C‑C chemokine receptor type 3 gene knockout alleviates inflammatory responses in allergic rhinitis model mice by regulating the expression of eosinophil granule proteins and immune factors

Abstract

The present study aimed to investigate the effects of C‑C chemokine receptor type 3 (CCR3) gene knockout on allergic rhinitis (AR) in mice, as well as the underlying molecular mechanisms. Ovalbumin was administrated to CCR3+/+ and CCR3‑/‑ BALB/c mice to establish an AR model. The mice were divided into four groups: i) Normal control (CG), ii) AR model (AR), iii) CCR3 knockout CG (CCR3‑/‑CG) and iv) AR model with CCR3 knockout (CCR3‑/‑AR). Histological sections of nasal mucosae were examined by hematoxylin and eosin staining, which revealed that CCR3 knockout suppressed the invasion of inflammatory cells and relieved the damage of nasal mucosae. Peripheral blood smear and nasal‑washing smears were evaluated by Wright's staining. Eosinophil (EOS) numbers in nasal mucosae, peripheral blood, and nasal washings of the various groups were ranked in the order: AR>CCR3‑/‑AR>CG>CCR3‑/‑. mRNA expression levels of CCR3, EOS peroxidase (EPO), EOS cationic protein (ECP), and major basic protein (MBP) in the peripheral serum and nasal washings were detected by reverse transcription‑polymerase chain reaction. Interferon‑γ (IFN‑γ), interleukin (IL)‑4, IL‑10, and immunoglobulin E (IgE) protein levels in the peripheral serum and nasal washings were investigated by ELISA. CCR3 mRNA expression was not detected in the CCR3‑/‑ and CCR3‑/‑AR groups, whereas expression levels in the AR group were markedly higher compared with expression in the CG group. Compared with the CG‑associated groups (i.e., the CG and CCR3‑/‑CG groups), the levels of EPO, ECP, MBP, IL‑4, and IgE were significantly increased in the AR‑associated groups (that is, R and CCR3‑/‑AR). In addition, the CCR3‑/‑AR group mice produced significantly lower levels of EPO, ECP, MBP, IL‑4 and IgE compared with the AR group, whereas the expression levels of IFN‑γ and IL‑10 were increased. CCR3 gene knockout may alleviate EOS invasion and the inflammatory response in AR model mice by reducing the expression levels of EPO, ECP, MBP, IL‑4, and IgE, and increasing the expression of IL‑10 and IFN‑γ.

Figure 1.

Identification of mice with CCR3 gene knockout by polymerase chain reaction. B6 was the genomic DNA (wild-type), which served as negative control. Target fragments of wild-type and mutant were 120 and 280 bp, respectively. N indicates the blank control. Template control was not set. The rightmost lane was the marker. There were no bands in the location of 120 bp in the left six lanes, but in the location of 280 bp Mut gene, notable bands were detected. Mut, mutant; WT, wild-type.

Figure 2.

Histological examination of nasal mucosae. Sections of nasal mucosae from mice in the (A) CG, (B) CCR3^−/−CG, (C) AR and (D) CCR3^−/−AR groups were examined by hematoxylin and eosin staining; Detachment of epithelial cells with irregular arrangement, structural disorder of the mucosal layer and invasion of numerous inflammatory cells, including EOS, are indicated by arrows (magnification, ×100). AR, allergic rhinitis model; CG, normal control; CCR3, C-C chemokine receptor 3; EOS, eosinophil.

Figure 3.

Analysis of peripheral blood smears. Blood smears from mice in the (A) CG, (B) AR, (C) CCR3^−/−CG and (D) CCR3^−/−AR groups were examined by Wright's staining; magnification, ×100. CG, normal control; AR, allergic rhinitis model, CCR3^−/−CG, C-C chemokine receptor-3 knockout control; CCR3^−/−AR, AR model with CCR3 knockout.

Figure 4.

Images of the nasal washings smear in the (A) CG, (B) AR, (C) CCR3^−/−CG, and (D) CCR3^−/−AR groups which were examined by Wright's staining (magnification, ×100). AR, allergic rhinitis model; CG, normal control; CCR3^−/−CG, C-C chemokine receptor-3 knockout control; CCR3^−/−AR, AR model with CCR3 knockout.

Figure 5.

mRNA expression levels of CCR3, EPO, ECP, and MBP in the peripheral serum of mice in various groups were determined by reverse transcription-quantitative polymerase chain reaction. *P<0.05, AR group vs. the CG group and the CCR3^−/−AR group vs. the CCR3^−/−CG group. ^#P<0.05, CCR3^−/−AR group vs. the AR group. AR, allergic rhinitis model; CCR3, C-C chemokine receptor-3; CG, normal control; CCR3^−/−CG, CCR3 knockout control; CCR3^−/−AR, AR model with CCR3 knockout; ECP, eosinophil cationic protein; EPO, eosinophil peroxidase; MBP, major basic protein.

Figure 6.

mRNA expression levels of CCR3, EPO, ECP, and MBP in the nasal washings of mice in various groups were determined by reverse transcription-quantitative polymerase chain reaction. *P<0.05, AR group vs. the CG group and the CCR3^−/−AR group vs. the CCR3^−/−CG group. ^#P<0.05, CCR3^−/−AR group vs. the AR group. AR, allergic rhinitis model; CCR3, C-C chemokine receptor-3; CG, normal control; CCR3^−/−CG, CCR3 knockout control; CCR3^−/−AR, AR model with CCR3 knockout; ECP, eosinophil cationic protein; EPO, eosinophil peroxidase; MBP, major basic protein.

Figure 7.

Expression levels of IFN-γ, IL-4, IL-10, and IgE in the peripheral serum of mice were evaluated by ELISA. *P<0.05, AR vs. CG and CCR3^−/−AR vs. CCR3^−/−CG; ^#P<0.05, CCR3^−/−AR vs. AR. AR, allergic rhinitis model; CCR3, C-C chemokine receptor-3; CCR3^−/−AR, AR model with CCR3 knockout; CCR3^−/−CG, CCR3 knockout control; CG, normal control; IFN-γ, interferon-γ; IgE, immunoglobulin E; IL-10, interleukin-10.

Figure 8.

Expression levels of IFN-γ, IL-4, IL-10, and IgE in the nasal washings of mice in various groups were evaluated by ELISA. *P<0.05, AR vs. CG and CCR3^−/−AR vs. CCR3^−/−CG. AR, allergic rhinitis model; CCR3, C-C chemokine receptor-3; CCR3^−/−AR, AR model with CCR3 knockout; CCR3^−/−CG, CCR3 knockout control; CG, normal control; IFN-γ, interferon-γ; IgE, immunoglobulin E; IL-10, interleukin-10.