Introduction

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The incidence and severity of asthma is increasing worldwide. Asthma is a syndrome characterized by intermittent and reversible airway obstruction, airway hyperresponsiveness (AHR), and airway inflammation. Today it is considered a chronic airway inflammatory disorder associated with the presence of activated cells, particularly T lymphocytes, eosinophils, mast cells, and epithelial cells. In susceptible individuals these cells release mediators that amplify the inflammatory cascade, increase AHR, and stimulate mucus secretion, which contribute to airway obstruction (1). As a result of this chronic inflammatory process, the airway tissue may suffer profound structural and functional changes referred as to "airway remodeling" (6).

Major advances in the understanding of the pathogenesis of asthma were provided by murine models which showed that interleukin (IL)-4 and IL-5, two cytokines secreted by T helper (Th) 2 lymphocytes, play a pivotal role in allergic airway inflammation. Thus, disruption of IL-4 production or suppression of its signaling pathway inhibit immunoglobulin (Ig) E production and attenuate airway eosinophilia and AHR (7). In addition, IL-5 deficiency or its overproduction may be associated with the suppression or induction of airway eosinophilia and bronchial hyperreactivity, respectively (10, 11). This is not surprising, inasmuch as IL-4 appears to be important for the development of Th2 cells. IL-4 and IL-13 are involved in IgE synthesis and mucus production (12), whereas IL-5 regulates the growth, differentiation, and activation of eosinophils (18). However, depending on the mouse strain, one cytokine may preponderate over the other for the induction of experimental asthma (19). We have demonstrated that the BP2 selection of Biozzi mice, once immunized and challenged with ovalbumin (OVA), became hyperresponsive to methacholine (MCh) inhalation whereas BALB/c mice reacted less markedly (20). Differences among mouse strains in antigen (Ag)-independent (inherent) airway responsiveness to MCh were also reported (21). Interestingly, this inherent airway responsiveness is T cell-dependent, because the passive transfer of naive T cells from a hyperreactive to a hyporeactive strain confers enhanced airway reactivity (21). Collectively, these experiments highlight the fundamental role of T cells in airway inflammation and AHR. Indeed, asthma-like responses are absent in T-cell receptor (TCR)- knockout (KO) mice (22). In contrast to conventional T cells, T cells appear to exert a negative regulation of airway responsiveness because TCR--deficient TCR-^/) animals present higher airway responsiveness to MCh and diminished airway eosinophilia when compared with conventional mice from the same genetic background (22).

Current therapeutics are effective for asthma control but do not cure the disease. Data from murine models indicate that attenuation or suppression of asthma can be achieved by targeting Th2 development (24, 25) or Th2 cytokines (19). However, continuous inhibition of Th2 cells or cytokines potentially may lead to immunosuppression, to exacerbated Th1 responses or to autoimmunity. Thus, it is more desirable to suppress specifically the immune responses to a given innocuous Ag.

Usually, mucosal administration of soluble proteins by nasal or oral routes results in the development of a state of peripheral immunologic tolerance. The mucosal-tolerance approach has proven effective in the suppression of a variety of experimental organ-specific autoimmune diseases (26). However, literature is scarce regarding the effect of oral tolerance on experimental asthma. Recently, Russo and colleagues have shown that oral tolerance can prevent the development of lung and bone marrow eosinophilia in B6 mice (27). Other studies using similar approaches showed that high-dose oral tolerance was effective in preventing Ag-induced eosinophil infiltration in the trachea (28, 29). However, the effect of oral tolerance on airway reactivity or mucus production has not yet been addressed. Moreover, the effect of oral tolerance on asthma-like responses using different mouse strains has not been studied.

For the present work we selected BALB/c, BP2, CBA/ Ca IL-5 transgenic, and BALB/c TCR-^/ mouse strains to study the effect of oral tolerance on the development of experimental asthma. Selection of these mouse strains was based on the following facts: BALB/c mice develop an IL-4-dependent asthma (7); BP2 animals present high AHR that is largely dependent on IL-5 production (22); IL-5 transgenic mice exhibit hypereosinophilia (18); and - deficient mice appear to be hyperreactive to MCh and refractory to oral tolerance induction (22, 30, 31).

This study shows that a 5-d continuous oral Ag administration before immunization suppressed asthma-like responses in all mouse strains tested. Moreover, oral treatment starting after the primary immunization (Day 0) or after the booster (Day 7) was still effective in suppressing key phenotypes of asthma, such as airway eosinophilia, Th2 cytokine production, anti-OVA IgE synthesis, AHR, and mucus production. However, OVA feeding after Ag challenge (Day 14) enhanced the allergic responses.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Mice

BP2 and BALB/c mice were obtained from Centre d'Elevage R. Janvier (Le Genest Saint-Isle, France). IL-5 transgenic mice on CBA/ca background were originally provided by Prof. C. Sanderson (Perth, Australia). TCR-^/ -deficient mice on BALB/c background were obtained from the Laboratoire d'Immuno-differenciation (Université Paris, Paris, France). BALB/c mice used in experiments designed to determine the time course of tolerance induction and mucus formation were obtained from the animal breeding unit of the Instituto de Ciências Biomédicas-USP, São Paulo, Brazil.

Oral Ag Administration

Oral tolerance to OVA was induced by offering to the animals, ad libitum, a 1% OVA (grade II) solution dissolved in sterile drinking water for 5 consecutive days. This solution was freshly prepared each 12 h. Each individual mouse usually consumed 3 to 5 mL of this solution per day.

Ag Immunization, Booster, and Airway Challenge

At weekly intervals, animals were immunized and boosted by a subcutaneous injection of 4 µg OVA (grade V)/1.6 mg of aluminum hydroxide gel in 0.4 mL saline followed by one or two intranasal OVA challenges with 10 µg OVA/50 mL saline delivered into the nostrils under light ether anesthesia with the aid of a micropipette. At 1 d after the last intranasal challenge the mice were analyzed for AHR, eosinophil infiltration, levels of cytokines, antibody production, and lung histology.

Determination of Airway Responsiveness

Airway responsiveness was assessed as previously described (5, 20). Unrestrained, conscious mice were placed in a plethysmographic chamber (Buxco Eletronics, Sharon, CT), and respiratory parameters were measured before (1 to 3 min) and after (3 to 10 min) an aerosol MCh (Sigma-Aldrich, Stonheim, Germany) delivered for 20 s at 3 or 10 × 10² M in the aerosolator. In experiments performed with PB2 mice, MCh at a dose of 3 × 10² M was used because this strain presents very high AHR (22). For the other strains tested, a dose of 10 × 10² M of MCh was delivered. The resistance was expressed as enhanced pause (Penh), calculated as {Expiratory time/40% of (Relation time)  1} × {(Peak expiratory flow)/(Peak inspiratory flow)} × 0.67, according to the manufacturer's recommendations.

Bronchoalveolar Lavage Fluid

Immediately after assessment of AHR, the mice were deeply anesthetized by the intreperitoneal injection of urethane (15 mg/ 10 g body wt), the abdominal cavity was opened, and blood samples from the inferior cava vein were collected for serum antibody determinations. The trachea was cannulated and lungs were lavaged twice with 0.5 and 1.0 mL saline. After total cell counting, cytospin preparations of bronchoalveolar lavage fluid (BALF) cells were stained with Diff-Quik (Baxter-Dade AG, Dudingen, Germany) and differential cell counts were performed on 200 cells on the basis of morphology and staining characteristics.

Cytokine Levels in BALF

The levels of IL-4, IL-5, IL-10, interferon (IFN)-, and IL-12 in the BALF were assessed by sandwich kit enzyme-linked immunosorbent assay (ELISA) in Nunc-Immuno plates Maxi-Sorb-coated with appropriate anticytokine capture monoclonal antibodies (mAbs) and second-step biotinylated mAbs according to manufacturer order (PharMingen, San Diego, CA). Values are expressed as picograms per milliliter deduced from standards run in parallel with recombinant cytokines.

ELISA for Transforming Growth Factor (TGF)- and Anti-TGF- Treatment

For TGF- quantification, immunoplates (Nunc, Naperville, IL) were coated with 100 ng/well of human TGF- sRII/Fc chimera (R&D Systems, Minneapolis, MN) in carbonate buffer, pH 8.2, at 4°C overnight. Plates were washed, blocked with 10% bovine serum albumin, washed again. Then BALF samples treated with 1 N HCl for 10 min at room temperature to acid-activate TGF- and pH-neutralized by addition of 1 N NaOH, or with standard human TGF-, were added for another overnight incubation at 4°C. Wells were washed and 1 µg/mL of byotinylated polyclonal anti- TGF-1 antibody (R&D Systems) was added. Plates were incubated for 1 hour at room temperature, washed again, and incubated with Avidin-peroxidase (Sigma, St. Louis, MO) for 30 min at room temperature. Color was developed by adding ortho-phenilenodiamino solution containing H₂O₂. The reaction was stopped by sodium dodecyl sulfate and absorbance of the samples was determined at 498 nm. For in vivo treatment, neutralizing anti-TGF- mAb (Genzyme, Cambridge, MA) was administered intraperitoneally before OVA challenge at a dose of 1 mg/mouse.

Determination of OVA-Specific IgG1 and IgG2a

OVA-specific IgG1 and IgG2a antibodies were assayed by sandwich ELISA as previously described (27) with slight modifications. Briefly, Nunc-Immuno Plates Maxi-Sorb (Inter-Med, São Paulo, Brazil) were coated overnight at 4°C with 2 µg of OVA per well, washed with phosphate-buffered saline (PBS)-Tween 0.05%, and blocked with 0.25% casein in PBS. After washing, the serum samples were added and incubated for 1 h. The bound antibodies were revealed by the addition of 1 mg/mL of goat antimouse IgG1 or IgG2a for 1 h followed by washings and the addition of peroxidase-conjugated rabbit antigoat IgG (H+L) antibodies (Southern Biotechnology, Birmingham, AL) for 1 h. After washings, the reaction was developed by the addition of 100 µL/well of substrate containing 0.5 mg/mL of o-orthophenylenediamine dihydrochloride (Sigma and 0.0015 % of H₂O₂ in 0.1 M citric acid/sodium citrate buffer (Merck S.A, São Paulo, Brazil), pH 5.0. The reaction was stopped with 4 M H₂SO₄ and the absorbance of the samples determined at 490 nm. The concentrations of IgG1 and IgG2a antibodies were estimated by comparison with IgG1 and IgG2a standards run in parallel on rabbit antimouse (Southern Biotechnology) Ig-coated plates.

Determination of OVA-Specific IgE

For OVA-specific serum IgE determinations, plates were coated overnight at 4°C with 2 µg/mL of goat-antimouse IgE antibodies (Southern Biotechnology). The serum samples were added and subsequently biotin-labeled OVA was added to the wells. The biotinylated OVA was prepared by reacting 1 mL OVA in PBS (1 mg/mL) that was dialyzed at 4°C overnight against 0.2 M borate buffer (pH 8.5) with 100 mL of N-hydroxysuccinimidobiotin in dimethyl sulfoxide (DMSO) (4 mg/mL) for 4 h at room temperature, followed by overnight dialysis against PBS at 4°C. The bound OVA-biotin was revealed by extrAvidin Peroxidase conjugate (Sigma) followed by 100 ml/well of substrate as described earlier. OVA-specific IgE levels of samples were related to an internal standard obtained from pooled sera of hyperimmunized BALB/c mice that was arbitrarily assigned to be 10,000 units.

Lung Histology

Lungs were removed after BALF collection, perfused via the right ventricle with 10 mL saline to remove residual blood and immersed in 10% phosphate-buffered formalin for 24 h and then in 70% ethanol until embedding in paraffin. Tissues were sliced and 5-µm sections were stained with hematoxylin/eosin for light microscopy examination or with periodic acid-Schiff (PAS)/hematoxylin for evaluation of mucus-producing cells. Mucus production was determined by counting the number of PAS-positive (PAS⁺) bronchi or the number of bronchi containing plugs of mucus. Values represent the sums of 25 bronchi scored randomly at ×250 magnification.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Oral OVA Administration Suppresses Ag-Induced Airway Inflammation and Airway Th2 Cytokine Production in BALB/c and BP2 Mice

We first examined the effect of continuous oral OVA administration on eosinophilic airway inflammation and Th2 cytokine production 24 h after OVA challenge. The content of eosinophils and of IL-4 and IL-5 cytokines was quantified in the BALF of BALB/c and BP2 mice. As shown in Figure 1A, the number of eosinophils increased significantly in immunized mice after intranasal OVA challenge when compared with the nonimmunized Control group. The number of eosinophils in BP2 mice was higher than that in BALB/c, confirming previous results (5, 20). The oral OVA administration for 5 consecutive days completely suppressed OVA-induced airway eosinophilia in both mouse strains (Figure 1A). The total numbers of cells in BALB/c and BP2 control groups were 1.8 ± 0.19 × 10⁵ and 2.9 ± 0.40 × 10⁵, respectively. These cells were mainly mononuclear cells (more than 95% macrophages and less than 5% lymphocytes). In immunized mice the total number of cells increased (3.1 ± 0.48 × 10⁵ for BALB/c and 6.6 ± 0.64 × 10⁵ for BP2). Differential counts showed that in both strains, eosinophils and neutrophils increased (~ 40% and 15%, respectively), macrophages decreased (~ 40%), and lymphocytes increased slightly (5%). Total cell count and differential in tolerized animals were roughly similar to that of the Control group (Figure 1A and data not shown). The cytokine content in the BALF of BALB/c and BP2 revealed that the Immune groups from both mouse strains produced significant levels of IL-4 and IL-5 when compared with the Control group (nonimmunized and OVA-challenged mice) (Figure 1B and 1C). The levels of IL-5 in BALF were roughly similar in BALB/c and BP2 mice (Figure 1B), whereas IL-4 levels were higher in BALB/c than in BP2 (Figure 1C). In contrast, mice that were fed OVA before immunization (the Oral group) failed to show significant amounts of IL-5 and IL-4 when compared with control mice (Figures 1B and 1C).

View larger version (17K): [in this window] [in a new window]   Figure 1.   The effect of oral OVA administration on Ag-induced airway eosinophilic inflammation and BALF Th2 cytokine levels in BALB/c and BP2 mice. Groups of five mice received a solution of 1% OVA dissolved in the drinking water for 5 consecutive days (Oral) or water (Immune). At 2 d later these groups of mice were immunized and boosted subcutaneously (1 wk apart) with 0.4 mL of a solution containing 4 µg of OVA and 1.6 mg of aluminum hydroxide. At 1 wk after booster the groups were challenged intranasally with 10 µg of OVA in 50 µL of saline. The Control group received only OVA intranasally. Experiments were performed 24 h after OVA challenge. Results are expressed as means ± standard error of the mean (SEM). *Significant difference (P < 0.05) between values of Immune versus Oral groups.

Oral OVA Administration Suppresses IgE and IgG1 Anti-OVA Antibodies of BALB/c and BP2 Mice

The serum levels of specific IgE and IgG1 antibodies were determined 24 h after the first or second OVA challenge. It was found that control BALB/c and BP2 mice (animals not immunized but challenged intranasally with OVA) produced undetectable levels of IgE whereas the Immune group produced low but detectable levels of specific IgE antibodies (Figure 2A). The levels of IgE anti-OVA antibodies were not statistically different when BALB/c and BP2 mice of the immune group were compared (Figure 2A). Animals fed OVA (oral group) did not produce detectable levels of specific IgE antibodies (Figure 2A). These results confirm and extend previous findings that oral tolerance suppresses IgE production (26). Figure 2B shows that sensitized BP2 mice produced very high amounts of IgG1 antibodies (~ 1,400 mg/mL). The concentration of IgG1 antibodies of BP2 mice was roughly 50 times more than that produced by BALB/c mice (~ 30 mg/mL). Despite these differences, oral tolerance suppressed IgG1 production in both strains (Figure 2B). Because IgE and IgG1 production by BALB/c mice after OVA challenge was very low, we investigated whether a second challenge with OVA, 7 d later, would increase antibody production. Indeed, BALB/c mice challenged twice with OVA produced higher amounts of IgE and IgG1 as compared was mice challenged only once. Again, oral OVA administration significantly suppressed IgE (Figure 2C) and IgG1 (Figure 2D) antibody production.

View larger version (20K): [in this window] [in a new window]   Figure 2.   The effect of oral OVA administration on Ag-induced anti-OVA IgG1 and IgE antibody production in BALB/c and BP2 mice. Groups of five mice received a solution of 1% OVA dissolved in the drinking water for 5 consecutive days (Oral) or water (Immune). At 2 d later these groups of mice were immunized and boosted subcutaneously (1 wk apart) with 0.4 mL of a solution containing 4 µg of OVA and 1.6 mg of aluminum hydroxide. At 1 wk after booster the groups were challenged intranasally, once or twice (1 wk apart), with 10 µg of OVA in 50 µL of saline. The Control group received only OVA intranasally. Experiments were performed 24 h after OVA challenge. Results are expressed as means ± SEM. *Significant difference (P < 0.05) between values of Immune versus Oral groups.

Oral OVA Administration Suppresses Ag-Induced Airway Hyperreactivity of BP2 Mice

As shown in Figures 3A and 3B, sensitized BP2, but not BALB/c, mice developed significant AHR after MCh inhalation when compared with the Control group, confirming previous results (5, 20). OVA feeding resulted in the suppression of AHR of BP2 mice (Figure 3B). Because BALB/ c mice challenged twice with OVA presented high production of OVA-specific IgE and IgG1 antibodies, we investigated whether these animals would develop AHR. As shown in Figure 3C, BALB/c mice challenged twice with OVA did not present enhanced bronchial response to MCh.

View larger version (20K): [in this window] [in a new window]   Figure 3.   The effect of oral OVA administration on Ag-induced airway reactivity in BALB/c and BP2 mice. Groups of five mice received a solution of 1% OVA dissolved in the drinking water for 5 consecutive days (Oral) or water (Immune). At 2 d later these groups of mice were immunized and boosted subcutaneously (1 wk apart) with 0.4 mL of a solution containing 4 µg of OVA and 1.6 mg of aluminum hydroxide. At 1 wk after booster the groups were challenged intranasally with 10 µg of OVA in 50 µL of saline. The Control group received only OVA intranasally. Experiments were performed 24 h after OVA challenge. Results are expressed as means ± SEM. *Significant difference (P < 0.05) between curves of Immune versus Oral groups.

Oral OVA Administration Suppresses Ag-Induced Airway Eosinophilia, Airway IL-5 Production, and Airway Hyperreactivity in Transgenic IL-5 Mice

To determine whether oral Ag administration would suppress allergic responses in mice presenting hypereosinophilia, we used IL-5 transgenic mice (18), which show very high levels of IL-5 in serum (~ 4,000 pg/mL) with more than 50% of their granulocytes in blood being eosinophils. As shown in Figure 4, after immunization and challenge IL-5 transgenic mice developed Ag-induced airway eosinophilia, increased production of IL-5 in airways, and AHR to MHc. However, they presented very low levels of IL-4 and of OVA-specific IgG1 and IgE antibodies (data not shown). Again, OVA feeding suppressed Ag-induced allergic responses (Figure 4).

View larger version (16K): [in this window] [in a new window]   Figure 4.   The effect of oral OVA administration on Ag-induced airway eosinophilic inflammation, BALF IL-5 levels, and airway hyperreactivity in IL-5 transgenic mice. Groups of four mice received a solution of 1% OVA dissolved in the drinking water for 5 consecutive days (Oral) or water (Immune). At 2 d later these groups of mice were immunized and boosted subcutaneously (1 wk apart) with 0.4 mL of a solution containing 4 µg of OVA and 1.6 mg of aluminum hydroxide. At 1 wk after booster the groups were challenged intranasally with 10 µg of OVA in 50 µL of saline. The Control group received only OVA intranasally. Experiments were performed 24 h after OVA challenge. Results are expressed as means ± SEM. *Significant difference (P < 0.05) between values or curves of Immune versus Oral groups.

Oral Tolerance Does Not Increase Immunosuppressive Cytokines (IL-10 and TGF-) or Type 1 Cytokines (IL-12 and IFN-)

It has been reported that OVA-specific unresponsiveness induced by oral or nasal Ag administration might be mediated by an increased secretion of immunosuppressive cytokines such as IL-10 and TGF- secreted by regulatory cells (reviewed in 26 and 32), or by an increased production of cytokines such as IL-12 and IFN- that inhibit Th2 responses (immune deviation) (26, 36). To study these possibilities, we determined the levels of IL-10, TGF-, IL-12, and IFN- in BALF of BALB/c mice from the Control, Immune, and Oral groups 24 h after the second intranasal OVA challenge. As shown in Figure 5, the levels of IL-12, IFN-, and TGF- were similar in all groups, whereas IL-10 titers were decreased in the Oral group as compared with the Immune group. In addition, we examined whether neutralizing anti-TGF- mAb (1 mg/ mouse) given during the second OVA challenge would reverse the inhibitory effect of oral tolerance on airway eosinophilia and AHR exhibited by sensitized BP2 mice. The anti-TGF- treatment failed to reverse these responses (data not shown). Globally, these experiments document that Ag-specific unresponsiveness cannot be ascribed to regulatory activities exerted by IL-10, TGF-, IFN-, or IL-12 cytokines produced in the BALF.

View larger version (18K): [in this window] [in a new window]   Figure 5.   The effect of oral OVA administration on Ag-induced IL-10, TGF-, IL-12, and IFN- production in BALF of BALB/c mice. Groups of five mice received a solution of 1% OVA dissolved in the drinking water for 5 consecutive days (Oral) or water (Immune). At 2 d later these groups of mice were immunized and boosted subcutaneously (1 wk apart) with 0.4 mL of a solution containing 4 µg of OVA and 1.6 mg of aluminum hydroxide. At 1 wk after booster the groups were challenged intranasally, twice (1 wk apart), with 10 µg of OVA in 50 µL of saline. The Control group received only OVA intranasally. Experiments were performed 24 h after the last OVA challenge. Results are expressed as means ± SEM. *Significant difference (P < 0.05) between values of Immune versus Oral groups.

Oral OVA Administration Suppresses Ag-Induced Airway Hyperreactivity, OVA-Specific IgG1 and IgE Responses, and Mucus Formation of -Deficient Mice

Finally, it has been reported that T cells play a critical active role (suppressor activity or immune deviation) in tolerance induced by orally or nasally administered Ag cells (26, 30, 36, 37) and that TCR- KO mice exhibited enhanced bronchial hyperreactivity to MCh inhalation (22). Thus, we decided to examine in -deficient mice with a BALB/c background the effect of our Ag feeding protocol on Ag-induced AHR and anti-OVA antibody production. For this, TCR-^/ BALB/c were fed with 1% OVA dissolved in the drinking water for 5 consecutive days; starting 2 d later, at weekly intervals, mice were immunized, boosted, and challenged with OVA as described in MATERIALS AND METHODS. The AHR was determined after the first intranasal OVA challenge, and antibody and mucus production was quantified after the second OVA challenge. Figure 6A shows that oral OVA administration to -deficient mice significantly suppressed the AHR to MCh inhalation as well as the specific IgG1 and IgE responses (Figures 6B and 6C) and mucus formation (Figure 7).

View larger version (15K): [in this window] [in a new window]   Figure 6.   The effect of oral OVA administration on Ag-induced airway hyperreactivity and anti-OVA IgG1 and IgE antibody production in -deficient BALB/c mice. Groups of five mice received a solution of 1% OVA dissolved in the drinking water for 5 consecutive days (Oral) or water (Immune). At 2 d later these groups of mice were immunized and boosted subcutaneously (1 wk apart) with 0.4 mL of a solution containing 4 µg of OVA and 1.6 mg of aluminum hydroxide. At 1 wk after booster, the groups were challenged intranasally, once (for AHR) or twice (for antibody production), with 10 µg of OVA in 50 µL of saline. The Control group received only OVA intranasally. Experiments were performed 24 h after OVA challenge. Results are expressed as means ± SEM. *Significant difference (P < 0.05) between values or curves of Immune versus Oral groups.

View larger version (152K): [in this window] [in a new window]   View larger version (148K): [in this window] [in a new window]   Figure 7.   Inhibition of mucus formation by oral Ag administration in -deficient mice. Representative lung sections stained with PAS and hematoxylin showing in Immune group (A) three PAS+ bronchi (stained red) and intense peribronchovascular inflammatory cell infiltration. Note the partial bronchial occlusion with a mucus plug (upper bronchiole); and in the Oral group (B), the absence of these alterations. Experimental protocol was identical to that described in Figure 6 caption.

The Effect of Oral OVA Administration Initiated after the Primary Immunization or after the Booster on Allergic Responses of BALB/c Mice

The experiments outlined earlier indicate that oral Ag administration has a preventive effect on the development of experimental asthma. To determine whether oral Ag administration would display a curative effect on experimental asthma, the oral OVA treatment was initiated soon after the primary immunization (Day 0) or after the booster (Day 7). Experiments were performed 24 h after the second intranasal OVA challenge. The asthma-like responses quantified were airway eosinophilic inflammation, Th2 cytokines present in BALF, and specific IgE and IgG1 production. As shown in Figure 8A, oral Ag administration after primary immunization (Day 0) or the booster (Day 7) significantly suppressed airway eosinophilic inflammation as revealed by total and eosinophil cell counts. The inhibition of eosinophil recruitment was paralleled by the inhibition of IL-5 production (Figure 8B). Although IL-4 levels were lower in animals treated with oral OVA at Day 0 and Day 7, statistical analysis revealed that IL-4 production was significantly suppressed by oral Ag administration after the primary immunization but not after the booster (Figure 8B). The specific IgE production presented the same pattern of inhibition as that observed for IL-4 (Figure 8C). However, the specific IgG1 responses were not significantly suppressed by oral Ag administration at Day 0 or Day 7 (Figure 8D).

View larger version (26K): [in this window] [in a new window]   Figure 8.   The effect of oral OVA administered after primary immunization or after booster on Ag-induced allergic responses in BALB/c mice. Groups of five mice were immunized and boosted subcutaneously (1 wk apart) with 0.4 mL of a solution containing 4 µg of OVA and 1.6 mg of aluminum hydroxide. OVA solution (1%) was offered to the animals immediately after primary immunization (Oral-Day 0) or after booster (Oral-Day 7) for 5 consecutive days. At 1 wk after booster the groups were challenged intranasally, twice (1 wk apart), with 10 µg of OVA in 50 µL of saline. The Control group received only OVA intranasally. Experiments were performed 24 h after the last OVA challenge. Results are expressed as means ± SEM. *Significant difference (P < 0.05) between values of Immune versus Oral groups.

Oral OVA Administration after Ag Challenge Increases Airway Eosinophilia and OVA-Specific IgG1 and IgE Antibody Production

Next we assessed the effect of oral Ag administration initiated after OVA challenge (Day 14) on the development of allergic-responses. As shown in Figure 9, oral Ag administration after the first OVA challenge for 5 consecutive days, instead of suppressing, substantially increased airway eosinophilic inflammation and IgE and IgG1 antibody production, but did not affect IL-4 and IL-5 levels present in BALF when compared with OVA-sensitized animals that did not receive oral OVA.

View larger version (12K): [in this window] [in a new window]   Figure 9.   The effect of oral OVA administration after intranasal OVA challenge on Ag-induced allergic responses in BALB/c mice. Groups of five mice were immunized and boosted subcutaneously (1 wk apart) with 0.4 mL of a solution containing 4 µg of OVA and 1.6 mg of aluminum hydroxide. At 1 wk after booster the groups were challenged intranasally with 10 µg of OVA in 50 µL of saline and immediately after mice received 1% of OVA solution (Oral) or water (Immune) for 5 consecutive days. At 2 d later the animals were challenged again with OVA. The Control group received only OVA intranasally. Experiments were performed 24 h after the last OVA challenge. Results are expressed as percent of increase comparing the immunized group without OVA feeding versus the immunized group with OVA feeding. *Significant difference (P < 0.05) between values of Immune versus Oral groups.

Oral OVA Administration after Immunization or Booster Suppresses Airway Hyperreactivity and Mucus Production

Finally, we determined whether oral Ag administration after immunization or booster would suppress the two major events responsible for airway obstruction. To do so, BP2 mice were challenged once with OVA for AHR determination and BALB/c mice challenged twice with OVA for analysis of mucus formation. As shown in Figure 10, immunized, boosted, and OVA-challenged BP2 mice developed an intense AHR whereas animals that received oral OVA after immunization or after the booster did not present AHR (Figure 10). Figure 10 also shows that 65% of airways of BALB/c mice from the immune group stained positively for PAS. Administration of oral OVA significantly decreased mucus formation being more effective when administered after immunization than after booster (Figure 10). Similar results were obtained in BP2 mice (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 10.   The effect of oral OVA administered after primary immunization or after booster on Ag-induced airway reactivity in BP2 mice and mucus production in BALB/c mice. Groups of five mice were immunized and boosted subcutaneously (1 wk apart) with 0.4 mL of a solution containing 4 µg of OVA and 1.6 mg of aluminum hydroxide. OVA solution (1%) was offered to the animals immediately after primary immunization (Oral-Day 0) or after booster (Oral-Day 7) for 5 consecutive days. At 1 wk after booster the groups were challenged intranasally, once for AHR determination or twice (1 wk apart) for mucus production, with 10 µg of OVA in 50 µL of saline. The Control group received only OVA intranasally. PAS-stained lung sections were examined at ×100 or ×250 magnification. The percentage of PAS+ bronchi was determined by counting 25 bronchi randomly. Experiments were performed 24 h after the last OVA challenge.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study we demonstrate that the oral administration of Ag for 5 consecutive days before immunization prevents the development of key phenotypes of experimental asthma in BP2, BALB/c, CBA IL-5 transgenic, and BALB -deficient mouse strains. As commented earlier, we selected these mouse strains because they outline particular features of allergic responses. In all strains used in our study we followed the same experimental protocol and found that airway eosinophilic inflammation, mucus production and specific anti-OVA IgE were always inhibited by continuous OVA feeding. Thus, our data reveal how potent and strain-independent is the phenomenon of oral tolerance.

As compared with BALB/c, BP2 mice presented higher levels of IL-5 and eosinophils and developed a very intense AHR, whereas BALB/c mice exhibited higher levels of IL-4 but failed to develop AHR, confirming previous results (20). These different phenotypes were abolished by oral tolerance. BP2 mice produced higher levels of IgG1 anti-OVA antibodies than did BALB/c mice, confirming that BP2 strain is a high antibody responder. Surprisingly, the levels of specific IgE antibodies were very low and similar in BP2 and BALB/c mice indicating that the BP2 strain cannot be classified as a high responder for specific IgE production, even though BP2 mice do produce high titers of total IgE, which increase after immunization (20). These findings suggest that enhanced AHR of the BP2 strain might be associated either with high IgG1 production or with total rather than specific IgE production. We favor the later hypothesis because BALB/c mice challenged twice with OVA produced higher amounts of specific IgE antibodies and equivalent amounts of specific IgG1 antibodies when compared respectively with those seen in BP2 after one OVA challenge, and yet did not develop AHR. Actually, the expression of asthma in humans is associated with total serum IgE (polyclonal responses) and not with specific IgE responses (39). It has been reported that strains of mouse selected for high antibody responsiveness (Biozzi selection III and IV A) are resistant to tolerance induction by one high dose oral Ag administration (40). For the induction of tolerance we used a protocol of continuous Ag feeding for 5 consecutive days and showed that BP2 high-responder mice were tolerized for antibody production as well as for Th2-associated responses. Thus it remains to be determined whether this protocol of Ag administration is effective in suppressing Ag-induced immune responses of other mouse strains selected for high antibody responses.

We also demonstrated that Ag-induced airway eosinophilia, Th2 cytokine production and AHR of transgenic of IL-5 mice were prevented by oral tolerance. Clearly, high levels of serum IL-5 or blood hypereosinophilia do not prevent Ag-specific tolerance induction.

Our study also revealed that oral OVA administration after primary immunization or after booster is still effective in suppressing the key features of asthma. Particularly, AHR andto a lesser degreemucus formation were significantly decreased in animals fed OVA after the booster (day 7). Because IL-13 appears to be the major mediator of these alterations (17), it will be important to measure the levels or the expression of IL-13 in these animals. Recent data points out that the production of IL-13 is inhibited in animals that received oral OVA before immunization (current authors' unpublished results).

The major mechanisms suggested to underlie T-cell tolerance are: (1) selective expansion of regulatory cells, producing immunosuppressive cytokines such as TGF-, IL-4, and IL-10 (26, 32, 35); (2) downregulation of Th2 immunity due to immune deviation toward Th1 pathway (36- 38); (3) suppressor activity of CD8⁺ or ⁺ T cells (30, 31, 37, 38); and (4) anergy or deletion of Ag-specific T cells (41). We addressed these issues by performing the following experiments: First, we examined the roles of TGF- and IL-10 in our model by measuring these cytokines in the BALF. The levels of these cytokines did not increase in tolerized mice. Actually, TGF- production was unaffected whereas IL-10 levels were decreased in mice fed OVA, indicating that these cytokines are not released at the site of Ag challenge in tolerized animals. Also, administration of anti-TGF- mAb did not abrogate the suppression of AHR and airway eosinophilia of tolerized mice. Our results are in line with those reported by Nakao and associates (29), showing that anti-TGF- antibodies had no significant effect on the inhibition of tracheal eosinophilia mediated by oral tolerance in BALB/c mice. However, our results contrast with those obtained by Haneda and colleagues (28) that showed that the suppressive effect exerted by CD4⁺ splenic T cells obtained from OVA-fed transgenic mice for OVA TCR was abrogated by anti-TGF- antibodies. These differences could be due either to different experimental designs or to the usage of transgenic versus nontransgenic animals. Second, we measured the content of IL-12 and IFN- cytokines in the BALF of BALB/c mice that are known to exert inhibitory effects on allergic responses (34, 36). We found that after OVA challenge, IL-12 and IFN- production was unaltered in OVA-fed animals as compared with control sensitized animals. Moreover, we measured OVA-specific IgG2a antibodies that reflect Th1-associated response and their production was very low in OVA-fed animals, as well as in immunized mice (data not shown). Thus, our results do not support the notion that active suppression exerted by immunosuppressive or regulatory cytokines (immune deviation) is operating in our asthma model. However, it should be pointed out that these cytokines were measured in BALF. Thus, it is possible that these cytokines might exert immunoregulatory effects at different sites, such as the spleen, or at different time points, such as during the induction phase of tolerance. Third, we assessed the role of T cells in our model by using mice genetically deficient in T cells (TCR-^/). Confirming previous findings, TCR-^/ mice developed higher AHR to MCh and lower (although significant) influx of eosinophils than BALB/c mice (22, 23). However, no differences between these strains were observed regarding anti-OVA IgE and IgG1 antibody production or mucus formation. All of these parameters were inhibited in sensitized mice by previous administration of Ag in the drinking water for 5 consecutive days. These experiments clearly indicate that T cells are not required for the induction of oral tolerance. Our data conflict with previous reports showing that T cells are required for induction and maintenance of oral tolerance (30, 31). It is likely that these discrepancies may be related to regimens of feeding. It was shown that multiple and continuous feeding of OVAL is more effective in suppressing Th2 responses than is a single dose of Ag feeding (26, 27, 43). In contrast to a report emphasizing the role of T cells in aerosol-induced IgE unresponsiveness (37), we found that OVA-specific IgE responses were completely suppressed in mice deficient in T cells. Our findings resemble those of Seymour and colleagues (44) demonstrating that inhalation tolerance develops normally in -deficient mice and does not require IFN-. In another study it was shown that -deficient mice sensitized to OVA using an adjuvant-free protocol present lower OVA-specific IgE and IgG1 responses when compared with -sufficient mice (23). Here, we showed that BALB/c -deficient or -sufficient mice immunized and boosted with OVA plus Alum adjuvant and challenged twice with intranasal OVA produced equivalent amounts of IgE and IgG1 anti-OVA antibodies. Thus, it appears that under subtle immunologic conditions such as adjuvant-free sensitization or single Ag feeding, the physiologic role of T cells is more apparent than under strong immunologic manipulations. Finally, the fact that all Ag-induced allergic responses were inhibited by previous oral Ag administration favors the hypothesis of anergy/deletion rather than active suppression. However, this hypothesis cannot accommodate the data obtained with oral Ag administration after primary immunization or after booster. In these situations anti-OVA IgG1 responses were not inhibited whereas airway eosinophilic inflammation, airway Th2 cytokine production, AHR to MCh, and mucus formation were significantly inhibited. Thus, it is hard to conceive that anergy/deletion is operating in mice where some responses mediated by Th2 cells were suppressed whereas others were not blocked at all. Nevertheless, these results indicate that local pulmonary responses are more prone to be inhibited by oral tolerance than by systemic antibody production. Our results, mainly those obtained with oral Ag administration after immunization, are encouraging in considering mucosal tolerance as an alternative to treat allergic diseases. Actually, on the basis of experimental data in mucosal tolerance and the safety of the approach, trials in humans are ongoing in multiple sclerosis, rheumatoid arthritis, uveitis, and diabetes. Here, we showed that oral Ag administration can be preventive and curative when initiated before immunization, after immunization, or soon after booster. However, oral Ag administration after challenge (Day 14) instead of protecting the animals, enhanced their allergic responses. These findings indicate that mucosal tolerance induction is related to timing and/or immunologic status and may represent a two-edged sword. Thus, more studies are needed to define the conditions of safe procedure to treat asthma on the basis of the tolerance concept.