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Examination of oral sensitization with ovalbumin in Brown Norway rats and three strains of mice
Immunology Letters 78 (2001) 1 – 5
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Examination of oral sensitization with ovalbumin in Brown Norway rats and three strains of mice Hiroshi Akiyama a,*, Reiko Teshima b, Jun-ichiro Sakushima a,c, Haruyo Okunuki b, Yukihiro Goda a, Jun-ichi Sawada b, Masatake Toyoda a b
a Di6ision of Foods, National Institute of Health Sciences, 1 -18 -1, Kamiyoga, Setagaya-ku, Tokyo 158 -8501, Japan Di6ision of Biochemistry and Immunochemistry, National Institute of Health Sciences, 1 -18 -1, Kamiyoga, Setagaya-ku, Tokyo 158 -8501, Japan c Domestic Research Fellow, Japan Science and Technology Corporation, 4 -18 -8, Hommachi, Kawaguchi-shi, Saitama 332 -0012, Japan
Received 8 January 2001; received in revised form 7 May 2001; accepted 7 May 2001
Abstract We studied the conditions needed to sensitize animals to the oral feeding of food allergens, without induction of tolerance, in order to investigate the allergenicity of orally ingested food proteins. Brown Norway (BN) rats were sensitized by daily OVA (ovalbumin)-gavage or by drinking OVA containing water ad libitum and the ASA (active systemic anaphylaxis) response, as the immediate hypersensitivity response to antigen stimulation after oral sensitization, was examined. The oral administration of OVA by gavage produced a higher OVA-specific IgE response and an increase in serum histamine after antigen challenge, as compared to those produced by drinking water. Next, we examined the effect of murine age, the oral feeding technique and the oral feeding dose on sensitization using BALB/c, B10A and ASK mice. Twenty-week-old mice showed the strongest OVA-specific IgE and IgG1 responses and ASA-associated serum histamine contents increased with gavage in the three different age groups of BALB/c mice. Administering 0.1 mg of OVA by gavage daily for 9 weeks appeared to induce a higher response than administering 1 mg of OVA, in terms of OVA-specific IgE and IgG1 antibody responses and ASA responses. Among the three strains of mice, B10A mice exhibited the highest response in terms of OVA-specific IgE and IgG1 antibody and ASA responses. These findings suggested BN rats and B10A mice were suitable models for oral sensitization with antigen protein and that oral sensitization in mice requires low dose, intermittent antigen intakes. © 2001 Elsevier Science B.V. All rights reserved. Keywords: B10A mice; BALB/c mice; BN rats; Oral sensitization; Ovalbumin
1. Introduction Protein specific immunoglobulin E (IgE)-mediated allergic reactions are known to play a major role in food allergy [1]. In recent years, new proteins have been artificially produced in special foods, such as genetically modified foods. Some of the new proteins might be strongly allergenic in humans. Nevertheless, at present there are only a few methods of predicting the allergenicity of food proteins. Several attempts have been made to develop animal models for investigating the allergenicity of food proteins, mainly in the mouse, guinea-pig and rat. * Corresponding author. Tel.: + 81-03-3700-9397; fax: + 81-033707-6950. E-mail address: [email protected] (H. Akiyama).
Many of these studies have been conducted using parenteral sensitization and enteral challenges [2–6]. Oral immunization studies using animals are an important means of evaluating the allergenicity of food proteins and the mechanism of food allergy induction. However, the dosage of allergens, the method of oral administration and type of mouse strain used are thought to be important in terms of avoiding the occurrence of tolerance to orally-administered food allergens [7–9]. Recently, Knippels et al. reported that Brown Norway (BN) rats showed antigen specific-IgG, as well as IgE responses, after intragastric administration and may provide a suitable model for oral sensitization to food proteins [10,11]. It was also shown that BN rats manifest specific T-cell mediated hypersensitivity (DTH) to ovalbumin (OVA) by oral sensitization. In our experiment, we investigated whether an active systemic ana-
0165-2478/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 1 ) 0 0 2 2 9 - 2
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H. Akiyama et al. / Immunology Letters 78 (2001) 1–5
phylaxis (ASA) response, as immediate hypersensitivity, is induced in orally immunized BN rats. The development of suitable murine models for oral sensitization is also desirable if only small amounts of food proteins are available, because the amount fed to mice is usually less than that given to rats. Sensitization of mice, leading to the induction of active anaphylaxis, not only induces an antigen-specific IgE response, but also results in the production of antigen-specific IgG1 antibodies [12]. IgE binding to FcoRI-bearing cells and IgG1 binding to FcgR-bearing cells are essential steps in active anaphylaxis in mice and mediators released by these cells appear to be responsible for the symptoms of active anaphylaxis. Thus, it is thought to be important to assess the protein-specific IgE response, as well as the IgG1 response. In the present study, we confirmed that BN rats exhibit a specific-IgE response to oral administration and examined whether or not ASA is induced in rats as an immediate hypersensitivity response. We also investigated an intragastric feeding protocol, without the use of an adjuvant, designed to induce specific IgE and IgG1 immune responses and an ASA response, as an immediate hypersensitivity reaction, in BALB/c mice, B10A mice and ASK mice, all three of which have been reported to be immunologically sensitive strains [13,14].
2. Materials and methods
2.1. Animals and reagents Female BN rats, female BALB/C mice and female B10A mice were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan), and female ASK mice from SEAC Yoshitomi (Fukuoka, Japan), at 6 weeks of age. All animals were kept in our animal facility on a CRF-1 diet for 1 week before use. OVA (grade V) was purchased from Sigma Chemical Co. (St. Louis, MO).
2.2. Immunization and induction of acti6e systemic anaphylaxis (ASA) BN rats and the three strains of mice were sensitized by administration of 0.1 or 1.0 mg OVA by gavage daily for 9 weeks, or in drinking water (5 mg/ml) daily for 9 weeks. ASA challenge was elicited by intraperitoneal (ip) injection of 1 mg of OVA 1 day later [15].
2.3. Antibody (IgE, and IgG1 isotypes) titer determination Serum titers (reciprocal of serum dilution with fluorescence intensity at 50% of the maximum level) of OVAspecific IgE and IgG1 were determined according to a previously reported method, with some modifications
[16]. A 50 ml volume of OVA (40 mg/ml) in 50 mM sodium carbonate buffer, pH 9.6, was added to each well of a 96-well microtiter plate, and incubated overnight at 4°C. The solutions were discarded, and each well was washed four times with 200 ml PBS containing 0.05% Tween 20 (PBS/Tween). To minimize the nonspecific binding of serum proteins to unoccupied solid-phase sites, 200 ml of 0.1% casein in PBS were added and incubation was continued for 1 h at room temperature. The casein solution was removed, and each well was washed in the same way as above. Fifty microliters of the diluted serum containing OVA-specific antibodies were added to each well, followed by incubation for another 20 h at 4°C. The solutions were removed, and each well was washed. Fifty microliters of rabbit anti-mouse IgE and IgG1 [10 − 3 dilution in PBS containing 0.1% casein (Nordic Immunology, Tilburg, the Netherlands)] were added to each well, and then incubated for 1 h at room temperature. The solution in each well was removed and washed. Fifty microliters of b-galactosidase-linked goat anti-rabbit Ig conjugate [10 − 3 dilution in PBS containing 0.1% casein, (Amersham, UK)] were added to each well, followed by a 1 h incubation at room temperature. The antibody–enzyme conjugate solution in each well was removed and washed. The wells were incubated for 1 h at 37°C with 100 ml PBS containing 0.1 mM 4-methylumbelliferyl-bgalactoside (Sigma). Finally, 25 ml of 1 M sodium carbonate were added to each well. The fluorescence intensity of the liberated 4-methylumbelliferone was monitored at 317 and 374 nm for excitation and emission, respectively, using a Titertek Fluoroscan reader (Flow Laboratories Inc., Costa Mesa, CA).
2.4. Determination of plasma serum histamine in ASA reaction Twelve minutes after antigen challenge, blood was collected from the eye, then left standing for 1 h at room temperature, before centrifugation to obtain the serum. Serum histamine levels were measured by the postcolumn HPLC method, as described previously [17].
2.5. Statistical analysis The statistical significance of the data was determined by Student’s t-test. A P-value of less than 0.05 was taken as significant.
3. Results
3.1. Examination of oral sensitization in BN rats As shown in Fig. 1, we confirmed that 1 mg administration of OVA by gavage or via drinking water (5mg/ml)
H. Akiyama et al. / Immunology Letters 78 (2001) 1–5
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Fig. 2. Determination of OVA-specific IgE titer, OVA-specific IgG1 titer and serum histamine level 12 min after antigen challenge in OVA-gavaged 20-week old BALB/c mice. OVA-specific IgE and IgG1 titers and the serum histamine level were determined 12 min after antigen challenge in 0.1 or 1.0 mg OVA-gavaged mice, according to the method described in Section 2.3 and Section 2.4. Each value represents the mean 9 SD for five mice. Fig. 1. Determination of OVA-specific IgE titer and serum histamine level 12 min after antigen challenge in BN rats exposed to OVA by gavage or via drinking water. OVA-specific IgE titer and the serum histamine level were determined in BN rats 12 min after antigen challenge, according to the method described in Section 2.3. Each value represents the mean 9 SD for three –seven rats. Asterisks indicate significant differences from the control group (*PB0.05).
increased serum titers of OVA-specific IgE (drinking water: 3609 169, per oval, p.o.: 4509 70) as compared with control (saline-treated) rats. The oral administration of OVA by gavage produced a stronger OVA-specific IgE response than that induced by drinking water. In addition, a significant increase in the serum histamine level (drinking water: 2679 92 mM, p.o.: 4909 194 mM) occurred after antigen challenge in both the rats sensitized by gavage and those sensitized via drinking water.
3.2. Effect of age on sensitization by oral administration in BALB/c mice As shown in Table 1, the serum titers of OVA-specific IgE and IgG1 were higher in 20-week old mice than in 7-week old mice for both 0.1 and 1 mg administration. However, OVA-specific IgE and IgG1 titers in 1-year old mice were much lower than those in the two
younger groups. The serum histamine level in ASA of 20-week old mice was higher than those seen in ASA of 7- and 1-year old mice. Thereafter, we used 20-week old mice, as they exhibited the most sensitive response in terms of both OVA-specific IgE and IgG1 antibody production and the ASA response. Fig. 2 shows the OVA-specific IgE titer, OVA-specific IgG1 titer and serum histamine level 12 min after antigen challenge in OVA-gavaged 20-week old BALB/c mice. The production of OVA-specific IgE (0.1 mg: 62.09 62.4, 1 mg: 75.89 93.2) and IgG1 (0.1 mg: 18089 2482.3, 1 mg: 1253.39 1689) was observed in both 0.1 and 1 mg OVA-gavaged mice. An increase in the serum histamine level after ASA (0.1 mg: 0.79 0.83 mM, 1 mg: 0.59 0.67 mM) was observed with both 0.1 and 1 mg administration. The increase in the serum histamine level after ASA appears to correlate mainly with the increase in OVA-specific IgG1 in BALB/c mice. Oral administration of OVA via drinking water to BALB/c mice produced no OVA-specific IgE or IgG1 responses (data not shown).
3.3. Examination of oral sensitization in B10A mice As shown in Fig. 3, the production of OVA-specific IgE and IgG1 was observed in 0.1 mg OVA-gavaged
Table 1 Effects of murine age on OVA-specific IgE, OVA-specific IgG1 and serum histamine levels 12 min after antigen challenge in OVA-gavaged BALB/c mice. Each value represents the mean 9 SD for five mice Anti-OVA-IgE titer 0.1 mg Age
(OVA/day, p.o.)
7-week old 20-week old 1-year old
27.4 9 29.4 62.0 962.4 6.7 92.9
Anti-OVA-IgGl titer 1 mg
0.1 mg
Histamine (mM) 1 mg
(OVA/day, p.o.) B10 75.89 93.2 50.0 9 69.3
588 9 919.5 1808 9 2482.3 53.3 95.8
0.1 mg
1 mg
(OVA/day, p.o.) 180 9 290.7 1253.3 9 1689.7 847.7 91172.7
0.2 90.09 0.7 90.83 0.04 90.02
0.06 9 0.03 0.50 9 0.67 0.18 9 0.04
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significant increase in serum histamine (1.1190.9 mM), after ASA, was observed only with 0.1 mg administration. The serum histamine increase following ASA appears to correlate mainly with the increase in OVA-specific IgG1 in ASK mice.
4. Discussion Fig. 3. Determination of OVA-specific IgE titer, OVA-specific IgG1 titer and serum histamine level 12 min after antigen challenge in OVA-gavaged 20-week old B10A mice. OVA-specific IgE and IgG1 titers and the serum histamine level were determined 12 min after antigen challenge in 0.1 or 1.0 mg OVA-gavaged mice, according to the method described in Section 2.3 and Section 2.4. Each value represents the mean 9SD for five mice. Asterisks indicate significant differences from the control group (**PB 0.01, *PB 0.05).
B10A mice. The serum titers of OVA-specific IgE (296.79 184.5) and IgG1 (4400 9 1039.2) of 0.1 mg OVA-gavaged mice were apparently higher than those (IgE: 53.39 58.6, IgG1: 240092425) of 1 mg OVAgavaged mice. Furthermore, a significant increase in serum histamine was observed after ASA with both 0.1 and 1 mg administration. The histamine level increase (3.129 2.27 mM) in 0.1 mg OVA-gavaged mice was much greater than that (0.8691.1 mM) seen in 1 mg OVA-gavaged mice. The serum histamine increase after ASA appears to correlate mainly with the increase in OVA-specific IgE in B10A mice.
3.4. Examination of oral sensitization in ASK mice Fig. 4 shows the results obtained using ASK mice. The production of OVA-specific IgE (78 9 170) and IgG1 (236492049) in serum was observed only in 0.1 mg OVA-gavaged ASK mice. The titers of OVA-specific IgE and IgG1 varied widely among the mice. A
Fig. 4. Determination of OVA-specific IgE titer, OVA-specific IgG1 titer and serum histamine level 12 min after antigen challenge in OVA-gavaged 20-week old ASK mice. OVA-specific IgE and IgG1 titers and the serum histamine level were determined 12 min after antigen challenge in 0.1 or 1.0 mg OVA-gavaged mice, according to Section 2.3 and Section 2.4. Each value represents the mean 9 SD for five mice.
We have been studying the conditions for oral feeding of food allergens to animals, without induction of tolerance, in order to investigate the allergenicity of orally ingested food proteins. Recently, Knippels et al. reported BN rats to be a suitable model for oral sensitization to food proteins [10,11]. Herein, we also confirmed that BN rats were sensitized by daily OVAgavage or via continuous ad libitum drinking of water, and showed BN rats to exhibit ASA with an increase of the concentration of serum histamine, as the immediate hypersensitivity response to antigen stimulation after oral sensitization. Therefore, we confirmed BN rats to be suitable models for assessing the allergenicity of food proteins in terms of immediate hypersensitivity. We have also examined the effect of murine age, the oral feeding dose and the oral feeding technique on sensitization in BALB/c mice. We found that 20week old mice showed the highest OVA-specific IgE and IgG1 responses among the three different age groups examined. Decreased susceptibility to tolerance with increasing age has been reported in New Zealand Brown (NZB), BALB/c and C57BL/6 mice [18,19]. In those experiments, however, the animals were more than 1 year old. There is also evidence that the B-cell and T-cell populations in 24–28-week-old mice are both more resistant to tolerance induction than those in 6–8-week-old mice [20,21]. Faria et al. described oral sensitization as being likely to occur in 20–38week-old mice using several murine strains, such as C3H/HeJ, B6D2F1 and A/J mice [22]. The present results using BALB/c mice showed 20-week-old mice are more sensitive to oral immunization than 7-weekold mice. This suggests that there might be a particular period in the mouse life time when oral tolerance is more likely to occur; that period seems to be after immunological maturation but before adulthood. We found that 0.1 mg administration of OVA by gavage induced much stronger OVA-specific IgE and IgG1 responses than 1 mg administration of OVA in all strains of the mice we examined. Also, whereas gavage administration stimulated antibody responses, the drinking water induced no detectable response. Low doses (generally B0.005 mg/g body weight) given orally have been shown to prime the animal for subsequent systemic and local immune responses [23]. In
H. Akiyama et al. / Immunology Letters 78 (2001) 1–5
addition, according to Strokes et al., oral tolerance induction is related to a gradual and continuous absorption of the antigen [24]. These results strongly suggest that oral immunization requires a low antigen dose and rapid antigen intake, whereas oral tolerance requires gradual and continuous administration of the antigen. Furthermore, we compared the OVA-specific IgE and IgG1 antibody responses, as well as the ASA response, of three strains of mice sensitized by gavage. As shown in Figs. 2–4, the IgE response of gavaged B10A mice was the highest among the three strains to which we administered 0.1 mg. In addition, the serum histamine increase after ASA appears to correlate mainly with the increase in OVA-specific IgE only in B10A mice. B10A mice have also been reported to be sensitive to the oral intake of OVA and the other antigens [13], a report which is in agreement with our results. In conclusion, we demonstrated that: (1) low doses of OVA (0.1mg OVA/day for mice and 1mg OVA/day for rats, which corresponds to about 0.004mg/g body weight) given orally have been shown to prime the animal for subsequent systemic and local immune responses; (2) monitoring the concentration of serum histamine was a good marker for quantifying the ASA reaction; and (3) BN rats and B10A mice were suitable animal models for investigating the IgE-mediated allergenicity of food proteins via oral sensitization.
Acknowledgements This work was supported in part by Grants-in-Aid for scientific research from the Ministry of Health and Welfare of Japan.
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Examination of oral sensitization with ovalbumin in Brown Norway rats and three strains of mice Hiroshi Akiyama a,*, Reiko Teshima b, Jun-ichiro Sakushima a,c, Haruyo Okunuki b, Yukihiro Goda a, Jun-ichi Sawada b, Masatake Toyoda a b
a Di6ision of Foods, National Institute of Health Sciences, 1 -18 -1, Kamiyoga, Setagaya-ku, Tokyo 158 -8501, Japan Di6ision of Biochemistry and Immunochemistry, National Institute of Health Sciences, 1 -18 -1, Kamiyoga, Setagaya-ku, Tokyo 158 -8501, Japan c Domestic Research Fellow, Japan Science and Technology Corporation, 4 -18 -8, Hommachi, Kawaguchi-shi, Saitama 332 -0012, Japan
Received 8 January 2001; received in revised form 7 May 2001; accepted 7 May 2001
Abstract We studied the conditions needed to sensitize animals to the oral feeding of food allergens, without induction of tolerance, in order to investigate the allergenicity of orally ingested food proteins. Brown Norway (BN) rats were sensitized by daily OVA (ovalbumin)-gavage or by drinking OVA containing water ad libitum and the ASA (active systemic anaphylaxis) response, as the immediate hypersensitivity response to antigen stimulation after oral sensitization, was examined. The oral administration of OVA by gavage produced a higher OVA-specific IgE response and an increase in serum histamine after antigen challenge, as compared to those produced by drinking water. Next, we examined the effect of murine age, the oral feeding technique and the oral feeding dose on sensitization using BALB/c, B10A and ASK mice. Twenty-week-old mice showed the strongest OVA-specific IgE and IgG1 responses and ASA-associated serum histamine contents increased with gavage in the three different age groups of BALB/c mice. Administering 0.1 mg of OVA by gavage daily for 9 weeks appeared to induce a higher response than administering 1 mg of OVA, in terms of OVA-specific IgE and IgG1 antibody responses and ASA responses. Among the three strains of mice, B10A mice exhibited the highest response in terms of OVA-specific IgE and IgG1 antibody and ASA responses. These findings suggested BN rats and B10A mice were suitable models for oral sensitization with antigen protein and that oral sensitization in mice requires low dose, intermittent antigen intakes. © 2001 Elsevier Science B.V. All rights reserved. Keywords: B10A mice; BALB/c mice; BN rats; Oral sensitization; Ovalbumin
1. Introduction Protein specific immunoglobulin E (IgE)-mediated allergic reactions are known to play a major role in food allergy [1]. In recent years, new proteins have been artificially produced in special foods, such as genetically modified foods. Some of the new proteins might be strongly allergenic in humans. Nevertheless, at present there are only a few methods of predicting the allergenicity of food proteins. Several attempts have been made to develop animal models for investigating the allergenicity of food proteins, mainly in the mouse, guinea-pig and rat. * Corresponding author. Tel.: + 81-03-3700-9397; fax: + 81-033707-6950. E-mail address: [email protected] (H. Akiyama).
Many of these studies have been conducted using parenteral sensitization and enteral challenges [2–6]. Oral immunization studies using animals are an important means of evaluating the allergenicity of food proteins and the mechanism of food allergy induction. However, the dosage of allergens, the method of oral administration and type of mouse strain used are thought to be important in terms of avoiding the occurrence of tolerance to orally-administered food allergens [7–9]. Recently, Knippels et al. reported that Brown Norway (BN) rats showed antigen specific-IgG, as well as IgE responses, after intragastric administration and may provide a suitable model for oral sensitization to food proteins [10,11]. It was also shown that BN rats manifest specific T-cell mediated hypersensitivity (DTH) to ovalbumin (OVA) by oral sensitization. In our experiment, we investigated whether an active systemic ana-
0165-2478/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 1 ) 0 0 2 2 9 - 2
2
H. Akiyama et al. / Immunology Letters 78 (2001) 1–5
phylaxis (ASA) response, as immediate hypersensitivity, is induced in orally immunized BN rats. The development of suitable murine models for oral sensitization is also desirable if only small amounts of food proteins are available, because the amount fed to mice is usually less than that given to rats. Sensitization of mice, leading to the induction of active anaphylaxis, not only induces an antigen-specific IgE response, but also results in the production of antigen-specific IgG1 antibodies [12]. IgE binding to FcoRI-bearing cells and IgG1 binding to FcgR-bearing cells are essential steps in active anaphylaxis in mice and mediators released by these cells appear to be responsible for the symptoms of active anaphylaxis. Thus, it is thought to be important to assess the protein-specific IgE response, as well as the IgG1 response. In the present study, we confirmed that BN rats exhibit a specific-IgE response to oral administration and examined whether or not ASA is induced in rats as an immediate hypersensitivity response. We also investigated an intragastric feeding protocol, without the use of an adjuvant, designed to induce specific IgE and IgG1 immune responses and an ASA response, as an immediate hypersensitivity reaction, in BALB/c mice, B10A mice and ASK mice, all three of which have been reported to be immunologically sensitive strains [13,14].
2. Materials and methods
2.1. Animals and reagents Female BN rats, female BALB/C mice and female B10A mice were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan), and female ASK mice from SEAC Yoshitomi (Fukuoka, Japan), at 6 weeks of age. All animals were kept in our animal facility on a CRF-1 diet for 1 week before use. OVA (grade V) was purchased from Sigma Chemical Co. (St. Louis, MO).
2.2. Immunization and induction of acti6e systemic anaphylaxis (ASA) BN rats and the three strains of mice were sensitized by administration of 0.1 or 1.0 mg OVA by gavage daily for 9 weeks, or in drinking water (5 mg/ml) daily for 9 weeks. ASA challenge was elicited by intraperitoneal (ip) injection of 1 mg of OVA 1 day later [15].
2.3. Antibody (IgE, and IgG1 isotypes) titer determination Serum titers (reciprocal of serum dilution with fluorescence intensity at 50% of the maximum level) of OVAspecific IgE and IgG1 were determined according to a previously reported method, with some modifications
[16]. A 50 ml volume of OVA (40 mg/ml) in 50 mM sodium carbonate buffer, pH 9.6, was added to each well of a 96-well microtiter plate, and incubated overnight at 4°C. The solutions were discarded, and each well was washed four times with 200 ml PBS containing 0.05% Tween 20 (PBS/Tween). To minimize the nonspecific binding of serum proteins to unoccupied solid-phase sites, 200 ml of 0.1% casein in PBS were added and incubation was continued for 1 h at room temperature. The casein solution was removed, and each well was washed in the same way as above. Fifty microliters of the diluted serum containing OVA-specific antibodies were added to each well, followed by incubation for another 20 h at 4°C. The solutions were removed, and each well was washed. Fifty microliters of rabbit anti-mouse IgE and IgG1 [10 − 3 dilution in PBS containing 0.1% casein (Nordic Immunology, Tilburg, the Netherlands)] were added to each well, and then incubated for 1 h at room temperature. The solution in each well was removed and washed. Fifty microliters of b-galactosidase-linked goat anti-rabbit Ig conjugate [10 − 3 dilution in PBS containing 0.1% casein, (Amersham, UK)] were added to each well, followed by a 1 h incubation at room temperature. The antibody–enzyme conjugate solution in each well was removed and washed. The wells were incubated for 1 h at 37°C with 100 ml PBS containing 0.1 mM 4-methylumbelliferyl-bgalactoside (Sigma). Finally, 25 ml of 1 M sodium carbonate were added to each well. The fluorescence intensity of the liberated 4-methylumbelliferone was monitored at 317 and 374 nm for excitation and emission, respectively, using a Titertek Fluoroscan reader (Flow Laboratories Inc., Costa Mesa, CA).
2.4. Determination of plasma serum histamine in ASA reaction Twelve minutes after antigen challenge, blood was collected from the eye, then left standing for 1 h at room temperature, before centrifugation to obtain the serum. Serum histamine levels were measured by the postcolumn HPLC method, as described previously [17].
2.5. Statistical analysis The statistical significance of the data was determined by Student’s t-test. A P-value of less than 0.05 was taken as significant.
3. Results
3.1. Examination of oral sensitization in BN rats As shown in Fig. 1, we confirmed that 1 mg administration of OVA by gavage or via drinking water (5mg/ml)
H. Akiyama et al. / Immunology Letters 78 (2001) 1–5
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Fig. 2. Determination of OVA-specific IgE titer, OVA-specific IgG1 titer and serum histamine level 12 min after antigen challenge in OVA-gavaged 20-week old BALB/c mice. OVA-specific IgE and IgG1 titers and the serum histamine level were determined 12 min after antigen challenge in 0.1 or 1.0 mg OVA-gavaged mice, according to the method described in Section 2.3 and Section 2.4. Each value represents the mean 9 SD for five mice. Fig. 1. Determination of OVA-specific IgE titer and serum histamine level 12 min after antigen challenge in BN rats exposed to OVA by gavage or via drinking water. OVA-specific IgE titer and the serum histamine level were determined in BN rats 12 min after antigen challenge, according to the method described in Section 2.3. Each value represents the mean 9 SD for three –seven rats. Asterisks indicate significant differences from the control group (*PB0.05).
increased serum titers of OVA-specific IgE (drinking water: 3609 169, per oval, p.o.: 4509 70) as compared with control (saline-treated) rats. The oral administration of OVA by gavage produced a stronger OVA-specific IgE response than that induced by drinking water. In addition, a significant increase in the serum histamine level (drinking water: 2679 92 mM, p.o.: 4909 194 mM) occurred after antigen challenge in both the rats sensitized by gavage and those sensitized via drinking water.
3.2. Effect of age on sensitization by oral administration in BALB/c mice As shown in Table 1, the serum titers of OVA-specific IgE and IgG1 were higher in 20-week old mice than in 7-week old mice for both 0.1 and 1 mg administration. However, OVA-specific IgE and IgG1 titers in 1-year old mice were much lower than those in the two
younger groups. The serum histamine level in ASA of 20-week old mice was higher than those seen in ASA of 7- and 1-year old mice. Thereafter, we used 20-week old mice, as they exhibited the most sensitive response in terms of both OVA-specific IgE and IgG1 antibody production and the ASA response. Fig. 2 shows the OVA-specific IgE titer, OVA-specific IgG1 titer and serum histamine level 12 min after antigen challenge in OVA-gavaged 20-week old BALB/c mice. The production of OVA-specific IgE (0.1 mg: 62.09 62.4, 1 mg: 75.89 93.2) and IgG1 (0.1 mg: 18089 2482.3, 1 mg: 1253.39 1689) was observed in both 0.1 and 1 mg OVA-gavaged mice. An increase in the serum histamine level after ASA (0.1 mg: 0.79 0.83 mM, 1 mg: 0.59 0.67 mM) was observed with both 0.1 and 1 mg administration. The increase in the serum histamine level after ASA appears to correlate mainly with the increase in OVA-specific IgG1 in BALB/c mice. Oral administration of OVA via drinking water to BALB/c mice produced no OVA-specific IgE or IgG1 responses (data not shown).
3.3. Examination of oral sensitization in B10A mice As shown in Fig. 3, the production of OVA-specific IgE and IgG1 was observed in 0.1 mg OVA-gavaged
Table 1 Effects of murine age on OVA-specific IgE, OVA-specific IgG1 and serum histamine levels 12 min after antigen challenge in OVA-gavaged BALB/c mice. Each value represents the mean 9 SD for five mice Anti-OVA-IgE titer 0.1 mg Age
(OVA/day, p.o.)
7-week old 20-week old 1-year old
27.4 9 29.4 62.0 962.4 6.7 92.9
Anti-OVA-IgGl titer 1 mg
0.1 mg
Histamine (mM) 1 mg
(OVA/day, p.o.) B10 75.89 93.2 50.0 9 69.3
588 9 919.5 1808 9 2482.3 53.3 95.8
0.1 mg
1 mg
(OVA/day, p.o.) 180 9 290.7 1253.3 9 1689.7 847.7 91172.7
0.2 90.09 0.7 90.83 0.04 90.02
0.06 9 0.03 0.50 9 0.67 0.18 9 0.04
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significant increase in serum histamine (1.1190.9 mM), after ASA, was observed only with 0.1 mg administration. The serum histamine increase following ASA appears to correlate mainly with the increase in OVA-specific IgG1 in ASK mice.
4. Discussion Fig. 3. Determination of OVA-specific IgE titer, OVA-specific IgG1 titer and serum histamine level 12 min after antigen challenge in OVA-gavaged 20-week old B10A mice. OVA-specific IgE and IgG1 titers and the serum histamine level were determined 12 min after antigen challenge in 0.1 or 1.0 mg OVA-gavaged mice, according to the method described in Section 2.3 and Section 2.4. Each value represents the mean 9SD for five mice. Asterisks indicate significant differences from the control group (**PB 0.01, *PB 0.05).
B10A mice. The serum titers of OVA-specific IgE (296.79 184.5) and IgG1 (4400 9 1039.2) of 0.1 mg OVA-gavaged mice were apparently higher than those (IgE: 53.39 58.6, IgG1: 240092425) of 1 mg OVAgavaged mice. Furthermore, a significant increase in serum histamine was observed after ASA with both 0.1 and 1 mg administration. The histamine level increase (3.129 2.27 mM) in 0.1 mg OVA-gavaged mice was much greater than that (0.8691.1 mM) seen in 1 mg OVA-gavaged mice. The serum histamine increase after ASA appears to correlate mainly with the increase in OVA-specific IgE in B10A mice.
3.4. Examination of oral sensitization in ASK mice Fig. 4 shows the results obtained using ASK mice. The production of OVA-specific IgE (78 9 170) and IgG1 (236492049) in serum was observed only in 0.1 mg OVA-gavaged ASK mice. The titers of OVA-specific IgE and IgG1 varied widely among the mice. A
Fig. 4. Determination of OVA-specific IgE titer, OVA-specific IgG1 titer and serum histamine level 12 min after antigen challenge in OVA-gavaged 20-week old ASK mice. OVA-specific IgE and IgG1 titers and the serum histamine level were determined 12 min after antigen challenge in 0.1 or 1.0 mg OVA-gavaged mice, according to Section 2.3 and Section 2.4. Each value represents the mean 9 SD for five mice.
We have been studying the conditions for oral feeding of food allergens to animals, without induction of tolerance, in order to investigate the allergenicity of orally ingested food proteins. Recently, Knippels et al. reported BN rats to be a suitable model for oral sensitization to food proteins [10,11]. Herein, we also confirmed that BN rats were sensitized by daily OVAgavage or via continuous ad libitum drinking of water, and showed BN rats to exhibit ASA with an increase of the concentration of serum histamine, as the immediate hypersensitivity response to antigen stimulation after oral sensitization. Therefore, we confirmed BN rats to be suitable models for assessing the allergenicity of food proteins in terms of immediate hypersensitivity. We have also examined the effect of murine age, the oral feeding dose and the oral feeding technique on sensitization in BALB/c mice. We found that 20week old mice showed the highest OVA-specific IgE and IgG1 responses among the three different age groups examined. Decreased susceptibility to tolerance with increasing age has been reported in New Zealand Brown (NZB), BALB/c and C57BL/6 mice [18,19]. In those experiments, however, the animals were more than 1 year old. There is also evidence that the B-cell and T-cell populations in 24–28-week-old mice are both more resistant to tolerance induction than those in 6–8-week-old mice [20,21]. Faria et al. described oral sensitization as being likely to occur in 20–38week-old mice using several murine strains, such as C3H/HeJ, B6D2F1 and A/J mice [22]. The present results using BALB/c mice showed 20-week-old mice are more sensitive to oral immunization than 7-weekold mice. This suggests that there might be a particular period in the mouse life time when oral tolerance is more likely to occur; that period seems to be after immunological maturation but before adulthood. We found that 0.1 mg administration of OVA by gavage induced much stronger OVA-specific IgE and IgG1 responses than 1 mg administration of OVA in all strains of the mice we examined. Also, whereas gavage administration stimulated antibody responses, the drinking water induced no detectable response. Low doses (generally B0.005 mg/g body weight) given orally have been shown to prime the animal for subsequent systemic and local immune responses [23]. In
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addition, according to Strokes et al., oral tolerance induction is related to a gradual and continuous absorption of the antigen [24]. These results strongly suggest that oral immunization requires a low antigen dose and rapid antigen intake, whereas oral tolerance requires gradual and continuous administration of the antigen. Furthermore, we compared the OVA-specific IgE and IgG1 antibody responses, as well as the ASA response, of three strains of mice sensitized by gavage. As shown in Figs. 2–4, the IgE response of gavaged B10A mice was the highest among the three strains to which we administered 0.1 mg. In addition, the serum histamine increase after ASA appears to correlate mainly with the increase in OVA-specific IgE only in B10A mice. B10A mice have also been reported to be sensitive to the oral intake of OVA and the other antigens [13], a report which is in agreement with our results. In conclusion, we demonstrated that: (1) low doses of OVA (0.1mg OVA/day for mice and 1mg OVA/day for rats, which corresponds to about 0.004mg/g body weight) given orally have been shown to prime the animal for subsequent systemic and local immune responses; (2) monitoring the concentration of serum histamine was a good marker for quantifying the ASA reaction; and (3) BN rats and B10A mice were suitable animal models for investigating the IgE-mediated allergenicity of food proteins via oral sensitization.
Acknowledgements This work was supported in part by Grants-in-Aid for scientific research from the Ministry of Health and Welfare of Japan.
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