−/− (n = 5) mice were fed 20 mg KLH (filled columns) or PBS vehicle control (open columns). On day 7, mice were immunized s.c. in the base of the tail with 100 μg KLH in CFA. On day 12, mice were challenged with 50 μg KLH in 50 μl PBS in the left foot pad and with 50 μl PBS in the right foot pad. On day 14, foot pad swelling was assessed by an observer blinded to the experimental design. ANOVA with Bonferroni/Dunn post-test; ∗, p < 0.0005. B, The effect of Ag feeding on draining lymph node proliferative response to Ag. Draining lymph nodes were taken from the mice described in A on day 14. Cellular proliferation assay was conducted at 1 × 106 cells/ml with the indicated concentrations of KLH. Cultures were pulsed with 1 μCi/well [3H]TdR on day 4 and incorporation assayed on day 5. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0013; ∗∗, p = 0.0919. C, Feeding induced alterations of the humoral immune response. Sera from the mice described in A were collected on day 14. KLH-specific Ig response was determined by ELISA. The OD values shown here are from a serum dilution determined to be on the linear part of the OD vs dilution curve. The same dilution was used for all samples. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0147; ∗∗, p = 0.0004; ∗∗∗, p = 0.0003; †, p = 0.0048.

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Feeding KLH resulted in suppression of the proliferative response to KLH in draining lymph node cells from both the wild-type and iNOS−/− mice (Fig. 1 B). The in vitro proliferative response following immunization with KLH/CFA is more robust in the iNOS−/− mice. We observed the expected tolerogenic effect of fed KLH in the wild-type mice. Despite the robust proliferative response following immunization with KLH/CFA in the iNOS−/− mice, feeding KLH to these mice reduced the proliferative response to near baseline values. Because KLH can be mitogenic to naive splenocytes at concentrations exceeding 10 μg/ml (data not shown), we restricted our in vitro assays to lower concentrations of KLH. We additionally assayed the proliferative responses from spleens from the same animals in these experimental groups. The proliferative response to Ag was markedly higher in spleens from the iNOS−/− mice, compared with those from wild-type mice. However, in the spleen, we did not observe any feeding-dependent alterations in the proliferative response to KLH (data not shown).

Fig. 1 C depicts the effect of Ag feeding on the Ab response to KLH in the wild-type and knockout mice. Feeding KLH augmented the total IgG, IgG1, and IgG2a responses in both wild-type and iNOS−/− mice, but the effect was more marked for each determination in the iNOS−/− mice. Only in the absence of iNOS did the augmentation of the Ab response reach statistical significance in all three groups. In particular, the increase in IgG1 was greatest in iNOS−/− mice fed KLH.

Cytokine determinations were performed on supernatants derived from draining lymph node cultures set up in parallel with the above-described proliferation assays (Fig. 2). We assayed the supernatants for IL-2 (Fig. 2,A), IL-4 (Fig. 2,B), IFN-γ (Fig. 2,C), and TGF-β1 (Fig. 2,D). The supernatants were not acid activated for the TGF-β1 ELISA. Thus, the observed concentrations reflect the levels of bioactive TGF- β1 produced in the cultures. Lymphocytes from immunized iNOS−/− mice produced far more IL-2 and IFN-γ, and less TGF-β1 in response to Ag than did wild-type mice. Again, despite the augmented TH1-like response in iNOS−/− mice, feeding KLH suppresses the production of IL-2 and IFN-γ to below the limits of detection. The role of iNOS or fed Ag on the production of IL-4 is less clear because none of the groups tested reached statistical significance (Fig. 2,B). Feeding KLH to iNOS−/− (but not wild-type) mice augments the production of TGF-β1; Fig. 2 D) in the draining lymph nodes, consistent with the paradigm that oral exposure to Ag induces TGF-β1-secreting T cells (TH3) (20, 21).

FIGURE 2.

Ag feeding induced alterations of the pattern of cytokine production. Cytokine concentrations were determined by ELISA on the supernatants from the draining lymph node cultures in Fig. 1. ELISAs in B, C, and D, are from supernatants sampled on day 5 of culture, whereas the ELISAs in A are from supernatants sampled at 24 h of culture. The dashed line and bold-faced concentrations indicate the limit of detection in those assays. The supernatants evaluated for TGF-β1 in D were not acid activated and thus represent the concentrations of bioactive TGF-β1. ND, Not detectable. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0024; ∗∗, p = 0.0026; ∗∗∗, p = 0.0165

FIGURE 2.

Ag feeding induced alterations of the pattern of cytokine production. Cytokine concentrations were determined by ELISA on the supernatants from the draining lymph node cultures in Fig. 1. ELISAs in B, C, and D, are from supernatants sampled on day 5 of culture, whereas the ELISAs in A are from supernatants sampled at 24 h of culture. The dashed line and bold-faced concentrations indicate the limit of detection in those assays. The supernatants evaluated for TGF-β1 in D were not acid activated and thus represent the concentrations of bioactive TGF-β1. ND, Not detectable. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0024; ∗∗, p = 0.0026; ∗∗∗, p = 0.0165

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We additionally analyzed cytokine expression in supernatants derived from activated splenocytes set up in parallel with the proliferation assays. The levels of IL-2 produced by activated splenocytes from the iNOS−/− are significantly higher than those of the wild type. This is consistent with the higher proliferative responses in these spleens. However, we did not detect any significant effects of feeding on the cytokine levels produced by Ag-activated splenocytes (data not shown).

Previously used protocols for assessing the role of IFN-γ in oral tolerance have included a single high dose feeding followed by two s.c. immunizations (11). Because such boosted immune responses may be fundamentally different from nonboosted responses, we treated wild-type and iNOS−/− mice as before (Fig. 1) but then boosted them 2 wk following the initial injection of KLH/CFA. Draining lymph node cells from boosted iNOS−/− mice have a dramatically heightened proliferative response relative to cells from boosted wild-type mice (Fig. 3 A). Despite this robust immune response, the effect of feeding persists and reduces the proliferative response in iNOS−/− mice to near baseline. In the wild type, the effect of feeding is unclear due to the lack of proliferative response to immunization alone. The failure in wild-type mice to make a proliferative immune response with boosting may reflect potent NO dependent negative regulation, because we have found that CFA immunization is a potent stimulus of iNOS expression in the lymph node (D.K. and C.K., unpublished observations).

FIGURE 3.

A, Persistence of oral tolerance induced suppression of draining lymph node proliferation. C57BL/6J NOS2+/+ (n = 4) and C57BL/6J NOS2−/− (n = 4) mice were fed 20 mg KLH or PBS vehicle control. On day 7, mice were immunized s.c. in the base of the tail with 100 μg KLH in CFA. On day 21, mice were given a boosting immunization of 100 μg KLH in CFA s.c. in the flank at a site distinct from the previous immunization. The mice were euthanized on day 28, and the draining lymph nodes were harvested and cultured for 5 days at 1 × 106 cells/ml with the indicated concentrations of KLH. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0106. B and C, Cytokine concentrations were determined by ELISA on the day-5 supernatants from the cultures in A. The dashed line and bold-faced concentrations indicates the limit of detection in those assays. ND, Not detectable. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0185; ∗∗, p = 0.0015. D, Sera from the mice described in A were collected on day 28. KLH-specific Ig response was determined by ELISA. The OD values shown here are from a serum dilution (1:3000) determined to be on the linear part of the OD vs dilution curve. ANOVA with Bonferroni/Dunn posttest; ∗, p = 0.0055.

FIGURE 3.

A, Persistence of oral tolerance induced suppression of draining lymph node proliferation. C57BL/6J NOS2+/+ (n = 4) and C57BL/6J NOS2−/− (n = 4) mice were fed 20 mg KLH or PBS vehicle control. On day 7, mice were immunized s.c. in the base of the tail with 100 μg KLH in CFA. On day 21, mice were given a boosting immunization of 100 μg KLH in CFA s.c. in the flank at a site distinct from the previous immunization. The mice were euthanized on day 28, and the draining lymph nodes were harvested and cultured for 5 days at 1 × 106 cells/ml with the indicated concentrations of KLH. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0106. B and C, Cytokine concentrations were determined by ELISA on the day-5 supernatants from the cultures in A. The dashed line and bold-faced concentrations indicates the limit of detection in those assays. ND, Not detectable. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0185; ∗∗, p = 0.0015. D, Sera from the mice described in A were collected on day 28. KLH-specific Ig response was determined by ELISA. The OD values shown here are from a serum dilution (1:3000) determined to be on the linear part of the OD vs dilution curve. ANOVA with Bonferroni/Dunn posttest; ∗, p = 0.0055.

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In both wild-type and iNOS−/− mice, the effect of feeding on the pattern of cytokines produced is consistent with deviation from a TH1- to a TH2-like profile (Fig. 3, B and C). The amount of IL-4 produced in the PBS-gavaged mice is similar between the two strains (Fig. 3,B). Draining lymph nodes from wild-type mice fed KLH make 3- to 4-fold higher levels of IL-4 compared with unfed wild-type mice. In the absence of iNOS, the feeding-induced augmentation of IL-4 is exaggerated. Feeding results in a 7- to 10-fold increase in the amount of IL-4 produced in draining lymph nodes. iNOS−/− mice produce ∼40 fold more IFN-γ than wild-type mice (Fig. 3 C). Despite the overwhelmingly TH1-like response in iNOS−/− mice, KLH feeding significantly reduces the amount of IFN-γ 4- to 5-fold.

Feeding-induced immune deviation in iNOS−/− mice is also evident in the composition of the Ag (KLH)-specific Ab response (Fig. 3 D). Both wild-type and iNOS−/− mice produced an Ab response of similar magnitude in terms of total IgG. In iNOS−/− mice fed KLH, the ratio of IgG1/IgG2a changed from 3.89 in unfed mice to 7.29 in KLH-fed mice. The shift in the ratio was due to statistically significant increases in IgG1 and decreases in IgG2a with KLH feeding. This trend was not observed in wild-type mice.

We next examined whether the effects of Ag feeding seen in iNOS−/− mice could be reproduced in animals made transiently iNOS deficient by in vivo administration of a selective iNOS inhibitor, l-NIL (22) (Fig. 4). In contrast to iNOS−/− mice, feeding KLH to mice treated with l-NIL did not significantly suppress Ag-specific proliferative responses in draining lymph node cells. However, consistent with our findings in iNOS−/− mice, feeding Ag to the l-NIL-treated mice substantially decreased the amount of IFN-γ produced by Ag-reactive cells (Fig. 5,C). Lymphocytes from KLH-fed mice treated with l-NIL additionally secreted three times more bioactive TGF-β1 as did unfed l-NIL-treated mice (Fig. 5 D).

FIGURE 4.

Altered induction of oral tolerance in mice treated with an iNOS inhibitor. C57BL/6J NOS2+/+ (n = 6) were fed 20 mg KLH or PBS vehicle control. Mice were treated with l-NIL (50 μg/ml) in the drinking water beginning on the day before gavage and discontinued on day 5 postgavage. Mice were immunized with 100 μg KLH/CFA s.c. in the base of the tail on day 7 and euthanized on day 14. Draining lymph nodes were harvested and cultured at 1 × 106 cells/ml with the indicated concentrations of KLH. Cultures were pulsed with 1 μCi/well [3H]TdR on day 4 and incorporation assayed on day 5. ANOVA with Bonferroni/Dunn post-test did not yield any statistically significant differences (p ≤ 0.05).

FIGURE 4.

Altered induction of oral tolerance in mice treated with an iNOS inhibitor. C57BL/6J NOS2+/+ (n = 6) were fed 20 mg KLH or PBS vehicle control. Mice were treated with l-NIL (50 μg/ml) in the drinking water beginning on the day before gavage and discontinued on day 5 postgavage. Mice were immunized with 100 μg KLH/CFA s.c. in the base of the tail on day 7 and euthanized on day 14. Draining lymph nodes were harvested and cultured at 1 × 106 cells/ml with the indicated concentrations of KLH. Cultures were pulsed with 1 μCi/well [3H]TdR on day 4 and incorporation assayed on day 5. ANOVA with Bonferroni/Dunn post-test did not yield any statistically significant differences (p ≤ 0.05).

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FIGURE 5.

Pharmacological inhibition of iNOS alters the humoral and cytokine response to feeding. A, Sera from the mice in Fig. 4 were collected on day 14 and evaluated for the composition of KLH-specific Ig by ELISA. The OD values shown here are from a serum dilution determined to be on the linear part of the OD vs dilution curve. B–D, Cytokine concentrations were determined by ELISA on the day 5 supernatants from the cultures in Fig. 4. The supernatants evaluated for TGF-β1 in D were not acid activated and thus represent the concentrations of bioactive TGF-β1. The dashed line and bold-faced concentrations indicates the limit of detection in those assays. ND, Not detectable. ANOVA with Bonferroni/Dunn post-test; †, p = < 0.0001; ∗, p = <0.0001.

FIGURE 5.

Pharmacological inhibition of iNOS alters the humoral and cytokine response to feeding. A, Sera from the mice in Fig. 4 were collected on day 14 and evaluated for the composition of KLH-specific Ig by ELISA. The OD values shown here are from a serum dilution determined to be on the linear part of the OD vs dilution curve. B–D, Cytokine concentrations were determined by ELISA on the day 5 supernatants from the cultures in Fig. 4. The supernatants evaluated for TGF-β1 in D were not acid activated and thus represent the concentrations of bioactive TGF-β1. The dashed line and bold-faced concentrations indicates the limit of detection in those assays. ND, Not detectable. ANOVA with Bonferroni/Dunn post-test; †, p = < 0.0001; ∗, p = <0.0001.

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Feeding significantly altered the IgG subtype composition. The ratio of IgG1:IgG2a was reduced nearly 3-fold by feeding. The observed reduction was primarily due to a feeding induced increase in IgG2a. The levels of IgG1 were unaffected by feeding (Fig. 5 A).

When wild-type mice that were deprived of food for 18 h were gavaged with 20 mg KLH, iNOS mRNA was up-regulated by 6 h in the Peyer’s patch (Fig. 6). However, by 12 h postgavage, iNOS mRNA had subsided to baseline levels. Single cell suspensions prepared from the Peyer’s patches 6 h after feeding were cultured for an additional 24–96 h, and the supernatants were analyzed for nitrite and nitrate by Greiss reaction. We could not detect any elevation in NO endproducts in the Peyer’s patches derived from fed animals (data not shown). This likely reflects the relative insensitivity of the assay to lower concentrations of NO endproducts. Because intestinal epithelia can produce iNOS in response to a variety of signals (23, 24), surrounding non-Peyer’s patch tissue was evaluated for iNOS expression to control for possible contamination of the sample. iNOS mRNA was below the limits of detection in non-Peyer’s patch intestine in all experimental groups (data not shown).

FIGURE 6.

Ag feeding induces the transient expression of iNOS mRNA in the Peyer’s patch. C57BL/6J NOS2+/+ mice had food taken away for 18 h before being fed 20 mg KLH or PBS vehicle control. At 6 and 12 h postgavage, groups of mice (n = 3) were euthanized and total RNA was isolated from Peyer’s patches. Total RNA was evaluated for iNOS and β-actin mRNA levels by competitive RT-PCR. ND, Not detectable. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0097; ∗∗,p = 0.0066.

FIGURE 6.

Ag feeding induces the transient expression of iNOS mRNA in the Peyer’s patch. C57BL/6J NOS2+/+ mice had food taken away for 18 h before being fed 20 mg KLH or PBS vehicle control. At 6 and 12 h postgavage, groups of mice (n = 3) were euthanized and total RNA was isolated from Peyer’s patches. Total RNA was evaluated for iNOS and β-actin mRNA levels by competitive RT-PCR. ND, Not detectable. ANOVA with Bonferroni/Dunn post-test; ∗, p = 0.0097; ∗∗,p = 0.0066.

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Previous studies have supported the notion that NO can be an important immunoregulator. However, such studies have largely supported the paradigm that, in the absence of functional iNOS, TH1-like responses are accentuated. In these experiments, we have found that functional iNOS is additionally an important modulator of a regulatory response, i.e., the induction of tolerance by oral Ag administration. iNOS−/− mice demonstrated augmented immune responses to KLH/CFA, as demonstrated by the enhanced DTH responses, enhanced Ag-specific proliferative responses, and enhanced production of IFN-γ and IL-2. However, the iNOS−/− mice similarly demonstrate augmented regulatory responses following Ag feeding. This is demonstrated by the partial inhibition of DTH in the iNOS−/− by Ag feeding, and the inhibition of proliferative responses and TH1 cytokine production, and augmented TGF-β production. Inhibition of TH1 cytokine production, and augmented TGF-β, is also seen when the animals are fed Ag while transiently treated with a selective iNOS inhibitor. Finally, we have shown that iNOS mRNA is transiently up-regulated within the Peyer’s patches following high dose Ag feeding.

In addition, significant alterations in Ig titers and composition with feeding were observed under conditions (genetic or pharmacological) of iNOS deficiency. Feeding increases the serum titers of KLH-specific Igs in both wild-type mice and iNOS knockout mice. Similar to the alternations in cytokine production, the increase in Ab concentrations is exaggerated in the knockout compared with the wild type. In the single feed and immunization protocol, the trend in both strains is for the Ab concentrations to increase. However, only in the knockout does the increase reach statistical significance. One plausible interpretation is that these changes reflect systemic immune deviation as a consequence of feeding. The more substantial increase in KLH-specific IgG1 concentrations observed in the knockout compared with the wild type is perhaps a reflection of the dramatic suppressive effect that feeding has on the production of IFN-γ in the knockout. In contrast, the levels of serum KLH-specific Abs are unaltered in the wild type in response to feeding when a boosting immunization is administered. Boosted iNOS−/− mice show a significant increase in IgG1 without the associated change in IgG2a observed in a single immunization protocol. Whether the increase is Ab titers is critical for tolerance or an epiphenomena of the altered cytokine milieu awaits further study.

A plausible conclusion from these studies is that the tolerogenic immune response generated by feeding is more robust in the absence of iNOS, suggesting that functional expression of iNOS in the gut limits the intensity of the response of the mucosal immune system to Ag feeding. This conclusion is supported by the results of the draining lymph node proliferation assays. First, it is clear that iNOS-derived NO effectively regulates the proliferative response to immunization in wild-type mice because in iNOS−/− mice the response is more vigorous (Fig. 1 B). Furthermore, feeding suppressed the response in the wild type as expected, but the subsequent level of proliferative suppression observed in KLH-fed iNOS−/− mice was surprising to us considering the intensity of the immune response to KLH in PBS-fed iNOS−/− mice.

It has been previously suggested that the absence of iNOS leads to heightened TH1-like responses, and that NO selectively inhibits TH1-like responses (2, 3). One of the hallmarks of oral tolerance is alteration of the pattern of cytokines produced by Ag-reactive T cells to a TH2- or TH3-like pattern. When the wild-type mice are fed Ag, the reduced effectiveness of oral Ag to modulate DTH and proliferative responses is matched by insignificant changes in the pattern of cytokines produced (Fig. 2). The effectiveness of fed Ag in iNOS−/− mice is most profoundly reflected in the suppression of IL-2 and IFN-γ and the significant augmentation of TGF-β1 production. Although IL-4 concentrations were not affected by feeding to statistically significant differences, a trend to increased IL-4 levels with feeding is present in iNOS−/− mice. The immune deviation is also reflected in the Ab response (Fig. 1 C). Although immune reactions in iNOS−/− mice are typically TH1-like, these mice significantly augment their IgG1 responses when fed Ag. Our results suggest that NO generated through the iNOS isoform may regulate a wider variety of immune responses than previously hypothesized.

In this study, a powerful example of NO-mediated immune regulation (independent of feeding Ag) is the draining lymph node proliferative response seen in wild-type mice given a boosting immunization of KLH/CFA (Fig. 3). The lack of proliferation of the lymph node cells is clearly related to iNOS-derived NO because immune lymphocytes from lymph nodes of similarly boosted iNOS−/− mice proliferate briskly. Again, feeding KLH to these iNOS−/− mice leads to significant attenuation of the immune response. Because the observed suppression is 2 wk later than in the single immunization protocol and is following a boosting immunization, the effectiveness of fed KLH to regulate the immune response is remarkable for both the duration and degree of the tolerance. In the boosted mice, the mechanism of tolerance is consistent with immune deviation from TH1 to TH2 (Fig. 3) and previous studies supporting a requirement for IL-4 and IL-10 in oral tolerance (25). In contrast with the single immunization protocol, the levels of TGF-β1 were below the limits of detection suggesting that perhaps short term tolerance is due to induction of TH3-like T cells, but that longer term tolerance is maintained by immune deviation.

Wild-type mice treated with the iNOS-specific inhibitor l-NIL only during the feeding period mount qualitatively similar responses to iNOS−/− mice. The failure of fed KLH to suppress the proliferative response of the draining lymph node cells to statistically significant levels may be due to incomplete iNOS suppression by l-NIL. Analogous to iNOS−/− mice, l-NIL-treated wild-type mice fed KLH suppress IFN-γ production, whereas augmenting TGF-β1 expression.

We were intrigued to see the transient nature of iNOS mRNA up-regulation following high dose Ag ingestion. Peyer’s patch expression of IFN-γ has been observed early (≤6 h) in response to high and low doses of oral Ags (11, 12). Others have found no increase in IFN-γ expression in the Peyer’s patch after low doses of Ag (6). However, the consequences of IFN-γ treatment and IFN-γ receptor deficiency on oral tolerance induction are clear (13, 14). Furthermore, the ability to augment oral tolerance with anti-IL-12 treatment in vivo suggests a functional role for IFN-γ as a negative regulator. Although it is possible that expression of IFN-γ in the Peyer’s patch impairs oral tolerance induction solely by antagonizing the expression of IL-4, IL-10, and TGF-β1 (6, 11, 12), IFN-γ may additionally impair oral tolerance via iNOS-dependent mechanisms. Whether the transient induction of iNOS mRNA expression in the Peyer’s patch following Ag feeding is an “innate” response or one that requires Ag-specific T cells is not yet known. In our preliminary studies, where the animals were not fasted before Ag feeding, we observed high levels of iNOS mRNA in Peyer’s patches and gut epithelium in both Ag-fed and PBS-fed mice (D.A.K. and C.K., unpublished observations). The levels of iNOS mRNA in both tissues under nonfasting conditions were comparable to those seen in Fig. 6 with fed Ag at 6 h. These results, coupled to the kinetics depicted in Fig. 6, suggest to us that the up-regulation of iNOS is likely an innate response and one that occurs in response to multiple dietary Ags.

NO donors, such as nitroglycerin derivatives and nitroprusside, are already in clinical use for the treatment of angina and hypertension. Therefore, it is a valid question to ask whether the administration of NO donors to iNOS−/− mice might restore their responses to resemble wild-type mice. In our experience, it is difficult, with the currently available agents, to administer a large enough dose of a NO donor to a small rodent, systemically and chronically, to achieve the high local concentrations of NO present at sites of inflammation. Whether more sophisticated delivery systems that target an organ directly might accomplish that goal remains to be seen.

Although there have not been any direct studies addressing the physiology of iNOS expression in human Peyer’s patches, it is clear that iNOS is expressed in human gut epithelium (24). Moreover, human Peyer’s patches express a pattern of cytokines similar to murine models including a preponderance of IFN-γ expression (26). The exaggerated nature of the tolerogenic immune response induced by oral Ag in the absence of iNOS suggests this could be useful as a model system for the dissection of the requirements and subtle characteristics of oral tolerance. Furthermore, the potential to augment tolerogenic immune responses to fed Ags by treatment with iNOS inhibitors may be important to hone oral tolerance treatment strategies of autoimmune disease.

We thank Daniel Gold (Sidney Kimmel Cancer Center, San Diego, CA) for his helpful suggestions and review of the manuscript.

1

This work was supported in part by research grants from the National Institutes of Health (DK42155) and the Office of Research and Development, Medical Research Service, Department of Veterans Affairs. C.J.K. is a recipient of a Clinical Investigator Award from the Medical Research Service, Department of Veterans Affairs. D.A.K. is supported by the Medical Scientist Training Program at University of California, San Diego (National Institutes of Health/National Institute of General Medical Sciences 5 T32 GM07198).

3

Abbreviations used in this paper: iNOS, inducible NO synthase; KLH, keyhole limpet hemocyanin; l-NIL, l-N6-(1-iminoethyl)-lysine · 2HCl; DTH, delayed-type hypersensitivity; RT, room temperature.

1
Faria, A. M., H. L. Weiner.
1999
. Oral tolerance: mechanisms and therapeutic applications.
Adv. Immunol.
73
:
153
2
Huang, F. P., W. Niedbala, X. Q. Wei, D. Xu, G. J. Feng, J. H. Robinson, C. Lam, F. Y. Liew.
1998
. Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages.
Eur. J. Immunol.
28
:
4062
3
Wei, X. Q., I. G. Charles, A. Smith, J. Ure, G. J. Feng, F. P. Huang, D. Xu, W. Muller, S. Moncada, F. Y. Liew.
1995
. Altered immune responses in mice lacking inducible nitric oxide synthase.
Nature
375
:
408
4
Chen, Y., J. Inobe, R. Marks, P. Gonnella, V. K. Kuchroo, and H. L. Weiner. 1995. Peripheral deletion of antigen-reactive T cells in oral tolerance. [Published erratum appears in 1995Nature377:257.] Nature376:177.
5
Melamed, D., A. Friedman.
1993
. Direct evidence for anergy in T lymphocytes tolerized by oral administration of ovalbumin.
Eur. J. Immunol.
23
:
935
6
Gonnella, P. A., Y. Chen, J. Inobe, Y. Komagata, M. Quartulli, H. L. Weiner.
1998
. In situ immune response in gut-associated lymphoid tissue (GALT) following oral antigen in TCR-transgenic mice.
J. Immunol.
160
:
4708
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
8
Kelly, K. A., C. C. Whitacre.
1996
. Oral tolerance in EAE: reversal of tolerance by T helper cell cytokines.
J. Neuroimmunol.
66
:
77
9
Miller, A., O. Lider, O. Abramsky, H. L. Weiner.
1994
. Orally administered myelin basic protein in neonates primes for immune responses and enhances experimental autoimmune encephalomyelitis in adult animals.
Eur. J. Immunol.
24
:
1026
10
Whitacre, C. C., I. E. Gienapp, A. Meyer, K. L. Cox, N. Javed.
1996
. Treatment of autoimmune disease by oral tolerance to autoantigens.
Clin. Immunol. Immunopathol.
80
:
S31
11
Kweon, M. N., K. Fujihashi, J. L. VanCott, K. Higuchi, M. Yamamoto, J. R. McGhee, H. Kiyono.
1998
. Lack of orally induced systemic unresponsiveness in IFN-γ knockout mice.
J. Immunol.
160
:
1687
12
Marth, T., W. Strober, B. L. Kelsall.
1996
. High dose oral tolerance in ovalbumin TCR-transgenic mice: systemic neutralization of IL-12 augments TGF-β secretion and T cell apoptosis.
J. Immunol.
157
:
2348
13
Kjerrulf, M., D. Grdic, L. Ekman, K. Schön, M. Vajdy, N. Y. Lycke.
1997
. Interferon-γ receptor-deficient mice exhibit impaired gut mucosal immune responses but intact oral tolerance.
Immunology
92
:
60
14
Zhang, Z. Y., J. G. Michael.
1990
. Orally inducible immune unresponsiveness is abrogated by IFN-γ treatment.
J. Immunol.
144
:
4163
15
Salkowski, C. A., G. Detore, R. McNally, N. van Rooijen, S. N. Vogel.
1997
. Regulation of inducible nitric oxide synthase messenger RNA expression and nitric oxide production by lipopolysaccharide in vivo: the roles of macrophages, endogenous IFN-γ, and TNF receptor-1-mediated signaling.
J. Immunol.
158
:
905
16
Gabbai, F. B., C. Boggiano, T. Peter, S. Khang, C. Archer, D. P. Gold, C. J. Kelly.
1997
. Inhibition of inducible nitric oxide synthase intensifies injury and functional deterioration in autoimmune interstitial nephritis.
J. Immunol.
159
:
6266
17
Gold, D. P., K. Schroder, H. C. Powell, C. J. Kelly.
1997
. Nitric oxide and the immunomodulation of experimental allergic encephalomyelitis.
Eur. J. Immunol.
27
:
2863
18
Sahrbacher, U. C., F. Lechner, H. P. Eugster, K. Frei, H. Lassmann, A. Fontana.
1998
. Mice with an inactivation of the inducible nitric oxide synthase gene are susceptible to experimental autoimmune encephalomyelitis.
Eur. J. Immunol.
28
:
1332
19
Fenyk-Melody, J. E., A. E. Garrison, S. R. Brunnert, J. R. Weidner, F. Shen, B. A. Shelton, J. S. Mudgett.
1998
. Experimental autoimmune encephalomyelitis is exacerbated in mice lacking the NOS2 gene.
J. Immunol.
160
:
2940
20
Khoury, S. J., W. W. Hancock, H. L. Weiner.
1992
. Oral tolerance to myelin basic protein and natural recovery from experimental autoimmune encephalomyelitis are associated with downregulation of inflammatory cytokines and differential upregulation of transforming growth factor β, interleukin 4, and prostaglandin E expression in the brain.
J. Exp. Med.
176
:
1355
21
Miller, A., O. Lider, A. B. Roberts, M. B. Sporn, H. L. Weiner.
1992
. Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor beta after antigen-specific triggering.
Proc. Natl. Acad. Sci. USA
89
:
421
22
Moore, W. M., R. K. Webber, G. M. Jerome, F. S. Tjoeng, T. P. Misko, M. G. Currie.
1994
. l-N6-(1-iminoethyl)lysine: a selective inhibitor of inducible nitric oxide synthase.
J. Med. Chem.
37
:
3886
23
Jones-Carson, J., A. Vazquez-Torres, H. C. van der Heyde, T. Warner, R. D. Wagner, E. Balish.
1995
. γδ T cell-induced nitric oxide production enhances resistance to mucosal candidiasis.
Nat. Med.
1
:
552
24
Gupta, S. K., J. F. Fitzgerald, S. K. Chong, J. M. Croffie, J. G. Garcia.
1998
. Expression of inducible nitric oxide synthase (iNOS) mRNA in inflamed esophageal and colonic mucosa in a pediatric population.
Am. J. Gastroenterol.
93
:
795
25
Rizzo, L. V., R. A. Morawetz, N. E. Miller-Rivero, R. Choi, B. Wiggert, C. C. Chan, H. C. Morse, III, R. B. Nussenblatt, R. R. Caspi.
1999
. IL-4 and IL-10 are both required for the induction of oral tolerance.
J. Immunol.
162
:
2613
26
Hauer, A. C., M. Bajaj-Elliott, C. B. Williams, J. A. Walker-Smith, T. T. MacDonald.
1998
. An analysis of interferon γ, IL-4, IL-5 and IL-10 production by ELISPOT and quantitative reverse transcriptase-PCR in human Peyer’s patches.
Cytokine
10
:
627