Comparison of Sensitization to Crude and Purified House Dust Mite Allergens in Inbred Mice

Masaaki Yasue,1* Toyokazu Yokota,1 Matsunobu Suko,2 Hirokazu Okudaira,2 and Yasushi Okumura1

Abstract ͉ To establish a murine model for house dust mite allergy to purified mite allergens, we studied the immune response to two major mite allergens, native Dermatophagoides farinae 1 (nDer f 1) and recombinant Der f 2 (rDer f 2), and crude mite extract in four mouse strains, A/J, BALB/c, C57BL/6, and C3H/He. Mice were immunized with mite extract, nDer f 1 or rDer f 2, three times at 2-week intervals. Then mice were examined to determine status of sensitization to the antigen. Anti-mite extract IgE production was induced in all strains, and plasma IgE concentration did not differ much among the four strains. In contrast, IgE response to nDer f 1 and rDer f 2 indicated an intra-strain difference. The A/J mice had high responses to both antigens, whereas BALB/c did not respond to rDer f 2. The C57BL/6 and C3H/He mice had moderate to low IgE responses to nDer f 1 and rDer f 2. Immediate airway constriction was provoked by inhalation of mite extract or rDer f 2 in sensitized mice, and the degree of the immediate response was almost proportional to antigen-specific IgE concentration. We concluded that immunization of inbred mice with nDer f 1 and rDer f 2 achieved sensitization to mite allergens. Among the four strains, A/J mice with H-2a haplotype were the highest responder to mite allergens.

In many countries, the house dust mite is an important Materials and Methods source of bronchial asthma and allergic rhinitis (1). Much Antigen: Recombinant Der f 2, clone 1 (9), was kindly of the new knowledge concerning the inflammatory pro- supplied by the Nikka Whisky Distilling Co., Ltd. (Chiba, cess and medical treatment of house dust mite allergy, as Japan). Crude body extract from house dust mite, D. farinae well as biochemical and immunologic properties of mite al- (mite extract), and native Der f 1 purified from mite culture lergens, has been obtained from animal studies of mice and extract were kindly supplied by Torii & Co., Ltd. (Chiba, guinea pigs that were immunized with crude body extract Japan). This mite extract contained about 0.3% (wt/wt) na- from house dust mites, Dermatophagoides farinae (Der f) tive Der f 1 and 0.3% (wt/wt) native Der f 2, as determined and D. pteronyssinus (Der p) (2–6). To date, several clini- by enzyme-linked immunosorbent assay. Allergens were dis- cally important allergens, such as Der f 1, Der p 1, Der f 2, solved in phosphate-buffered saline (PBS) before use. and Der p 2, have been identified and purified from crude Animals: Specific-pathogen-free male inbred mice and mite extract (7, 8). Genes for several mite allergens have Sprague Dawley (SD) rats were purchased from Japan SLC, been cloned, and a number of the products of the cloned Inc. (Shizuoka, Japan). We used four mouse strains, A/J genes expressed in microorganisms, including recombinant (H-2a), BALB/c (H-2d), C57BL/6 (H-2b), and C3H/He (H-2k). Der f 2, have been shown to have allergic activities compa- Animals were housed in an air-conditioned room main- rable to native allergens purified from mite extract (9–11). tained at 24 Ϯ 2ЊC and 55 Ϯ 5% relative humidity, with Purified and recombinant mite allergens have recently been controlled illumination (lights on from 0600 to 1800 h). A marketed as reagents; thus, these allergens can be substi- commercial diet and potable water were available ad libi- tuted for crude mite extract in immunologic research. tum. All mice were housed without any treatments for at The aim of the study reported here was to establish a mu- least 1 week after arrival at our laboratory. rine model of house dust mite allergy by using purified mite The animal maintenance and all experiments were con- allergen. The IgE antibody response and allergic reaction to ducted in accordance with guidelines instituted by the Japa- mite allergens were compared in several mouse strains. Na- nese government and Japanese Society of Laboratory Ani- tive Der f 1 (nDer f 1), recombinant Der f 2 (rDer f 2), and mals (12–14). crude mite extracts were used as sensitizing and provok- Immunization: Groups of five 7-week-old mice were ing antigens. sensitized by intraperitoneal injection of 4 mg of alumi- num hydroxide (alum) and 10 ␮g of either mite extract, Bioscience Research and Development Laboratory, Asahi Breweries Ltd., nDer f 1, or rDer f 2 three times at intervals of 2 weeks. Ibaraki, Japan,1 and Department of Medicine and Physical Therapy, Fac- ulty of Medicine, University of Tokyo, Tokyo, Japan2 Several mice not immunized (“naive mice”) were used as *Address correspondence to Masaaki Yasu, Bioscience Research and De- negative controls. The experimental schedule is shown velopment Laboratory, Asahi Breweries Ltd., 1-21, Midori 1-chome, Moriya- in Figure 1. cho, Kitasoma-gun, Ibaraki 302-0106, Japan.

346 Anti-Mite IgE Production in Inbred Strains of Mice

Measurement of antibody titers

i.p. injection of 10 ␮g of antigen with 4.5 mg of alum Induction of active paw anaphylaxis Determination of immediate airway constriction

Induction of late-phase airway inflammation

Figure 1. Experimental schedule. Groups of five mice were sensitized with either crude mite extract, native or recombinant Dermatophagoides farinae (nDer f 1 or rDer f 2, respectively). Finally, mice were examined for immediate bronchoconstriction Figure 2. Scheme of the Konzett-Rössler method. Mite antigen or late-phase airway inflammation provoked by sensitizing mite was aerosolized, using an ultrasonic nebulizer, to induce immedi- allergen. ate bronchoconstriction in mice that inhaled the antigen.

Determination of plasma antibody titers: On the provocation tests, using sensitizing antigen, were carried day before the first, second, and third (last) immunizations, out 7 days after the foot pad anaphylaxis assay by the and 2 weeks after the last immunization, heparinized blood Konzett-Rössler method. This assay is used to determine was obtained from the retroorbital plexus of individual mice airway constriction provoked by sensitizing antigen or under anesthesia with ethyl ether (Figure 1). Plasma from chemical mediators in immunologic and pharmacologic five mice was pooled and frozen until determination of an- studies with small experimental animals (17, 18). This tibody titer. The IgE concentration specific for sensitizing method measures ventilation overflow (VO, volume of air antigen was determined by mouse-rat heterogeneous 72-h that cannot enter an animal’s lung when the respirator passive cutaneous anaphylaxis (PCA), using SD rats at inflates the lung [Figure 2]), which is known to accurately 8 weeks of age as recipients; this is a standard method to reflect severity of airway constriction (19, 20). Mice were determine antigen-specific IgE concentration in murine anesthetized by intraperitoneal injection of pentobarbital plasma (15). Briefly, serial dilutions of plasma (1:10 to sodium (50 mg/kg) and were immobilized by i.v. injection 1:3,000) were intracutaneously injected into the back of re- of pancuronium bromide (0.5 mg/kg). Tracheal cannulation cipient rats under anesthesia with ethyl ether. After 72 h, was performed by use of an 18-gauge needle connected to Evans Blue and 0.1 mg of provoking antigen were admin- an animal respirator. The lung was inflated with a fixed istered intravenously (i.v.) to the rats to provoke a PCA volume of air under 5 cm H2O pressure at a rate of 60 reaction. The maximal dilution at which a blue spot >5 mm breaths/min, and VO was continuously measured by use of in diameter was induced was determined as the PCA titer, a pneumotachograph. The ventilation volume (7 to 10 ml/ an index of antigen-specific IgE concentration. kg of body weight) was adjusted so that VO was about 3 Active foot pad anaphylaxis: Three weeks after the ml/min. A 60-W glow lamp was placed over anesthetized last immunization, immediate skin reaction in the foot pad mice to prevent decrease in body temperature during mea- was examined by use of the previously described method, surement of bronchoconstriction. with some modifications (16). Under light anesthesia with When VO was stabilized, challenge with sensitizing an- ethyl ether, thickness of each foot pad was measured by tigen, either mite extract or rDer f 2, was started. An aero- use of a dial thickness gauge (Ozaki Seisakusyo Co., To- sol mist of 10 mg of antigen solution/ml was generated by kyo, Japan) as “base thickness,” then 10 ␮l of 0.01, 0.1, 1, an ultrasonic nebulizer and was inhaled by the mice for 8 and 10 ␮g of sensitizing antigen/ml (mite extract, nDer f 1, min. Ventilation overflow was recorded for at least 15 min or rDer f 2) solutions were sequentially injected intrader- from initiation of antigen inhalation. Delta VO, the differ- mally into the right foot pad of mice at intervals of 60 min. ence between the VO value at each time point and the Ten microliters of saline was injected into the left foot pad base value before antigen challenge, was used as an in- four times as a negative control. Fifteen to 20 min after dex of the degree of bronchoconstriction. When ∆VO ex- each injection, the thickness of each foot pad was re-mea- ceeded 0.2 ml/min, the immediate airway reaction was con- sured. A food pad reaction was considered to be positive sidered to be positive. Because of difficulty in preparing a when thickness after injection of the provocating solution large amount of nDer f 1, inhalation challenge with nDer f exceeded 1.20-fold of the base thickness value. The mini- 1 was not performed. mal concentration of antigen at which a positive reaction Induction of late-phase airway inflammation: An was provoked was determined. inhalation challenge with sensitizing antigen followed by Determination of airway constriction: Bronchial bronchial alveolar lavage (BAL) was carried out in an in-

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A A

Figure 4. Immediate bronchoconstriction provoked by inhalation of sensitizing antigen in sensitized A/J (Ⅵ), BALB/c (□), C57BL/6 (ⅷ), and C3H/He (ࠗ) mice. (A) Mite extract-sensitized mice were challenged with mite extract, and (B) rDer f 2-sensitized mice were challenged with rDer f 2.

Figure 3. Induction of antigen-specific IgE production in A/J ( Ⅵ ), Figure 5. Leukocyte count in bronchoalveolar lavage fluid (BALF) 123 BALB/c (123 ), C57BL/6 ( ), and C3H/He ( ) mice. Plasma from 24 h after inhalation of rDer f 2 and mite extract by naive (N) and five mice was pooled, and IgE titer was determined by passive sensitized (S) mice. Sensitized mice were challenged with sensi-

cutaneous anaphylaxis (PCA). (A) Anti-mite extract IgE titers in tizing antigen, either rDer f 2 or mite extract. Macrophages123 (Ⅵ), 123 mice immunized with mite extract. (B) Anti-nDer f 1 IgE titers in eosinophils ( ), neutrophils ( ), and lymphocytes (123 ). mice immunized with nDer f 1. (C) Anti-rDer f 2 IgE titers in mice immunized with rDer f 2.

348 Anti-Mite IgE Production in Inbred Strains of Mice

Table 1. Minimal provocative concentration in the IgE measurement were examined for skin re- of foot pad anaphylaxis in sensitized mice activity 2 weeks after the last immunization. Posi- Antigen concentrationa tive reactions were observed only in the foot pads of Antigenb Strain 0.01 0.1 1 10 >10c sensitized mice injected with mite allergens, and not Mite extract A/J ⅥⅥⅥ ⅥⅥ in the foot pads of naive mice injected with mite BALB/c Ⅵ ⅥⅥⅥ Ⅵ C57BL/6 ⅥⅥ ⅥⅥ Ⅵ allergens or in that of sensitized mice challenged C3H/He ⅥⅥ ⅥⅥ Ⅵ with saline. The minimal concentration of antigen nDer f 1 A/J ⅥⅥⅥⅥ Ⅵ provoking a positive reaction in the foot pad is shown BALB/c Ⅵ ⅥⅥⅥⅥ C57BL/6 ⅥⅥ ⅥⅥⅥ in Table 1. Provoking concentration reflected plasma C3H/He Ⅵ ⅥⅥⅥ Ⅵ IgE titer in all antigens. The higher the IgE titer, the rDer f 2 A/J ⅥⅥ ⅥⅥ Ⅵ lower the provoking concentration. Crude mite extract- BALB/c ⅥⅥⅥⅥⅥ C57BL/6 Ⅵ ⅥⅥⅥ Ⅵ induced reaction did not indicate interstrain difference, C3H/He ⅥⅥ ⅥⅥⅥ whereas nDer f 1- and rDer f 2-induced reactions did. Each mouse in respective antigen-strain groups is represented by “Ⅵ”. The A/J mice with high IgE production in response a Data are expressed in micrograms per milliliter. to nDer f 1 and rDer f 2 were exposed to the lowest bUsed for immunization and provocation of the reaction. cDid not react to antigen concentration <10 ␮g/ml. provoking concentration of these antigens. Foot pads of BALB/c mice, in which little anti-rDer f 2 IgE pro- dependent experiment. A group of four mice were newly duction was elicited, did not react to rDer f 2. immunized with either mite extract or rDer f 2 by use of Immediate airway constriction provoked by anti- the aforementioned method. Mice were examined for allergen inhalation: Bronchoconstrictive response to antigen gic airway inflammation 4 weeks after the last immuniza- provocation was determined 7 days after induction of foot tion. Leukocytes in the BAL fluid (BALF) were enumer- pad anaphylaxis. As indicated in Figure 4A, all mouse ated as an index of allergic airway inflammation. strains immunized with mite extract had positive reaction An aerosol mist of 10 mg of mite extract/ml or rDer f 2 after inhalation of 10 mg of mite extract/ml for 8 min. Such solution was generated by an ultrasonic nebulizer and in- reaction was not observed in naive mice challenged by mite haled by a mouse confined in a small chamber for 30 min. allergens or sensitized mice challenged with PBS. The de- After 24 h, mice were anesthetized with an excess amount gree of airway constriction was comparable among the four of pentobarbital sodium (100 mg/kg, given intraperito- mouse strains. In contrast, a strain difference was observed neally), and were euthanized by bleeding from the femoral in the reaction provoked by rDer f 2 inhalation (Figure 4B). artery to avoid the influx of shed blood into the airway The A/J and C3H/He mouse strains, which had higher IgE during the surgical process. Tracheal cannulation was per- production, reacted intensely, whereas BALB/c mice did not formed, using a PE-50 polyethylene tube; 0.7 ml of PBS respond to the rDer f 2 inhalation challenge. was injected into the lung through the tube, and the BALF Late-phase airway inflammation: Mice were newly was recovered. This procedure was repeated once more to immunized with a mite allergen to induce airway inflam- yield 1.0 to 1.2 ml of BALF from each mouse. After count- mation 6 to 48 h after inhalation of sensitizing antigen. ing total cell number in the BALF, preparations for cyto- Leukocyte counts in the BALF collected from sensitized A/J logic examination were made from 0.2-ml aliquots of the and BALB/c mice 24 h after inhalation of mite extract or BALF. The numbers of macrophages, neutrophils, eosino- rDer f 2 are shown in Figure 5. Only macrophages (1.8 to phils, and lymphocytes were determined by differential leu- 2.3 x 104 cells/ml) were found in the BALFs obtained from kocyte counting after staining, and about 300 cells/prepa- mice not subject to inhalation challenge, irrespective of ration were examined. strain and whether they were sensitized (data not shown). After mite extract inhalation challenge, severe inflamma- Results tion was induced in A/J and BALB/c mice immunized with Production of IgE in response to mite allergens: mite extract. The major infiltrating cells were neutrophils Antibody response to a certain antigen is considered to be in A/J and eosinophils in BALB/c mice. Some increase in genetically regulated in mice. Production of IgE in response the number of neutrophils was observed in naive BALB/c to mite allergens was examined in four strains of mice of mice. Inhalation of rDer f 2 provoked a weak reaction in A/J different H-2 haplotypes. Anti-mite extract IgE titers in mice sensitized with rDer f 2, but had little or no effect on mice immunized with mite extract are shown in Figure 3A. BALB/c and naive A/J mice. No reaction was observed in Although a strain difference in IgE titer was observed at 2 sensitized C57BL/6 and C3H/He mice (data not shown). to 4 weeks when second and third immunizations were car- Response to mite allergens by inbred strains of ried out, the IgE titer was 100 to 300 in all strains at 6 mice: Allergic response to mite allergens in four mouse weeks, 2 weeks after the last immunization. In contrast, strains, deduced from IgE production and allergic reaction IgE titer in mice sensitized with purified allergen were after immunization, is summarized in Table 2. The sensiti- widely distributed even at 6 weeks (Figures 3B and 3C). zation studies in the four inbred strains of mice indicated Anti-rDer f 2 IgE response in A/J mice was very high, unlike that the immune response to crude mite extract was al- that in BALB/c mice. An order (A/J > BALB/c > C57/BL6 Ϸ most comparable among H-2a, H-2d, H-2b, and H-2k C3H/He) also appeared in the anti-nDer f 1 IgE response. haplotypes. The A/J (H-2a) and BALB/c (H-2d) mice are high Active foot pad anaphylaxis: The sensitized mice used and very low responders to rDer f 2, respectively. We found

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Table 2. Summary of allergic response to mite inbred mice strains is little understood. In the study re- allergens in four mice strains ported here, to establish murine allergic models by using Mite allergens purified mite allergens, we compared IgE production and Mouse allergic reaction to nDer f 1, rDer f 2, and crude mite ex- strain H-2 Crude extract nDer f 1 rDer f 2 tract among four inbred strains of mice. A/J a High High High BALB/c d High Medium Low Although the immune response to a foreign antigen is ge- C57BL/6 b Medium Low Medium netically regulated and differs considerably from strain to C3H/He k Medium Low Medium strain with many antigens (34), immunization of mice with crude mite extract induced sufficient IgE production in all later that the CBA/N strain, having the same H-2 haplotype strains and the response did not differ much among strains, as C3H/He (H-2k), was highly responsive to rDer f 2, and as reported previously (35). Foot pad anaphylaxis, immedi- anti-rDer f 2 PCA titer increased to 1:1,000 after immuniza- ate bronchoconstriction, and airway inflammation provoked tion (data not shown). Because PCA titer in C3H/He mice by mite inhalation also were comparable among strains. In also reached 1:300 after immunization, H-2k haplotype was contrast, IgE response to nDer f 1 and rDer f 2 indicated a considered to be a high responder, similar to H-2a. Response clear interstrain difference. The lack of interstrain differ- to nDer f 1 was high in A/J (H-2a) mice but was medium to ence in the response to mite extract might come from the low in C57BL/6 (H-2b)and C3H/He (H-2k) mice. fact that mite extract contains many antigens. All mice used in this study were subjected to necropsy. The A/J (H-2a), a minor strain in immunologic research, Mice examined for immediate bronchoconstriction under compared with the other three strains, responded strongly anesthetization with pentobarbital sodium were euthanized to nDer f 1 and rDer f 2. The BALB/c strain is known to by bleeding from the femoral artery, as were mice subjected respond well to many antigens and is used most frequently to BAL. In some mice, plasma anti-Sendai virus (HVJ) and in production of monoclonal antibodies. However, we could anti-Mycoplasma pulmonis IgG titers also were measured. not induce anti-rDer f 2 IgE production in the strain. It has Throughout the studies, no evidence of pulmonary infection been reported that mice with H-2b haplotype are high re- or parasitic infection, which could modulate immune responders to Der p 1 and Der p 2, which are counterparts of sponses and airway reactivity, was found (data not shown). Der f 1 and Der f 2, as determined by use of the lymphocyte proliferation assay in animals immunized with the allergen Discussion and Freund’s adjuvant (36, 37). This was not the case in our The house dust mite is one of the most important caus- study. Concerning IgE production, responses to nDer f 1 and ative allergens of bronchial asthma and allergic rhinitis rDer f 2 in C57BL/6 (H-2b) mice sensitized with alum adju- (21, 22). Prevalence of positive skin reactions to mite ex- vant were not intense. Several studies have indicated that tract is 50 to 80% in asthmatics (23). Asthma mortality the kind of adjuvant (alum, Freund’s adjuvant, or other) and has increased worldwide, chiefly in the age ranges of 5 to dosage of antigen could influence the immune response in 34 and >50 years (24, 25). Any approach to the experimen- mice (38–40); thus, immunization experiments with several tal study of house dust mite allergy to improve medical dosages of mite allergens (0.01 to 100 ␮g/head) or an adju- treatment requires a suitable animal model, and two mod- vant other than alum remain to be performed. els with severe eosinophil infiltration of the airway have Foot pad anaphylaxis, immediate airway constriction, and recently been established, using mice and guinea pigs with airway inflammation after inhalation of rDer f 2 reflected crude mite extract as sensitizing allergen (5, 6). However, the plasma anti-rDer f 2 titers, and A/J mice were the high- because mite extract contains many allergens at various est responders. The results indicated that A/J is a suitable concentrations (26, 27), it is difficult to standardize. Ex- strain for developing a murine model of mite allergy, using perimental reproducibility may be lost when allergen is purified mite allergen. The degree of the allergic reaction obtained from different suppliers. In addition, mite extract in airway to rDer f 2 was somewhat weaker than that to contains antigenic proteins and substances that can cause mite extract. An inflammatory substance in the extract nonallergic inflammation by activating complement or might promote the reaction in the airway tissue. A more platelet-activating factor (28–30). This property can make intense reaction in A/J and the other strains would be ob- analysis of experimental results difficult by masking aller- tained by local immunization, such as intranasal injection gic reactions with nonallergic inflammatory reactions, of antigen, or boostering with killed Bordetella pertussis, which we observed in naive BALB/c mice inhaling mite known to induce IgE synthesis and organ sensitivity to for- extract (Figure 5). Since Der p 1 was purified from a house eign antigens (41–43). dust mite, D. pteronyssinus, in 1980 (7), several major al- Although mice have not been used frequently in studies lergens have been purified, and their genes have been of allergic reactions in the airway caused by soluble anti- cloned from Dermatophagoides species (11). Purified mite gens, compared with guinea pigs and rats, the immuno- allergens have been used for production of monoclonal an- logic background of mice has been well characterized, and tibodies and determination of epitopes for T-cell recogni- many antibodies with which to investigate the immune tion and IgE-binding in laboratories (30–32). Despite sev- function of mouse cells are available. This is not the case eral interstrain comparisons of immune response to puri- in rats and guinea pigs. Our allergic model of mice with fied mite allergens in mice (7, 33), inducibility of IgE pro- bronchoconstriction after inhalation of rDer f 2 is expected duction or immediate allergic reaction with the allergen in to enable studies of the relationships between allergic re-

350 Anti-Mite IgE Production in Inbred Strains of Mice

actions in the airway and leukocyte function in vitro, such 11. Thomas, W. R., W. Smith, B. J. Hales, et al. 1997. Func- as expression of cytokines and cell-surface molecules. Use tional effects of polymorphisms of house dust mite allergens. Int. Arch. Allergy Immunol. 113:96–98. of purified mite allergens, rDer f 2 and nDer f 1, in immu- 12. Law No. 105: Law Concerning the Protection and Control of nologic studies of mice sensitized to these allergens will Animals. October 1, 1973. eliminate the problems associated with use of crude mite 13. Notification No. 6 of the Prime Minister’s Office: Standard extract (contamination of inflammatory substances and Relating to the Care and Management etc. of Experimental variation in the contents of respective antigens) and will Animals. March 27, 1980. 14. Japanese Society of Laboratory Animals. Guidelines for Ani- provide experimental reproducibility. In addition, availabil- mal Experiments. 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