Figure 1.
Generation of BasoDTR mice for the selective and inducible ablation of basophils.
(A) Construct of a transgene for BasoDTR mice. The alb promoter of the original TRECK cassette was replaced with the CD203c promoter to allow the exclusive expression of hDTR on basophils [29]. (B) Basophils were purified as IgE-positive cells from the peripheral blood of BasoDTR and WT mice by using the MACS system. RT-PCR analysis was performed to examine the specific expression of hDTR mRNA in basophils. (C) Cytospin slides of the peripheral leukocytes of BasoDTR mice were stained with antibodies against hDTR (green), IgE (red), and DAPI (nuclei, blue). Scale bar = 20 µm.
Figure 2.
Basophil-specific ablation after DT administration in BasoDTR mice.
BasoDTR and WT mice were treated once i.p. with DT (50 µg/kg body weight). After 4 days, basophils in the peripheral blood (A), spleen (B), bone marrow (C), and peritoneal mast cells (D) were analyzed by flow cytometry. The percentages of basophils (IgE+CD49b+) and mast cells (IgE+c-kit+) are indicated.
Table 1.
Percentage of leukocytes in peripheral blood and mast cells in peritoneal exudate cells.
Figure 3.
IgE-CAI and the expression of cytokines and chemokines in antigen-challenged ears.
(A) BasoDTR (n = 4) and WT mice (n = 4) were treated once i.p. with 25 µg/kg DT or with PBS 2 days before and were injected i.v. with 300 µg TNP-specific IgE 1 day before challenge with 10 µg TNP-OVA. Ear thickness was measured at the indicated time points. The kinetics of changes in ear thickness after antigen challenge are shown. Data have been expressed as mean ± SEM and are representative of 4 repeated experiments. P values of DT-administered BasoDTR mice versus WT mice are indicated. *p<0.05, **p<0.01. (B) WT and BasoDTR mice were passively sensitized with i.v. injections of 200 µl diluted ascites containing 300 µg of TNP-specific IgE at 1 day before 50 µg of TNP13-OVA was administered. The change in rectal temperature over time after allergen challenge is shown. Data have been expressed as mean ± SEM and are representative of 4 repeated experiments. (C) The ears of WT (n = 4), BasoDTR (n = 4), and EoDTR (n = 4) mice were excised and homogenized in PBS at 4 days after antigen challenge. The major cytokines and chemokines in 200 µg of extracted total proteins were analyzed semiquantitatively by using antibody arrays. Relative concentrations have been indicated in terms of mean ± SEM. The p values of BasoDTR mice versus WT mice and of EoDTR mice versus WT mice are indicated. *p<0.05, **p<0.01. Statistical analysis was performed using the Student’s t test.
Figure 4.
IgG-mediated systemic anaphylaxis decreased in BasoDTR mice.
(A) BasoDTR mice (n = 4 per group) were treated once i.p. with 25 or 50 µg/kg of DT or PBS at 4 days prior to and were i.v. injected with 500 µg rat anti-FcγRII/III (2.4G2) antibodies in 200 µl PBS. Body temperature was measured at the indicated time points. Data have been expressed as mean ± SEM and are representative of 4 repeated experiments. The p values of DT-administered mice versus control mice are indicated. *p<0.05, **p<0.01. (B) The maximum decrease in the body temperature of BasoDTR and WT mice (n = 4 per group) with or without DT treatment were compared. Data have been expressed in terms of mean ± SEM and are representative of 4 repeated experiments. *p<0.05. Statistical analysis was performed using the Student’s t test.
Figure 5.
Generation of EoDTR mice for the selective and inducible ablation of eosinophils.
(A) The alb promoter of the original TRECK cassette was replaced with the EPO promoter to identify the exclusive expression of hDTR on eosinophils [29]. (B) Cytospin slides of the peripheral leukocytes of EoDTR mice were stained with antibodies against hDTR (green), CCR3 (red), and DAPI (nuclei, blue). Scale bar = 20 µm. (C) EoDTR and WT mice were treated once i.p. with DT (5 µg/kg body weight). After 3 days, eosinophils in the peripheral blood, spleen, and bone marrow were analyzed by flow cytometry. The percentage of eosinophils (CCR3+Siglec-F+) is indicated.
Table 2.
Percentage of leukocytes in peripheral blood after DT administration.
Figure 6.
Ear swelling in IgE-CAI in mice receiving PBS (A, n = 5) or 5 µg/kg of DT at 2 days before (B, n = 5), at the same time as (C, n = 7), or 2 days after (D, n = 6) the antigen challenge were measured at the indicated time points. Data have been expressed in terms of mean ± SEM and are representative of 2 repeated experiments. The p values of DT-treated EoDTR mice versus DT-treated WT mice are indicated. *p<0.05, **p<0.01. Statistical analysis was performed using the Student’s t test.
Figure 7.
Histopathological analysis of the ear skin in chronic-phase swelling in IgE-CAI in EoDTR mice.
WT (A and B) and EoDTR (C and D) mice were treated once i.p. with 5 µg/kg DT at 2 days before and were injected i.v. with 300 µg TNP-specific IgE at 1 day before challenge with 10 µg TNP-OVA. Ear specimens prepared 4 days after antigen challenge were incubated with the CCR3-specific antibody, stained with DAB (brown), and counterstained with hematoxylin (blue). Scale bars = 50 µm. Data are representative of 6 independent experiments. (E) The number of eosinophils and other cells in a 100-µm square was counted. Data have been expressed in terms of mean ± SD and are representative of 3 repeated experiments. The p values of DT-treated EoDTR mice versus DT-treated WT mice are indicated. ***p<0.005, **p<0.001. Statistical analysis was performed using the Student’s t test.