Uteroglobin-related protein 1 expression suppresses allergic airway inflammation in mice

Abstract

Rationale: Uteroglobin-related protein (UGRP) 1, which is highly expressed in the epithelial cells of the airways, has been suggested to play a role in lung inflammation.

Objectives: The aim of study was to understand the effect of overexpressed UGRP1 on lung inflammation in a mouse model of allergic airway inflammation.

Methods: Ovalbumin-sensitized and -challenged mice, a model for allergic airway inflammation, were used in conjunction with recombinant adenovirus expressing UGRP1.

Measurements and main results: We demonstrated that intranasal administration of adeno-UGRP1 successfully delivered UGRP1 to the epithelial cells of airways and markedly reduced the number of infiltrating inflammatory cells, particularly eosinophils, in lung tissue as well as the level of proinflammatory cytokines such as interleukin (IL)-4, IL-5, and IL-13 in bronchoalveolar lavage fluids. The healed phase of inflammation was clearly seen in the peripheral areas of adeno-UGRP1-treated mouse lungs.

Conclusion: These results demonstrate that UGRP1 can suppress inflammation in the mouse model of allergic airway inflammation. Based on this result, we propose UGRP1 as a novel therapeutic candidate for treating lung inflammation such as is found in asthma.

Figure 1.

Eosinophilic inflammation in airways of a murine model of allergic inflammation. (A) Inflammatory cell counts in bronchoalveolar lavage fluids (BALF) from ovalbumin (OVA)-sensitized mice determined by Diff-Quick staining 24 h after saline inhalation (control mice) and OVA inhalation (challenged mice). *p < 0.05 versus sensitized control group. (B) Levels of inflammatory cytokines, interleukin (IL)-4, IL-5, and IL-13, in BALF. Significant increase was observed in the OVA-challenged mice. *p < 0.05 versus sensitized control group. (C–F) Histologic examinations of formalin-fixed lung sections stained with hematoxylin and eosin from sensitized control mice (C, E) and from OVA-challenged mice (D, F). A marked eosinophilia was found in the OVA-challenged mice. (E, F) Higher magnification of the area marked with * in C, D. Reactive hyperplasia due to inflammation is seen in the OVA-challenged mouse bronchial epithelial cells (indicated by an arrow in F). Original magnifications: ×100 (C, D), ×400 (E, F). Br = bronchus; E = bronchial epithelial cells; V = vein.

Figure 2.

Time-course analysis of uteroglobin-related protein 1 (UGRP1) and cytokines during 24 h after OVA challenge. Lung UGRP1 mRNA (A), UGRP1 protein in BALF (B), and IL-4 (C), IL-5 (D), IL-10 (E), and IL-13 (F) levels in BALF were determined 1, 3, 6, 12, and 24 h after OVA challenge in OVA-sensitized mice by use of quantitative polymerase chain reaction (A) and ELISA (B–F). The values of Sens are 24 h after saline inhalation. The cytokine levels in the Sens group in C–F are statistically significantly higher than those of the Nor group, which are not shown in the figure to avoid complexity. *p < 0.05 versus Sens group. Nor = naive control mice; Sens = sensitized control mice.

Figure 3.

Induction of UGRP1 in mouse lungs by adenovirus (Ad)-UGRP1. Mice were intranasally administered Ad-UGRP1 (20 μl of 10⁹ pfu/ml). (A) Northern blot analysis revealing a drastic increase of UGRP1 expression in lungs of naive mice 3 d after adenovirus infection; 3.5 μg of total RNA per lane. (B) Relative UGRP1 expression in A plotted against days after infection. (C) Immunohistochemistry for UGRP1 in lungs of sensitized mice infected with empty adenovirus vector (Ad-empty). (D) Immunohistochemistry demonstrating a significant increase of UGRP1 expression in epithelial cells of sensitized, Ad-UGRP1–infected mice. Sensitized mice were infected with Ad-empty or Ad-UGRP1 3 d before saline inhalation, and lungs were examined 24 h later. (E, F) Higher magnification of the area marked with * in C, D. Slight immunostaining surrounding the veins is likely to be an artifact because these cells are not always stained positive, whereas UGRP1 staining in epithelial cells is always positive. Original magnifications: ×100 (C, D), ×400 (E, F). Br = bronchus; E = bronchial epithelial cells; V = vein.

Figure 4.

UGRP1 mRNA and protein levels in lungs of inflammation model mice after adenovirus treatment. Lung UGRP1 mRNA (A) and protein (B) levels and protein concentration in BALF (C) were determined using real-time polymerase chain reaction, quantification of UGRP1-positive immunostaining, and ELISA, respectively. All groups of mice were OVA sensitized and subjected to analysis after 24 h of saline (Sens) or OVA challenge (Chal). Adenovirus was administered 3d before challenge. Results of quantification of UGRP1- positive immunostaining are shown as the average of five randomly selected sections per mouse from five mice per group. The differences in lanes 4, 5, and 6 versus 1 and 6 versus 3 in A, lanes 4, 5, and 6 versus 1 in B, and lanes 4, 5, and 6 versus 1 in C are also statistically significant, which is not shown in the figure to avoid complexity. (D) UGRP1 in situ hybridization was performed using lungs of OVA-challenged, Ad-empty–infected mice. (E) In situ hybridization of lungs showing the increased UGRP1 expression in OVA-challenged, Ad-UGRP1–infected mice. Right panels of D, E show the results obtained with a sense-strand probe. Relative expression was 1.0 for D versus 1.25 for E, a difference similar to that shown in B. Original magnification: ×200. *p < 0.05; **p < 0.01.

Figure 5.

Inhibition of allergic airway inflammation by Ad-UGRP1. OVA-sensitized mice were infected with Ad-empty or Ad-UGRP1 3d before OVA challenge. The eosinophil counts (A) and cytokine levels (B) in BALF collected 24 h after the OVA challenge were significantly inhibited in Ad-UGRP1–infected mice when compared with Ad-empty–infected animals (*p < 0.05 vs. Ad-empty–infected group). Histologic examinations revealed a marked decrease in eosinophil infiltration in the Ad-UGRP1–infected mice (D, F) when compared with the Ad-empty–infected animals (C, E). (E, F) Higher magnification of the area marked with * in C, D. Reactive hyperplasia due to inflammation is seen in the Ad-empty–treated mouse bronchial epithelial cells (indicated by an arrow in E). In the peripheral areas of lungs, Ad-empty–infected animals exhibited marked infiltration of inflammatory cells such as eosinophils, neutrophils, and lymphocytes in alveoli and alveolar walls (G, H). In contrast, healed phase of inflammation was seen in the peripheral areas of Ad-UGRP1–infected mouse lungs (I), characterized by thickened alveolar wall (arrows) and occasional diminished alveolar cavities (asterisks), Mononuclear cells infiltrated slightly (arrowheads), but no eosinophils were found. Original magnifications: ×40 (C, D), ×200 (G), ×400 (E, F, H, I). Br = bronchus; E = bronchial epithelial cells.