Enhanced airway inflammation and remodeling in adenosine deaminase-deficient mice lacking the A2B adenosine receptor

Abstract

Adenosine is a signaling nucleoside that is generated in response to cellular injury and orchestrates the balance between tissue protection and the progression to pathological tissue remodeling. Adenosine deaminase (ADA)-deficient mice develop progressive airway inflammation and remodeling in association with adenosine elevations, suggesting that adenosine can promote features of chronic lung disease. Furthermore, pharmacological studies in ADA-deficient mice demonstrate that A(2B)R antagonism can attenuate features of chronic lung disease, implicating this receptor in the progression of chronic lung disease. This study examines the contribution of A(2B)R signaling in this model by generating ADA/A(2B)R double-knockout mice. Our hypothesis was that genetic removal of the A(2B)R from ADA-deficient mice would lead to diminished pulmonary inflammation and damage. Unexpectedly, ADA/A(2B)R double-knockout mice exhibited enhanced pulmonary inflammation and airway destruction. Marked loss of pulmonary barrier function and excessive airway neutrophilia are thought to contribute to the enhanced tissue damage observed. These findings support an important protective role for A(2B)R signaling during acute stages of lung disease.

FIGURE 1

A_2BR expression in the lungs of ADA^−/− mice. Transcript levels for the A_2BR were measured in whole-lung RNA extracts from postnatal day 18 ADA-containing (ADA⁺) and ADA-deficient (ADA^−/−) mice using quantitative RT-PCR. Data are presented as mean percentage of β-actin transcripts ± SEM; n = 4 for each. *, p ≤ 0.05 compared with ADA⁺.

FIGURE 2

Lung histopathology. Lungs were collected on postnatal day 35 and prepared for sectioning and H&E staining. A, Lung section from an ADA⁺ A_2BR^+/+ mouse. B, Lung section from an ADA⁺ A_2BR^−/− mouse. C, Lung section from an ADA^−/−A_2BR^+/+ mouse. D, Lung section from an ADA^−/−A_2BR^−/− mouse. Sections are representative of eight mice from each genotype. Bars, 100 μm.

FIGURE 3

Pulmonary inflammation. A, BAL fluid was collected on postnatal day 35, and total cell numbers were determined. B, BAL cells were cytospun and stained with Diff-Quick, allowing for determination of cellular differentials. Data are mean cell counts ± SEM. *, p ≤ 0.05 vs ADA⁺ mice; #, p ≤ 0.05 vs ADA^−/− A_2BR^+/+ mice; n = 6 (ADA⁺ A_2BR^+/+ and ADA⁺ A_2BR^−/−), n = 8 (ADA^−/−A_2BR^+/+ and ADA^−/−A_2BR^−/−). C, Cytospun BAL cells stained with Diff-Quick. Bar, 10 μm. D, Lung sections from an ADA^−/−A_2BR^−/− mouse and an ADA^−/−A_2BR^−/− mouse (E) were stained with an Ab against neutrophils to visualize infiltrated tissue neutrophils (brown). Bars, 100 μm.

FIGURE 4

Production of cytokines and chemokines. Transcript levels of various proinflammatory cytokines and chemokines were measured in whole-lung extracts from postnatal day 35 mice using quantitative RT-PCR. Shown are levels of TNF-α (A), IL-6 (B), CXCL1 (C), and MCP-1 (D). Results are presented as mean fold increase compared with controls or percent of 18S RNA ± SEM. *, p ≤ 0.05 vs ADA⁺ mice; #, p ≤ 0.05 vs ADA^−/−A_2BR^+/+ mice. n = 4 (ADA⁺ A_2BR^+/+ and ADA⁺A_2BR^−/−), n = 8 (ADA^−/− A_2BR^+/+ and ADA^−/− A_2BR^−/−).

FIGURE 5

Western blot analysis of E-selectin, ICAM-1, and IκB-α in whole-lung extracts. Samples from postnatal day 22 and 35 mice were subjected to Western blotting with the indicated Abs. Antiactin was used as a loading control.

FIGURE 6

Vascular permeability analysis. A, BAL fluid was collected from postnatal day 35 mice, and total BAL fluid protein levels were determined by Bradford assay. Data are mean total protein content ± SEM. *, p ≤ 0.05 vs ADA⁺ mice; #, p ≤ 0.05 vs ADA^−/− A_2BR^+/+ mice. n = 6 (ADA⁺), 8 (ADA^−/−). B, Lungs from postnatal day 35 mice were collected, and lung water content was determined by lung wet-dry weight ratio. Data are mean ratios ± SEM. *, p ≤ 0.05 vs ADA⁺ mice; #, p ≤ 0.05 vs ADA^−/− A_2BR^+/+ mice. n = 4 (ADA⁺ A_2BR^+/+ and ADA⁺A_2BR^−/−), n = 6 (ADA^−/−A_2BR^+/+ and ADA^−/−A_2BR^−/−). Mice were given i.p. Evans blue dye (0.2 ml of 0.5% in PBS) and sacrificed 4 h later, and the hearts and lungs were harvested. C, Representative images of lungs. Evans blue dye concentrations were quantified in lung (D). Data are mean dye content in micrograms per gram of lung tissue ± SEM. *, p ≤ 0.05 vs ADA⁺ mice; #, p ≤ 0.05 vs ADA^−/− A_2BR^+/+ mice. n = 4 (ADA⁺ A_2BR^+/+ and ADA⁺ A_2BR^−/−), n = 8 (ADA^−/− A_2BR^+/+ and ADA^−/−A_2BR^−/−).

FIGURE 7

Mucus metaplasia. Lungs were collected on postnatal day 35 and prepared for sectioning and PAS staining. A, Lung section from an ADA⁺ A_2BR^+/+ mouse. B, Lung section from an ADA⁺ A_2BR^−/− mouse. C, Lung section from an ADA^−/− A_2BR^+/+ mouse. D, Lung section from an ADA^−/− A_2BR^−/− mouse. Sections are representative of eight different mice from each genotype. Bars, 100 μm. E, A mucus index was determined, and data are presented as mean mucus index ± SEM. *, p ≤ 0.05 vs ADA⁺ mice; #, p ≤ 0.05 vs ADA^−/− A_2BR^+/+ mice. n = 4 (ADA⁺ A_2BR^+/+ and ADA⁺ A_2BR^−/−), n = 8 (ADA^−/− A_2BR^+/+ and ADA^−/− A_2BR^−/−).

FIGURE 8

Alveolar airway enlargement. Lungs from postnatal day 35 mice were infused with fixative under constant pressure (25 cm H₂O) and processed for H&E or TUNEL staining. A, H&E-stained lung section from an ADA^−/− A_2BR^+/+ mouse. B, H&E-stained lung section from an ADA^−/−A_2BR^+/+ mouse. C, TUNEL-stained lung section from an ADA^−/− A_2BR^+/+ mouse. D, TUNEL-stained lung section from an ADA^−/− A_2BR^−/− mouse. Images are representative of eight animals from each genotype. E, Alveolar airspace size was calculated using ImagePro analysis software; data are mean chord length ± SEM. F, TUNEL-positive cells were counted, and data are presented as mean TUNEL scores ± SEM. *, p ≤ 0.05 vs ADA⁺ mice; #, p ≤ 0.05 vs ADA^−/−A_2BR^+/+ mice. n = 4 (ADA⁺ A_2BR^+/+ and ADA⁺ A_2BR^−/−), n = 8 (ADA^−/− A_2BR^+/+ and ADA^−/− A_2BR^−/−). Bars, 100 μm.

FIGURE 9

Collagen levels. A, Transcript levels for α1-procollagen were measured in lung extracts from postnatal day 35 mice using quantitative RT-PCR, and results are presented as mean fold increase compared with controls or mean pg transcript/μg RNA ± SEM. B, Collagen protein levels in BAL fluid from postnatal day 35 mice were quantified using the Sircol assay. Data are presented as mean collagen levels ± SEM. *, p ≤ 0.05 vs ADA⁺ mice; #, p ≤ 0.05 vs ADA^−/− A_2BR^+/+ mice. n = 4 (ADA⁺ A_2BR^+/+ and ADA⁺ A_2BR^−/−), n = 8 (ADA^−/− A_2BR^+/+ and ADA^−/− A_2BR^−/−).

FIGURE 10

Tracheal angiogenesis. A, Tracheas were removed from postnatal day 35 mice and analyzed by whole-mount CD31 immunostaining for the visualization of vessels. Results are representative of four mice from each genotype. Bars, 100 μm. B, Tracheal vascularity was quantified by counting the number of vessels intersecting a line down the length of the cartilage ring. At least 12 cartilage rings were analyzed per sample. Data are represented as mean vessels (in millimeters) ± SEM; n = 4. *, p ≤ 0.05 vs ADA⁺ mice; #, p ≤ 0.05 vs ADA^−/− A_2BR^+/+ mice.