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Induction of active systemic anaphylaxis by oral sensitization with ovalbumin in mast-cell-deficient mice
Immunology Letters 74 (2000) 233 – 237
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Induction of active systemic anaphylaxis by oral sensitization with ovalbumin in mast-cell-deficient mice Haruyo Okunuki a, Reiko Teshima a,*, Jun-ichiro Sakushima b, Hiroshi Akiyama b, Yukihiro Goda b, Masatake Toyoda b, Jun-ichi Sawada a a
Di6ision of Biochemistry and Immunochemistry, National Institute of Health Sciences, 1 -18 -1, Kamiyoga, Setagaya-ku, Tokyo 158 -8501, Japan b Di6ision of Food, National Institute of Health Sciences, 1 -18 -1, Kamiyoga, Setagaya-ku, Tokyo 158 -8501, Japan Received 12 June 2000; accepted 9 July 2000
Abstract Mast-cell-deficient W/Wv mice were sensitized by oral administration of 0.1 and 1.0 mg ovalbumin (OVA) by gavage every day for 9 weeks, and active systemic anaphylaxis (ASA) was induced by intraperitoneal injection of OVA. The production of OVA-specific IgE and IgG1 by oral immunization of the W/Wv mice was high, and the production of IL-4 by splenocytes re-stimulated with OVA in vitro was increased. In contrast, production of OVA-specific IgG2a and IgG2b was low, and production of IFN-g by splenocytes after re-stimulation with OVA in vitro was rather decreased. These findings suggest that Th2-dominant helper T-cell activation had occurred. No increase in serum histamine level was observed following ASA induction. However, the plasma platelet-activating factor (PAF) levels of the mice sensitized with 0.1 and 1.0 mg OVA by gavage increased significantly. The increases in plasma PAF correlated well with the ASA-associated decreases in body temperature, suggesting that PAF plays an important role in ASA in W/Wv mice. Taken together the above findings indicate that W/Wv mice are a good model not only for studying induction of food allergy but also for examining the role of PAF in food-induced hypersensitivity. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Ovalbumin; Oral-immunization; W/Wv mice; PAF; ASA
1. Introduction The oral immunization studies in mice are an important means of evaluating the allergenicity of food and the mechanism of induction of food allergy. However, determination of the dosage of allergens and the duration of immunization are important to avoid the occurrence of tolerance to orally-administered food allergens [1 – 3]. Recently, Knippels et al. [4,5] reported that Brown Norway (BN) rats may provide a suitable model for oral sensitization to food proteins. We have been studying the conditions for oral feeding of food allergens to mice without induction of tolerance. In this paper, we report an intra-gastric feeding protocol, without the use of an adjuvant, for inducing specific humoral (IgG and IgE) immune responses in mast-cell* Corresponding author. Tel.: + 81-3-37009437; fax: +81-337076950. E-mail address: [email protected] (R. Teshima).
deficient (W/Wv) mice [6,10] by using the well-defined chicken-egg-white allergen ovalbumin (OVA) as a model antigen. IgE-dependent mast-cell activation has long been regarded as one of the essential steps in the development of the broncho-constriction and other physiological changes associated with active anaphylaxis [8]. Sensitization of mice to induce active anaphylaxis not only induces an antigen-specific IgE response but also results in the production of antigen-specific IgG1 antibodies as well [9]. 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 seem to cause the symptoms of active anaphylaxis. It has been reported that anaphylactic reactions can be induced in normal and mast-cell-deficient mice by parenteral sensitization and intraperitoneal challenge with specific antigens, such as OVA [6,7], and the importance of antigen-specific IgG1 production and its
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binding to FcgRIII on the cells has been discussed [9]. However, there have been no reports on oral sensitization to food proteins in such mast-cell-deficient mice. In this study, we used mast-cell-deficient W/Wv mice to investigate the possibility of oral sensitization to OVA and to identify the role of cells other than mastcells and the mediators involved in the active anaphylaxis induced in orally-immunized mice.
2. Materials and methods
2.1. Animals and reagents Female mast-cell-deficient WBB6F1-W/Wv(W/Wv) mice were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan) and kept in our animal facility for at least 1 week before use. All mice were used at 8 weeks of age. OVA (grade V) was purchased from Sigma Chemical Co. (St Louis, MO).
2.2. Immunization and induction of acti6e systemic anaphylaxis (ASA) Mice were sensitized by administration of 0.1 and 1.0 mg OVA by gavage every day for 9 weeks. ASA challenge was elicited by intraperitoneal injection of 1 mg of OVA 1 day later.
rabbit Ig conjugate (10 − 3 dilution in PBS containing 0.1% casein, Amersham, UK) were added to each well, and the plates were incubated for 1 h 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-b-galactoside (Sigma). Finally, 25 ml of 1 M sodium carbonate was added to each well. The fluorescence intensity of the liberated 4-methylumbelliferone was monitored at 317 and 374 nm for excitation and emission, respectively, by a Titertek Fluoroscan reader (Flow Laboratories Inc., Costa Mesa, CA).
2.4. Measurement of IL-4 and INF-g production by splenocytes Spleen cells were collected from the OVA-immunized mice (four mice per group), and the cells (5× 106 cells per ml) were re-stimulated with OVA in vitro at a final concentration of 100 mg/ml in a 24-well culture plate at 37°C for 3 days [11]. The levels of IL-4 and INF-g in the culture medium (RPMI 1640) after 3 days of coculture with OVA were measured with an ELISA kit (Pharmingen Co. Ltd., San Diego, CA). Absorbance was measured at 450 nm with a microplate reader (EL 340, Bio-Tek Instruments, Winooski, VT).
2.5. Determination of plasma PAF and serum histamine 2.3. Antibody (IgE, IgA, and IgG isotype) titer determination Serum titers of OVA-specific IgE, IgG1, IgG2a, IgG2b, and IgA were determined according to the previous method, with some modifications [10]. 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 the plate was 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 was added and the plates were incubated 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 and the plates were incubated for 20 h at 4°C. The solutions were removed, and each well was washed. Fifty microliters of rabbit anti-mouse IgE, IgG1, IgG2a, IgG2b, and IgA (10 − 3 dilution in PBS containing 0.1% casein, Nordic Immunology, Tilburg, The Netherlands) were added to each well, and the plates were incubated for 1 h at room temperature. The solution in each well was removed and washed. Fifty microliters of b-galactosidase-linked goat anti-
Post-challenge blood collected from the eye was mixed with a 0.1 volume of 3.8% ice-chilled citrate solution (pH 4.0) and centrifuged immediately with an Eppendorf microfuge. Plasma PAF was measured by the method described by Choi et al. [12]. In brief, 50 ml of plasma was vortexed with 3 volume of 2-propanol and centrifuged, and lipid was extracted from the samples with 2-propanol twice. The supernatants were applied to a reverse phase, octadecyl column (100 mg; Amprep minicolumn; Amersham, UK), and the column was washed with 2 ml of 30% 2-propanol solution, followed by 2 ml of 55% ethanol solution. PAF was eluted with 3 ml of 67% ethanol solution and extracted by the method of Bligh and Dyer [13]. The extract was evaporated under nitrogen, and PAF was quantified with a PAF[3H]SPA kit (Amersham, UK). The results are expressed as concentrations obtained from a standard curve of known PAF concentrations. Serum histamine levels were assayed by the post-column high performance liquid chromatography (HPLC) method described previously [14].
2.6. Body temperature measurement The body temperature changes associated with ASA were monitored with a rectal thermometer for mice
H. Okunuki et al. / Immunology Letters 74 (2000) 233–237
235
Table 1 Summary of body weight and spleen and thymus weight of W/W mice after OVA treatmenta mg 9 S.D.
g9 S.D.
OVA per day p.o. (0.1 mg) OVA per day p.o. (1 mg) Control a
Body weight
Liver
Spleen
Thymus
24.189 2.836 23.239 1.316 22.339 0.894
1.042 90.106 1.005 90.020 0.943 90.144
76.7590.015 92.00 90.023 79.75 90.022
29.00 90.006 26.75 90.003 23.50 90.005
n= 7.
(Shibaura Electronics Co. Ltd., Japan) without general anesthesia.
3. Results
3.1. Body and organ weight and OVA-specific antibody (IgE, IgA and IgG isotype) titers after OVA ga6age
3.3. Determination of plasma PAF and histamine concentrations As shown in Fig. 2, the concentration of plasma PAF was significantly higher in the 0.1 and 1.0-mg OVA groups, reaching 24.4 and 27.1 ng/ml, respectively, compared with 11.7 ng/ml in the non-immunized mice,
As shown in Table 1, the body weight and the immune organ (thymus, spleen and liver) weights of W/Wv mice after 9-week gavage of 0.1 or 1.0 mg OVA per day were not significantly different from the value in the saline-gavaged controls, and thus there seem to have been no nutritional differences between the saline and OVA-gavaged mice. To determine the level of antibody production in response to OVA gavage, the serum titers (the reciprocal of a serum dilution whose fluorescence intensity was 50% of the maximum level) of OVA-specific IgE, IgA and IgG isotype were determined by an indirect ELISA. Significant OVA-specific IgE and IgA production was observed in the sera of the 0.1 and 1.0 mg OVA-gavaged mice (titers were 70 – 400), and the level of production of OVA-specific IgG1 was very high (serum titers of 2.9 – 4.7× 104). By contrast, the titers of the OVA-specific IgG2a and IgG2b (150–180) were much lower than those of IgG1.
3.2. Measurement of IL-4 and INF-g production by splenocytes Fig. 1 shows the in vitro production of IL-4 and INF-g by splenocytes stimulated with OVA. As shown in Fig. 1a, a tendency for IL-4 production to increase was observed in the OVA-gavaged mice, and the increase was clearer in the 1.0-mg OVA-gavaged mice. By contrast, as shown in Fig. 1b, a tendency to decrease was observed for the production of INF-g. The results described in Sections 3.1 and 3.2 suggested that oral sensitization of W/Wv mice results in a state of Th2dominant helper T-cell activation.
Fig. 1. IL-4 and IFN-g production by splenocytes from W/Wv mice in response to re-stimulation with 100 mg/ml of OVA for 3 days was measured, as described in Section 2. Each value represents the mean 9S.D. for seven mice.
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4. Discussion
Fig. 2. Determination of plasma PAF concentrations in W/Wv mice after ASA. Plasma PAF concentrations in 0.1 or 1.0 mg-OVA-gavaged mice after ASA were determined according to the method described in Section 2. Each value represents the mean 9 S.D. for seven mice; *, indicate significant differences from the control group (*, P B 0.05; **, P B 0.01).
and, as described in the previous section (Section 3.1), a marked increase in the production of OVA-specific IgG1 in serum was observed in the 0.1 and 1.0 mg OVA-gavaged mice. Thus, the increase in plasma PAF concentration after ASA seems to be mainly dependent on the increase in OVA-specific IgG1. By contrast, no histamine could be detected in the serum of either the saline or the OVA-gavaged groups (data not shown).
3.4. Body temperature measurement As shown in Fig. 3, body temperature was measured every minute after intraperitoneal challenge with 1 mg of OVA. Gradual decreases in body temperature were observed in both the 0.1 and 1.0-mg OVA-gavaged W/Wv mice. Their body temperature decreased 2 –2.5°C within 10 min after OVA challenge. In contrast, no or only slight decreases in body temperature were observed in the control mice.
Fig. 3. Changes in body temperature after ASA. Body temperature was monitored every minute after challenge with OVA. Each value represents the mean 9S.D. for seven mice.
Ha and colleagues [6,7] reported that fatal anaphylaxis can occur in mast-cell-deficient mice upon challenge with specific antigens, such as OVA, BSA or chicken g-globulin (CGG) to a similar degree in both normal and mast-cell-deficient mice. Furthermore, recent studies have demonstrated that anaphylactic reactions can be induced in normal and mast-cell-deficient mice after passive transfer of antigen-specific IgG1Abs, and that these reactions reflect the binding of these Abs to FcgRIII, which can be expressed on the surface of mast cells as well as other cell types in the mouse [9]. Thus, it is likely that other cells that bind to IgGAbs, besides mast cells, such as macrophages, platelets and eosinophils, represent the source of the mediators in protein-induced fatal anaphylaxis. Moreover, the involvement of antigen-specific IgE in fatal anaphylaxis has been reported when W/Wv mice were sensitized with low-molecular-weight substances [12]. All of these studies were conducted by using parenteral sensitization and intraperitoneal challenge. However, there have been no reports on oral sensitization to food proteins in W/Wv mice. In the present study, we investigated oral dosing protocols for sensitizing W/Wv mice to OVA without using an adjuvant. Investigation of OVA-specific-antibody production, shown in Table 2, revealed a high level of the production of OVA-specific IgG1. We also tried to sensitize other strains of mice by oral dosing, and we observed OVA-specific IgE and IgG1 production in BALB/c, B10A and ASK mice (unpublished data). The titer of OVA-specific IgE was 60–300 and that of OVA-specific IgG1 was 2–5× 103. Comparison of the antibody titers of the W/Wv and other strains of mice revealed almost the same OVA-specific IgE titers in every strain, however, the titer of OVA-specific IgG1 of W/Wv mice was five to ten times higher than in the other strains. Assessment of in vitro cytokine production by splenocytes showed an increase in Th2-type cytokine (IL-4) production (Fig. 1). Thus, our intermittent feeding did not induce the tolerance of Th2 lymphocytes described in the report by Melamed et al. [2]. Fig. 3 shows a gradual decrease in body temperature after intraperitoneal OVA challenge in both 0.1 and 1.0-mg OVA-fed W/Wv mice and the decreases in body temperature were accompanied by the occurrence of ASA. Therefore, the ASA induced in OVA-gavage-sensitized W/Wv mice seems to be mainly dependent on high level production of antigen-specific IgG1 antibody. PAF also seems to be involved in the induction of ASA in W/Wv mice, because the level of plasma PAF, but not histamine, increased after OVA challenge in both the 0.1 and 1.0-mg OVA-fed W/Wv mice. PAF has been reported to be involved in antigen-induced bronchial
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Table 2 Serum OVA-specific antibody of W/Wv mice orally-sensitized with OVAa Group
0.1 mg per day p.o. 1 mg per day p.o. Control a
OVA-specific antibody (ELISA titer) IgE
IgG1
IgG2a
IgG2b
IgA
74.429 89.78 351.89 537.2 B50
29 179923402 47 00097937 B50
157.6 9186.2 150.0 9223.6 B50
180.0 9167.1 B50 B50
391.6 9 160.4 343.0 9193.0 B50
n= 7.
hyperreactivity in PAF-receptor overexpressing mice and cytosolic phospholipase A2 knockout mice [15,16]. PAF is produced by a variety of cells involved in inflammatory reactions, including neutrophils, basophils, mast cells, monocytes/macrophages, platelets, and endothelial cells. In W/Wv mice, FcgRbearing cells, except for mast cells, seem to be involving in the production of PAF. Since we succeeded in orally sensitizing W/Wv mice to OVA, W/Wv mice seem to be a good model for examining the effect of PAF in food-induced hypersensitivity.
Acknowledgements This work was supported in part by grants-in-aid for scientific research from the Ministry of Health and Welfare of Japan.
References [1] S. Strobel, A.M. Mowat, Immunol. Today 19 (1998) 173 – 181. [2] D. Melamed, J. Fishman-Lobell, Z. Uni, H.L. Weiner, A. Friedman, Int. Immunol. 8 (1996) 717–724.
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[3] S.J. Challaombe, T.B. Tomasi, J. Exp. Med. 152 (1980) 1459– 1472. [4] L.M.J. Knippels, A.H. Penninks, S. Spanhaak, G.F. Houben, Clin. Exp. Allergy 28 (1998) 368 – 375. [5] L.M. Knippels, A.H. Penninks, J.J. Smit, G.F. Houben, Toxicol. Appl. Pharmacol. 156 (1999) 161 – 169. [6] T.Y. Ha, N.D. Reed, Exp. Cell Biol. 55 (1987) 63 – 68. [7] T.Y. Ha, N.D. Reed, P.K. Crowle, Int. Arch. Allergy Appl. Immunol. 80 (1986) 85 – 94. [8] T. Ishizaka, K. Ishizaka, Prog. Allergy 34 (1984) 188 –235. [9] I. Miyajima, D. Dombrowicz, T.R. Martin, J.V. Ravetch, J.-P. Kinet, S.J. Galli, J. Clin. Invest. 99 (1997) 901 – 914. [10] R. Teshima, M. Kawase, T. Tanaka, K. Hirai, M. Sato, J. Sawada, H. Ikebuchi, M. Ichinoe, T. Terao, J. Agric. Food Chem. 38 (1990) 1618 – 1622. [11] H. Akiyama, K. Hoshino, M. Tokuzumi, R. Teshima, H. Mori, T. Inakuma, Y. Ishiguro, Y. Goda, J. Sawada, M. Toyoda, Biol. Pharm. Bull. 22 (1999) 551 – 555. [12] I.H. Choi, Y.M. Shin, J.S. Park, M.S. Lee, E.H. Han, O.K. Chai, S.Y. Im, T.Y. Ha, H.K. Lee, J. Exp. Med. 188 (1998) 1587 – 1592. [13] E.G. Bligh, W.J. Dyer, Can. J. Biochem. Physiol. 37 (1959) 911 – 917. [14] M. Kawasaki, M. Toyoda, R. Teshima, J. Sawada, Y. Saito, Biol. Pharm. Bull. 17 (1994) 1321 – 1325. [15] N. Uozumi, K. Kume, T. Nagase, N. Nakatani, S. Ishii, F. Tashiro, Y. Komagata, K. Maki, K. Ikuta, Y. Ouchi, J. Miyazaki, T. Shimizu, Nature 390 (1997) 618 – 622. [16] S. Ishii, T. Nagase, F. Tashiro, K. Ikuta, S. Sato, I. Waga, K. Kume, J. Miyazaki, T. Shimizu, EMBO J. 16 (1997) 133–142.
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Induction of active systemic anaphylaxis by oral sensitization with ovalbumin in mast-cell-deficient mice Haruyo Okunuki a, Reiko Teshima a,*, Jun-ichiro Sakushima b, Hiroshi Akiyama b, Yukihiro Goda b, Masatake Toyoda b, Jun-ichi Sawada a a
Di6ision of Biochemistry and Immunochemistry, National Institute of Health Sciences, 1 -18 -1, Kamiyoga, Setagaya-ku, Tokyo 158 -8501, Japan b Di6ision of Food, National Institute of Health Sciences, 1 -18 -1, Kamiyoga, Setagaya-ku, Tokyo 158 -8501, Japan Received 12 June 2000; accepted 9 July 2000
Abstract Mast-cell-deficient W/Wv mice were sensitized by oral administration of 0.1 and 1.0 mg ovalbumin (OVA) by gavage every day for 9 weeks, and active systemic anaphylaxis (ASA) was induced by intraperitoneal injection of OVA. The production of OVA-specific IgE and IgG1 by oral immunization of the W/Wv mice was high, and the production of IL-4 by splenocytes re-stimulated with OVA in vitro was increased. In contrast, production of OVA-specific IgG2a and IgG2b was low, and production of IFN-g by splenocytes after re-stimulation with OVA in vitro was rather decreased. These findings suggest that Th2-dominant helper T-cell activation had occurred. No increase in serum histamine level was observed following ASA induction. However, the plasma platelet-activating factor (PAF) levels of the mice sensitized with 0.1 and 1.0 mg OVA by gavage increased significantly. The increases in plasma PAF correlated well with the ASA-associated decreases in body temperature, suggesting that PAF plays an important role in ASA in W/Wv mice. Taken together the above findings indicate that W/Wv mice are a good model not only for studying induction of food allergy but also for examining the role of PAF in food-induced hypersensitivity. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Ovalbumin; Oral-immunization; W/Wv mice; PAF; ASA
1. Introduction The oral immunization studies in mice are an important means of evaluating the allergenicity of food and the mechanism of induction of food allergy. However, determination of the dosage of allergens and the duration of immunization are important to avoid the occurrence of tolerance to orally-administered food allergens [1 – 3]. Recently, Knippels et al. [4,5] reported that Brown Norway (BN) rats may provide a suitable model for oral sensitization to food proteins. We have been studying the conditions for oral feeding of food allergens to mice without induction of tolerance. In this paper, we report an intra-gastric feeding protocol, without the use of an adjuvant, for inducing specific humoral (IgG and IgE) immune responses in mast-cell* Corresponding author. Tel.: + 81-3-37009437; fax: +81-337076950. E-mail address: [email protected] (R. Teshima).
deficient (W/Wv) mice [6,10] by using the well-defined chicken-egg-white allergen ovalbumin (OVA) as a model antigen. IgE-dependent mast-cell activation has long been regarded as one of the essential steps in the development of the broncho-constriction and other physiological changes associated with active anaphylaxis [8]. Sensitization of mice to induce active anaphylaxis not only induces an antigen-specific IgE response but also results in the production of antigen-specific IgG1 antibodies as well [9]. 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 seem to cause the symptoms of active anaphylaxis. It has been reported that anaphylactic reactions can be induced in normal and mast-cell-deficient mice by parenteral sensitization and intraperitoneal challenge with specific antigens, such as OVA [6,7], and the importance of antigen-specific IgG1 production and its
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binding to FcgRIII on the cells has been discussed [9]. However, there have been no reports on oral sensitization to food proteins in such mast-cell-deficient mice. In this study, we used mast-cell-deficient W/Wv mice to investigate the possibility of oral sensitization to OVA and to identify the role of cells other than mastcells and the mediators involved in the active anaphylaxis induced in orally-immunized mice.
2. Materials and methods
2.1. Animals and reagents Female mast-cell-deficient WBB6F1-W/Wv(W/Wv) mice were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan) and kept in our animal facility for at least 1 week before use. All mice were used at 8 weeks of age. OVA (grade V) was purchased from Sigma Chemical Co. (St Louis, MO).
2.2. Immunization and induction of acti6e systemic anaphylaxis (ASA) Mice were sensitized by administration of 0.1 and 1.0 mg OVA by gavage every day for 9 weeks. ASA challenge was elicited by intraperitoneal injection of 1 mg of OVA 1 day later.
rabbit Ig conjugate (10 − 3 dilution in PBS containing 0.1% casein, Amersham, UK) were added to each well, and the plates were incubated for 1 h 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-b-galactoside (Sigma). Finally, 25 ml of 1 M sodium carbonate was added to each well. The fluorescence intensity of the liberated 4-methylumbelliferone was monitored at 317 and 374 nm for excitation and emission, respectively, by a Titertek Fluoroscan reader (Flow Laboratories Inc., Costa Mesa, CA).
2.4. Measurement of IL-4 and INF-g production by splenocytes Spleen cells were collected from the OVA-immunized mice (four mice per group), and the cells (5× 106 cells per ml) were re-stimulated with OVA in vitro at a final concentration of 100 mg/ml in a 24-well culture plate at 37°C for 3 days [11]. The levels of IL-4 and INF-g in the culture medium (RPMI 1640) after 3 days of coculture with OVA were measured with an ELISA kit (Pharmingen Co. Ltd., San Diego, CA). Absorbance was measured at 450 nm with a microplate reader (EL 340, Bio-Tek Instruments, Winooski, VT).
2.5. Determination of plasma PAF and serum histamine 2.3. Antibody (IgE, IgA, and IgG isotype) titer determination Serum titers of OVA-specific IgE, IgG1, IgG2a, IgG2b, and IgA were determined according to the previous method, with some modifications [10]. 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 the plate was 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 was added and the plates were incubated 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 and the plates were incubated for 20 h at 4°C. The solutions were removed, and each well was washed. Fifty microliters of rabbit anti-mouse IgE, IgG1, IgG2a, IgG2b, and IgA (10 − 3 dilution in PBS containing 0.1% casein, Nordic Immunology, Tilburg, The Netherlands) were added to each well, and the plates were incubated for 1 h at room temperature. The solution in each well was removed and washed. Fifty microliters of b-galactosidase-linked goat anti-
Post-challenge blood collected from the eye was mixed with a 0.1 volume of 3.8% ice-chilled citrate solution (pH 4.0) and centrifuged immediately with an Eppendorf microfuge. Plasma PAF was measured by the method described by Choi et al. [12]. In brief, 50 ml of plasma was vortexed with 3 volume of 2-propanol and centrifuged, and lipid was extracted from the samples with 2-propanol twice. The supernatants were applied to a reverse phase, octadecyl column (100 mg; Amprep minicolumn; Amersham, UK), and the column was washed with 2 ml of 30% 2-propanol solution, followed by 2 ml of 55% ethanol solution. PAF was eluted with 3 ml of 67% ethanol solution and extracted by the method of Bligh and Dyer [13]. The extract was evaporated under nitrogen, and PAF was quantified with a PAF[3H]SPA kit (Amersham, UK). The results are expressed as concentrations obtained from a standard curve of known PAF concentrations. Serum histamine levels were assayed by the post-column high performance liquid chromatography (HPLC) method described previously [14].
2.6. Body temperature measurement The body temperature changes associated with ASA were monitored with a rectal thermometer for mice
H. Okunuki et al. / Immunology Letters 74 (2000) 233–237
235
Table 1 Summary of body weight and spleen and thymus weight of W/W mice after OVA treatmenta mg 9 S.D.
g9 S.D.
OVA per day p.o. (0.1 mg) OVA per day p.o. (1 mg) Control a
Body weight
Liver
Spleen
Thymus
24.189 2.836 23.239 1.316 22.339 0.894
1.042 90.106 1.005 90.020 0.943 90.144
76.7590.015 92.00 90.023 79.75 90.022
29.00 90.006 26.75 90.003 23.50 90.005
n= 7.
(Shibaura Electronics Co. Ltd., Japan) without general anesthesia.
3. Results
3.1. Body and organ weight and OVA-specific antibody (IgE, IgA and IgG isotype) titers after OVA ga6age
3.3. Determination of plasma PAF and histamine concentrations As shown in Fig. 2, the concentration of plasma PAF was significantly higher in the 0.1 and 1.0-mg OVA groups, reaching 24.4 and 27.1 ng/ml, respectively, compared with 11.7 ng/ml in the non-immunized mice,
As shown in Table 1, the body weight and the immune organ (thymus, spleen and liver) weights of W/Wv mice after 9-week gavage of 0.1 or 1.0 mg OVA per day were not significantly different from the value in the saline-gavaged controls, and thus there seem to have been no nutritional differences between the saline and OVA-gavaged mice. To determine the level of antibody production in response to OVA gavage, the serum titers (the reciprocal of a serum dilution whose fluorescence intensity was 50% of the maximum level) of OVA-specific IgE, IgA and IgG isotype were determined by an indirect ELISA. Significant OVA-specific IgE and IgA production was observed in the sera of the 0.1 and 1.0 mg OVA-gavaged mice (titers were 70 – 400), and the level of production of OVA-specific IgG1 was very high (serum titers of 2.9 – 4.7× 104). By contrast, the titers of the OVA-specific IgG2a and IgG2b (150–180) were much lower than those of IgG1.
3.2. Measurement of IL-4 and INF-g production by splenocytes Fig. 1 shows the in vitro production of IL-4 and INF-g by splenocytes stimulated with OVA. As shown in Fig. 1a, a tendency for IL-4 production to increase was observed in the OVA-gavaged mice, and the increase was clearer in the 1.0-mg OVA-gavaged mice. By contrast, as shown in Fig. 1b, a tendency to decrease was observed for the production of INF-g. The results described in Sections 3.1 and 3.2 suggested that oral sensitization of W/Wv mice results in a state of Th2dominant helper T-cell activation.
Fig. 1. IL-4 and IFN-g production by splenocytes from W/Wv mice in response to re-stimulation with 100 mg/ml of OVA for 3 days was measured, as described in Section 2. Each value represents the mean 9S.D. for seven mice.
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4. Discussion
Fig. 2. Determination of plasma PAF concentrations in W/Wv mice after ASA. Plasma PAF concentrations in 0.1 or 1.0 mg-OVA-gavaged mice after ASA were determined according to the method described in Section 2. Each value represents the mean 9 S.D. for seven mice; *, indicate significant differences from the control group (*, P B 0.05; **, P B 0.01).
and, as described in the previous section (Section 3.1), a marked increase in the production of OVA-specific IgG1 in serum was observed in the 0.1 and 1.0 mg OVA-gavaged mice. Thus, the increase in plasma PAF concentration after ASA seems to be mainly dependent on the increase in OVA-specific IgG1. By contrast, no histamine could be detected in the serum of either the saline or the OVA-gavaged groups (data not shown).
3.4. Body temperature measurement As shown in Fig. 3, body temperature was measured every minute after intraperitoneal challenge with 1 mg of OVA. Gradual decreases in body temperature were observed in both the 0.1 and 1.0-mg OVA-gavaged W/Wv mice. Their body temperature decreased 2 –2.5°C within 10 min after OVA challenge. In contrast, no or only slight decreases in body temperature were observed in the control mice.
Fig. 3. Changes in body temperature after ASA. Body temperature was monitored every minute after challenge with OVA. Each value represents the mean 9S.D. for seven mice.
Ha and colleagues [6,7] reported that fatal anaphylaxis can occur in mast-cell-deficient mice upon challenge with specific antigens, such as OVA, BSA or chicken g-globulin (CGG) to a similar degree in both normal and mast-cell-deficient mice. Furthermore, recent studies have demonstrated that anaphylactic reactions can be induced in normal and mast-cell-deficient mice after passive transfer of antigen-specific IgG1Abs, and that these reactions reflect the binding of these Abs to FcgRIII, which can be expressed on the surface of mast cells as well as other cell types in the mouse [9]. Thus, it is likely that other cells that bind to IgGAbs, besides mast cells, such as macrophages, platelets and eosinophils, represent the source of the mediators in protein-induced fatal anaphylaxis. Moreover, the involvement of antigen-specific IgE in fatal anaphylaxis has been reported when W/Wv mice were sensitized with low-molecular-weight substances [12]. All of these studies were conducted by using parenteral sensitization and intraperitoneal challenge. However, there have been no reports on oral sensitization to food proteins in W/Wv mice. In the present study, we investigated oral dosing protocols for sensitizing W/Wv mice to OVA without using an adjuvant. Investigation of OVA-specific-antibody production, shown in Table 2, revealed a high level of the production of OVA-specific IgG1. We also tried to sensitize other strains of mice by oral dosing, and we observed OVA-specific IgE and IgG1 production in BALB/c, B10A and ASK mice (unpublished data). The titer of OVA-specific IgE was 60–300 and that of OVA-specific IgG1 was 2–5× 103. Comparison of the antibody titers of the W/Wv and other strains of mice revealed almost the same OVA-specific IgE titers in every strain, however, the titer of OVA-specific IgG1 of W/Wv mice was five to ten times higher than in the other strains. Assessment of in vitro cytokine production by splenocytes showed an increase in Th2-type cytokine (IL-4) production (Fig. 1). Thus, our intermittent feeding did not induce the tolerance of Th2 lymphocytes described in the report by Melamed et al. [2]. Fig. 3 shows a gradual decrease in body temperature after intraperitoneal OVA challenge in both 0.1 and 1.0-mg OVA-fed W/Wv mice and the decreases in body temperature were accompanied by the occurrence of ASA. Therefore, the ASA induced in OVA-gavage-sensitized W/Wv mice seems to be mainly dependent on high level production of antigen-specific IgG1 antibody. PAF also seems to be involved in the induction of ASA in W/Wv mice, because the level of plasma PAF, but not histamine, increased after OVA challenge in both the 0.1 and 1.0-mg OVA-fed W/Wv mice. PAF has been reported to be involved in antigen-induced bronchial
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Table 2 Serum OVA-specific antibody of W/Wv mice orally-sensitized with OVAa Group
0.1 mg per day p.o. 1 mg per day p.o. Control a
OVA-specific antibody (ELISA titer) IgE
IgG1
IgG2a
IgG2b
IgA
74.429 89.78 351.89 537.2 B50
29 179923402 47 00097937 B50
157.6 9186.2 150.0 9223.6 B50
180.0 9167.1 B50 B50
391.6 9 160.4 343.0 9193.0 B50
n= 7.
hyperreactivity in PAF-receptor overexpressing mice and cytosolic phospholipase A2 knockout mice [15,16]. PAF is produced by a variety of cells involved in inflammatory reactions, including neutrophils, basophils, mast cells, monocytes/macrophages, platelets, and endothelial cells. In W/Wv mice, FcgRbearing cells, except for mast cells, seem to be involving in the production of PAF. Since we succeeded in orally sensitizing W/Wv mice to OVA, W/Wv mice seem to be a good model for examining the effect of PAF in food-induced hypersensitivity.
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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