Establishment of antigen-specific IgE transgenic mice to study pathological and immunobiological roles of IgE in vivo

Kunie Matsuoka1, Choji Taya2, Shuichi Kubo1, Noriko Toyama-Sorimachi1, Fujiko Kitamura1, Chisei Ra1,3, Hiromichi Yonekawa2 and Hajime Karasuyama1

1 Department of Immunology and
2 Department of Laboratory Animal Science, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japan
3 Department of Immunology, Juntendo University School of Medicine, Tokyo 113, Japan

Correspondence to: H. Karasuyama, Department of Immunology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have established transgenic mice that carry the genes coding for heavy and light chains of TNP-specific IgE. They produced high titers of TNP-specific IgE (20–40 µg/ml in serum) and their mast cells were heavily loaded with IgE. The level of Fc{epsilon}RI expression on their mast cells was 6–8 times higher than that in non-transgenic littermates. The expression of low-affinity IgE receptor Fc{epsilon}RII (CD23) on splenic B cells was also 6–8 times higher in the transgenic mice. Consistent with this, substantial amounts of IgE were detected on B cells in the transgenic mice. When challenged with i.v. administration of the corresponding antigen, the transgenic mice exhibited systemic anaphylactic symptoms such as a drastic drop of body temperature and extravasation of administered dye. Biphasic (immediate and delayed) ear swelling response was also elicited in a TNP-specific manner by epicutaneous antigen challenge without any prior sensitization. Thus, IgE produced in the transgenic mice was found to be biologically active to induce both local and systemic allergic reactions in vivo upon the challenge of the corresponding antigen. Taken together, the antigen-specific IgE transgenic mice established for the first time in this study appear to provide an attractive model system to study the pathological roles of IgE in acute and chronic phases of allergic inflammation as well as their immunobiological roles in vivo. They may also be useful to develop novel therapeutic strategies for atopic disorders.

Keywords: allergen, allergy, CD23, Fc{varepsilon}RI, immediate hypersensitivity, systemic anaphylaxis, sensitization


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The overproduction of IgE is frequently observed in allergic patients in response to common environmental antigens or allergens that are otherwise innocuous, such as those present in house dust mites and pollen. It is now clear that allergen-specific IgE is directly responsible for immediate hypersensitivity reactions when cross-linked by antigen on the surface of effector cells such as mast cells and basophils (13). This can be easily demonstrated by passive sensitization of animals with antigen-specific IgE antibodies and subsequent challenge with the corresponding antigen (4). However, the immediate hypersensitivity reaction alone cannot account for the pathogenesis of many allergic diseases such as asthma and atopic dermatitis.

The late phase response occurs hours later following the immediate reaction and is characterized by infiltration of inflammatory cells such as eosinophils, generating a new wave of symptoms (5). The perpetuated late phase reaction appears to play a major role in the pathogenesis of chronic allergic inflammation. In contrast to the critical role of IgE in the immediate allergic reaction, its role in the chronic allergic reaction and inflammation has not yet been defined well (69). Passive sensitization of animals with exogenous IgE would not be appropriate for studying the role of IgE in such a long-term process of allergic reaction. Instead, it might be necessary for such studies to induce the constitutive production of antigen-specific IgE in experimental animals with or without T cell priming with antigens.

Recently, a novel mutant mouse strain NC/Nga was reported as a model of atopic dermatitis (10). When maintained under non-sterile (conventional) conditions but not under specific pathogen-free conditions, the mice spontaneously develop dermatitis which is clinically and histologically similar to human atopic dermatitis, and show high titers of serum IgE. Although this is an attractive animal model to explore a gene(s) causative of atopic dermatitis, an allergen(s) that triggers allergic reactions in these mice has not yet been identified. Therefore, in this model, allergic responses cannot be provoked by challenge with a given allergen, unlike in patients with allergic rhinitis and asthma.

In the present study, we have established antigen-specific IgE transgenic mice for the first time. They constitutively produced high titers of IgE, and showed local and systemic allergic reactions upon challenge with the corresponding antigen. They appear to provide an attractive model system to study pathological and immunobiological roles of IgE in vivo.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Construction of transgenes
cDNA fragments corresponding to variable regions of the H and L chains (VH and VL) of anti-TNP IgE were cloned from an anti-TNP IgE producing hybridoma IGEL b4 (11) (ATCC, Rockville, MD; TIB141) by RT-PCR. In brief, total RNA was prepared from IGEL b4 cells and incubated with oligo-dT15 primer and Superscript II RNaseH reverse transcriptase (Gibco/BRL, Rockville, MD) for 50 min at 42°C. Subsequently PCR was performed with 40 cycles amplification (0.5 min at 96°C, 1 min at 55°C, 1 min at 72°C) by using the heavy primer mix and the light primer mix (Pharmacia, Uppsala, Sweden) which were designed for cloning of most variable regions of H and L chains respectively. A 4.1 kbp EcoRI–EcoRI genomic DNA fragment hybridized with both the VH cDNA probe and the JH–Cµ intron probe [1.5 kbp HindIII–XbaI fragment from pVH167µ (12)] was isolated from the IGEL b4 genomic DNA library prepared with {lambda}gt10 vector (Stratagene, La Jolla, CA). The DNA sequence analysis revealed that this fragment contained a promoter, a leader exon, a rearranged VHDHJH exon and a part of the intronic enhancer of the anti-TNP Ig H chain gene. A missing part of the intronic enhancer (0.3 kbp EcoRI–XbaI fragment) as well as a 3.5 kbp XbaI–XbaI fragment of a germline C{varepsilon} gene (13) without two membrane exons were isolated from the BALB/c liver genomic DNA library based on their published sequences. Those three fragments were ligated and cloned into pBluescript II SK+ to obtain a recombinant genomic DNA coding for {varepsilon}H chain of the anti-TNP IgE, designated pTNP{varepsilon}. A 2.3 kbp HindIII–HindIII genomic DNA fragment hybridized with both the VL cDNA probe and the J{kappa}–C{kappa} intron probe [0.8 kbp SacI–SacII fragment from pVL167{kappa} (12)] was isolated from IGEL b4 genomic DNA library prepared with ZAP Express vector (Stratagene). A 1.3 kbp fragment carrying a leader exon and a rearranged V{kappa}J{kappa} gene segment of the anti-TNP Ig L chain gene was isolated by PvuII and PstI digestion of the 2.3 kbp fragment. A 5 kbp SalI–PvuII fragment containing a promoter region of a {kappa}L chain gene and a 12 kbp PstI–NotI fragment containing an intronic enhancer, C{kappa} and a 3' enhancer of a {kappa}L chain gene were isolated from pMM222 (14). Those three fragments were ligated and cloned into pBluescript II SK+ to obtain a recombinant genomic DNA coding for {kappa}L chain of the anti-TNP IgE, designated pTNP{kappa}.

Generation of transgenic mice
The anti-TNP IgE H and L chain constructs were mixed at equimolar concentration and microinjected into fertilized eggs of BALB/cCrSlc mice (Japan SLC, Hamamatsu, Japan), followed by transfer of viable eggs into the oviducts of pseudopregnant Slc:ICR mice (Japan SLC). Three founder lines of transgenic mice were established which carried both H and L chain transgenes, designated BALB/cCrSlc-TgN(TNPIgE)1602Rin, BALB/cCrSlc-TgN(TNPIgE)1603Rin and BALB/cCrSlc-TgN(TNPIgE)1649Rin. For convenience, they were named TNP-E2, TNP-E3 and TNP-E49 respectively. Mice were used for experiments at 8–16 weeks of age. The animal experiments were conducted in accordance with the Guidelines for Animal Use and Experimentation as set out by our institutions.

Northern blot analysis
Total cellular RNA was isolated from kidney, heart, liver, brain, spleen and thymus of mice by using Isogen (Nippon gene, Toyama, Japan). Poly(A)+ mRNA was further purified by Oligotex-dT30 (Daiichi Pure Chemicals, Tokyo, Japan), separated on a formaldehyde gel and transferred to nylon membranes (Hybond-N+; Amersham-Pharmacia Biotech, Little Chalfont, UK). {varepsilon}H chain transcripts were detected by hybridization with the 1.9 kb genomic BamHI–HindIII fragment containing C{varepsilon}H1 and C{varepsilon}H2 coding sequences as a probe. ß-Actin probe (Clontech, Palo Alto, CA) was used for control. The radioactive bands were visualized by the phosphoimager Fuji BAS2000 (Fuji Photo Film, Tokyo, Japan).

ELISA
Total IgE in serum was measured by ELISA as described previously (15). For measurement of TNP-specific IgE in serum, 96-well microtiter plates were coated with TNPconjugated BSA. Levels of total IgE and TNP-specific IgE in serum were calculated by comparison with mouse anti-TNP IgE standard (PharMingen, San Diego, CA).

Flow cytometric analysis
Bone marrow cells and spleen cells were isolated from mice. Cells were stained with phycoerythrin (PE)-conjugated anti-CD45R (B220/RA3-6B2) in conjunction with FITC-conjugated antibodies specific to µH chain, {varepsilon}H chain, CD23 or TCR ß chain. All the mAb were purchased from PharMingen. Stained cells were applied to the flow cytometer FACSCalibur equipped with an argon laser and calibrated using CaliBRITE beads and FACS COMP software (Becton Dickinson, Mountain View, CA). Data of cells present in the lymphocyte gate defined by light scatter were analyzed with CellQuest software (Becton Dickinson).

For the analysis of Fc{varepsilon}RI on mast cells, peritoneal cells were isolated from mice and preincubated with anti-Fc{gamma}RII/III mAb 2.4G2 and normal rat serum to block low-affinity binding and non-specific binding of IgE. Cells were then incubated at 4°C with 20 µg/ml of mouse IgE anti-TNP mAb (PharMingen) for 50 min to saturate IgE receptors expressed on the surface of cells. After washing out unbound IgE, cells were stained with FITC-conjugated anti-IgE and biotin-conjugated anti-c-kit antibodies (PharMingen) in conjunction with PE–streptavidin. Stained cells were applied to FACSCalibur and mast cells were identified as c-kit+ IgE+ cells. Under these experimental conditions, IgE binding to mast cells has been shown to reflect largely binding of IgE to Fc{varepsilon}RI (16).

Histochemistry
Ear specimens were embedded in OCT compound (Miles, Elkhart, IN) and frozen in liquid nitrogen. Frozen sections (6 µm) were fixed in acetone for 3 min. Staining of mast cells with Astra blue (Merck Led, Poole, UK) was described previously (17). For detection of IgE bound to mast cells, sections were incubated with biotin-conjugated anti-IgE mAb HMK-12 (15) for 60 min, and subsequently with streptavidin–horseradish peroxidase and its substrate TrueBlue (KPL, Gaithersburg, MD). The sections were counterstained with Contrast RED (KPL).

Analysis of systemic anaphylaxis
Mice were challenged with i.v. administration of 1 mg of TNP-conjugated or unconjugated BSA in 0.2 ml of PBS. Their rectal temperature was monitored with a rectal probe (Shibaura Electronics, Tokyo, Japan) before and at the indicated time points after the antigen challenge. To visualize the fluid extravasation in systemic anaphylaxis, 0.5% Evans blue dye (Sigma, St Louis, MO) was concomitantly injected with the antigens and photographs of the mice were taken 1h after the challenge.

Analysis of ear swelling responses
Mice were challenged epicutaneously with 10 µl of 0.1% picryl chloride (1-chloro-2,4,6-trinitrobenzene; Nakalai Tesque, Kyoto, Japan) in acetone on their right ears and with 10 µl of 0.1% oxazolone (4-ethoxymetholene-2-phenyl-2-oxazolin-5-one; Sigma) in acetone on their left ears. Ear thickness (in units of 10–2 mm) was measured with a dial thickness gauge G-1A (Ozaki, Tokyo, Japan) at the indicated time points before and after the antigen challenge.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Establishment of antigen-specific IgE transgenic mice
Recombinant genes coding for {varepsilon}H and {kappa}L chains of IgE specific for a hapten TNP were constructed as shown in Fig. 1Go(A), and co-injected into BALB/c fertilized eggs to generate transgenic mice. Transcription of the transgenes is under the control of native promoters and enhancers of Ig genes. In the construct of the {varepsilon}H chain transgene, the exons coding for the transmembrane portion of the {varepsilon}H chain were intentionally deleted so that only the secreted form of recombinant IgE would be produced in mice carrying the transgenes (Fig. 1AGo). In the construct of the {kappa}L chain transgene, not only intronic enhancer (E{kappa}) but also the 3' enhancer (E3') were included, which has been shown to regulate both the recombination and transcription of {kappa}L chain genes (14).



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Fig. 1. Constructs of transgenes coding for {varepsilon}H and {kappa}L chains of TNP-specific IgE and their expression in the transgenic mice. (A) A {varepsilon}H chain transgene construct (pTNP{varepsilon}) and a {kappa}L chain transgene construct (pTNP{kappa}) were prepared as described in Methods. Exons and enhancers are shown by closed boxes and striped ovals respectively. Arrow heads indicate the position of primers used for PCR to detect the transgenes. Restriction sites shown are: E, EcoRI; X, XbaI; S, SalI; N, NotI; H, HindIII; Pv, PvuII; Ps, PstI. Only one PvuII site among others is indicated which was used for construction of pTNP{kappa}. (B) Poly(A)+ mRNA was prepared from kidney, heart, liver, brain, spleen and thymus of the transgenic and non-transgenic mice, and subjected to Northern blot analysis with probes for constant region of {varepsilon}H chain (C{varepsilon}) and ß actin.

 
Three founder lines of transgenic mice (TNP-E2, TNP-E3 and TNP-E49) were established which carried both H and L chain transgenes detected by PCR analysis of tail DNA. Both transgenes were co-segregated in male and female F1 progeny between these transgenic founders and non-transgenic BALB/c mice, suggesting that the H and L chain transgenes are co-integrated into a single locus in an autosome. In the transgenic mice, {varepsilon}H chain transcripts were detected strongly in spleen and thymus and faintly in brain, but undetectable in kidney and liver (Fig. 1BGo). In contrast, in non-transgenic littermates, no {varepsilon}H chain transcript was detected in any of these organs.

Immunological characterization of the transgenic mice
The presence and absence of the transgenes in the progenitors of the founder mice were perfectly correlated with the IgE levels in their sera. In the transgenic mice 20–40 µg/ml of IgE was detected at 10 weeks of age, whereas <0.1 µg/ml of IgE was detected in non-transgenic littermates (Fig. 2Go). Levels of other Ig isotypes were comparable in the transgenic and non-transgenic littermates. Splenocytes from the transgenic mice secreted ~140 ng IgE/106 cells during 18 h culture in vitro, whereas IgE secretion was undetectable in culture of thymocytes from the transgenic mice or splenocytes from non-transgenic littermates. Importantly, virtually all of the serum IgE in the transgenic mice turned out to be specific for TNP, while no TNP-specificity was detected in serum of NC/Nga mice containing the comparable amount of total IgE (Fig. 2Go). Therefore, IgE produced in the transgenic mice was considered to be all derived from the transgenes.



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Fig. 2. High titers of TNP-specific IgE in serum of the transgenic mice. Serum levels of total and TNP-specific IgE were determined by ELISA in the three transgenic lines (TNP-E2, TNP-E3 and TNP-E49), non-transgenic littermates (non-Tg) and NC/Nga mice (10) at 10 weeks of age. The mean values of data from five mice in each group were shown.

 
In accord with the high titer of serum IgE in the transgenic mice, mast cells in their ear skin were loaded with IgE as detected by immunohistochemical staining (Fig. 3Go, upper left panel). In contrast, such IgE-loaded mast cells were undetectable in non-transgenic littermates (Fig. 3Go, upper right panel) even though the comparable number of mast cells stained with Astra blue existed in ear skin of transgenic and non-transgenic littermates (Fig. 3Go, lower panels). To estimate levels of Fc{varepsilon}RI expression on mast cells, peritoneal cells were stained with anti-IgE and anti-c-kit antibodies after incubation with excess amounts of monomeric IgE at 4°C to saturate their Fc{varepsilon}RI. Flow cytometric analysis revealed that amounts of IgE bound to c-kit+ mast cells were 6–8 times more in the transgenic mice than in non-transgenic littermates (Fig. 4CGo). Since the IgE binding detected under these experimental conditions has been shown to reflect the binding of IgE to Fc{varepsilon}RI (16), our result indicates that the expression of Fc{varepsilon}RI on mast cells is highly up-regulated in the transgenic mice.



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Fig. 3. Substantial amounts of IgE bound on mast cells in ear skin of the transgenic mice. Histological sections of ears from the transgenic mice (left panels) and non-transgenic littermates (right panels) were stained with either anti-IgE antibody (upper row) or astra blue (lower row). IgE was stained in dark blue (upper left panel) and mast cells were stained in violet (lower panels). Examination of sequential sections indicated that IgE+ cells were astra blue-stained mast cells (data not shown).

 


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Fig. 4. Normal B cell development in bone marrow, up-regulation of CD23 on mature B cells and up-regulation of Fc{varepsilon}RI on mast cells in the transgenic mice. (A) Bone marrow and spleen cells from the transgenic and non-transgenic littermates were stained with PE-conjugated anti-CD45R(B220) antibody in conjunction with FITC-conjugated antibody specific to either µH chain, TCR ß chain (TCRß), CD23, {varepsilon}H chain or control antibody. The two-color staining profiles of cells within lymphocyte gate are shown. (B) CD45R(B220)+ splenic B cells were gated, and their expression of CD23 and {varepsilon}H chain (thick solid lines) is shown in histograms as overlaid with control staining (thin dotted lines). (C) Peritoneal cells from the transgenic and non-transgenic littermates were stained with anti-IgE and anti-c-kit antibodies after incubation with anti-Fc{gamma}RII/III mAb 2.4G2 and excess amounts of monomeric IgE at 4°C to saturate their Fc{varepsilon}RI. c-kit+ mast cells were gated, and levels of IgE bound to their surface (thick solid lines) are shown in histograms as overlaid with control staining without anti-IgE antibody (thin dotted lines).

 
B cell development in bone marrow of the transgenic mice appeared normal as judged by flow cytometric analysis of bone marrow cells stained for CD45R (B220) and µH chain (Fig. 4AGo). The number of B and T cells in spleen of the transgenic mice was also comparable to that of the non-transgenic mice (Fig. 4AGo). Notably, the expression of the low-affinity Ig E receptor (Fc{varepsilon}RII, CD23) on splenic B cells was 6–8 times higher in the transgenic mice when compared to the non-transgenic mice (Fig. 4BGo). Furthermore, substantial amounts of IgE were detected on the surface of most splenic B cells in the transgenic mice, but little if any in the non-transgenic mice (Fig. 4BGo). The percentages of IgE+ cells and CD23+ cells in the transgenic splenic B cells were comparable. Furthermore, IgE but not CD23 detected on these B cells was easily detached from the cell surface by treating cells with lower pH on ice (data not shown), in accord with the fact that the transgenes encode the secreted but not membrane-anchored form of IgE. These results indicate that the IgE molecules detected on B cells bind to the cell surface through CD23.

Induction of systemic anaphylaxis
For the systemic challenge of the antigen, TNP-conjugated BSA (TNP–BSA) was injected i.v. to the transgenic and non-transgenic mice (Fig. 5Go). While the non-transgenic mice did not show any apparent change, the transgenic mice exhibited loss of mobility and tachycardia soon after injection. Rectal temperature of the transgenic mice dropped quickly from 38 to 35°C within 30 min after the antigen challenge and then gradually recovered thereafter (Fig. 5AGo). These symptoms of systemic anaphylaxis were not observed when unconjugated BSA was injected to the transgenic mice (Fig. 5AGo) or when TNP–BSA was injected to the non-transgenic littermates (Fig. 5BGo).



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Fig. 5. Drastic drop of body temperature in systemic anaphylaxis induced by i.v. injection of the antigen in the transgenic mice. The transgenic mice (A) and non-transgenic littermates (B) were challenged with i.v. injection of TNP-conjugated BSA (closed circle) or unconjugated BSA (open square). The kinetics of their rectal temperature after the antigen challenge is shown. Each symbol and bar shows the mean ± SD of four mice.

 
When Evans blue dye was co-injected with TNP–BSA to the transgenic and non-transgenic mice, the feet, nose and ears of the transgenic mice became bluish as compared to those of the non-transgenic littermates (Fig. 6Go). In contrast, the injection of unconjugated BSA with Evans blue did not elicit such a change (data not shown). This suggested that the challenge with the relevant antigen increased vascular permeability in the transgenic mice. Thus, the typical systemic anaphylaxis could be induced in an antigen-specific manner in the transgenic mice.



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Fig. 6. Dye extravasation during systemic anaphylaxis in the transgenic mice. A transgenic mouse (right) and a non-transgenic littermate (left) were challenged with i.v. injection of TNP-conjugated BSA together with 0.5% Evans blue dye. The photograph of the mice was taken 1 h after the challenge.

 
Induction of local allergic reaction
For the local challenge with the antigen, picryl chloride carrying a TNP group was painted on the right ears of the transgenic and non-transgenic mice. As a control, the left ears of these mice were challenged with an irrelevant antigen, ozaxolone. Biphasic skin swelling was elicited in the ears painted with picryl chloride in the transgenic mice (Fig. 7AGo). The first phase of ear swelling started within 30 min, reaching its maximal level at 1–3 h after the antigen challenge and then subsided by 10 h. Between 24 and 32 h after the challenge, the second peak of ear swelling was observed in the same ears, in which the magnitude of swelling was almost comparable with the first one. In contrast, no overt ear swelling response was observed in the ears painted with picryl chloride in the non-transgenic littermates (Fig. 7BGo). Moreover, the left ear challenged with ozaxolone did not show any significant swelling in either transgenic or non-transgenic littermates (Fig. 7A and BGo). Thus, both immediate and delayed reactions of allergy could be induced locally in an antigen-specific manner in the transgenic mice without any prior sensitization with the antigen.



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Fig. 7. Biphasic ear swelling response was elicited in an antigen-specific manner by epicutaneous antigen challenge in the transgenic mice but not in the non-transgenic littermates. The transgenic mice (A) and non-transgenic littermates (B) were challenged epicutaneously with picryl chloride on their right ears and with oxazolone on their left ears. The thickness of right ears (closed circle) and left ears (open square) was measured at the indicated times after the antigen challenge, and the kinetics of increase in ear thickness is plotted. Data shown are representative of six repeated analyses.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The establishment of IgE transgenic mice was previously reported by Adamczewski et al. (18,19). However, their transgenic mice produced polyclonal and non-specific IgE, since only {varepsilon}H chain gene but not L chain gene was introduced as the transgene into their mice. Therefore, it was impossible to elicit allergic reactions in their transgenic mice by means of antigen challenge. In contrast, our mice carry the transgenes coding for both {varepsilon}H chain and {kappa}L chain of IgE specific to TNP, and indeed produce constitutively high titers of TNP-specific IgE. Virtually all of IgE in sera of the transgenic mice showed the specificity to TNP. This clearly indicated that IgE produced in the mice was all derived from the introduced H and L chain transgenes, and that the endogenous L chain genes were almost completely excluded by the expression of the {kappa}L chain transgene. In contrast, the expression of the {varepsilon}H chain transgene did not appear to inhibit the rearrangement and expression of endogenous H chain genes and B cell development, since B cells expressing µH chains were generated in bone marrow of the transgenic mice as efficiently as in non-transgenic littermates. This could be attributed to the fact that the membrane exons had been deleted from the {varepsilon}H chain transgene to prevent a possible allelic exclusion.

Importantly, both local and systemic reactions of allergy could be elicited in our transgenic mice by simply challenging them with the corresponding antigen. This clearly indicates that IgE produced in the transgenic mice is biologically active in vivo. Interestingly, in the cutaneous allergic reaction, not only immediate phase (1–3 h post-challenge) but also delayed phase (24–32 h post-challenge) ear swelling was observed. Timing of this second ear swelling is much later than that of the typical late phase reaction which usually occurs at 6–12 h after the antigen challenge (5). Since the mice were not immunized with any antigen before challenging them with the antigen, it is unlikely that antigen-primed T cells were involved in the second peak of ear swelling. Notably, the flow cytometric analysis of peritoneal mast cells revealed that the surface expression of Fc{varepsilon}RI on mast cells was markedly up-regulated in the transgenic mice as compared to that in non-transgenic mice. A recent study demonstrated that the incubation of mast cells with exogenous IgE in vitro and in vivo enhanced Fc{varepsilon}RI expression on mast cells (16). Furthermore, this IgE-dependent up-regulation of Fc{varepsilon}RI expression was shown to significantly enhance the ability of mast cells to produce pro-inflammatory and immunoregulatory mediators such as IL-6 and IL-4 in response to antigen challenge (16). Therefore, it is reasonable to consider that high levels of serum IgE in our transgenic mice induce and maintain elevated levels of Fc{varepsilon}RI expression on mast cells. The delayed phase of ear swelling observed in the transgenic mice could be related to the overproduction of mediators by mast cells expressing high levels of Fc{varepsilon}RI. Although a cutaneous basophil hypersensitivity reaction characterized by prominent infiltration of basophils has been shown in man and guinea pig as a delayed inflammatory reaction (20,21), the involvement of basophils in the delayed cutaneous reaction in mice remains to be determined (22).

Our transgenic model system is simplified to focus on the effector phase of IgE-mediated allergic reactions. The induction (sensitization) phase of allergic reactions is intentionally skipped, in which antigen processing and presentation to T cells usually take place followed by differentiation and activation of Th2 cells, and subsequent IgE switching of B cells (23). In our transgenic model, prior sensitization of mice with antigens is not necessary to induce the production of antigen-specific IgE. Therefore, antigentriggered IgE-mediated allergic reactions in vivo can be analyzed in a straightforward manner without any possible complication accompanying the prior sensitization which is often achieved by repeated injections of high dose of antigens with adjuvants. For example, IgG-mediated allergic reactions are excluded in our transgenic system.

It has been demonstrated that antigen-triggered IgE-mediated allergic reactions can be elicited in unprimed animals by passive transfer of exogenous IgE followed by challenge with the corresponding antigen (4). Since the half-life of IgE in vivo is extremely short as compared to other isotypes of Ig (24), one of the advantages of our transgenic model over passive IgE sensitization of animals would be the constitutive production of endogenous IgE with the known antigen specificity in vivo as observed in allergic patients. Indeed, the constitutive elevation of Fc{varepsilon}RI expression on mast cells was observed in the transgenic mice. Therefore, our transgenic system could provide a superior way to study the role of IgE not only in the immediate phase but also in the late and chronic phases of allergic reactions following repeated antigen challenge, which appears to be implicated in chronic allergic diseases such as asthma and atopic dermatitis.

Substantial amounts of IgE were detected not only on mast cells but also on most splenic B cells in the transgenic mice. The IgE molecules detected on B cells most likely bind to the cell surface through CD23. The surface expression of CD23 was found to be up-regulated 6–8 times higher in the transgenic mice than in non-transgenic littermates. This could be explained by the decreased sensitivity of CD23 to proteolytic cleavage when associated with IgE as shown previously (25). The up-regulated expression of IgE-loaded CD23 on B cells may facilitate uptake, processing and presentation of antigens to T cells. Thus, our transgenic model system could be useful to study not only pathological but also immunobiological roles of IgE in vivo, which might not be addressed by passive IgE sensitization of animals.

It would be intriguing to cross the IgE transgenic mice established in this study into TCR transgenic mice, or T cell-, mast cell- or complement-deficient backgrounds for better understanding of the pathogenesis of allergy. These model animals may also be useful to develop a new generation of therapeutic strategies for the intervention of allergic diseases.


    Acknowledgments
 
We thank Dr K. Ishizaka for critical reading of the manuscript, Dr H. Sakano for kindly providing us with the plasmid pMM222, Dr U. Storb for the plasmids pVH167µ and pVL167{kappa}, Dr T. Hirano for the hybridomas 6HD5 and HMK12, and Dr H. Matsuda for NC/Nga mice. We are also grateful to Ms M. Yamagishi for excellent secretarial assistance. This research was supported in part by the grant for Specially Promoted Research on Atopic Disorders from The Tokyo Metropolitan Government and grant no. 10010109 from the Ministry of Health and Welfare, Japan.


    Abbreviations
 
PEphycoerythrin

    Notes
 
Transmitting editor: M. Miyasaka

Received 29 January 1998, accepted 4 March 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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