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. 2005 Jan;115(1):35-43.
doi: 10.1172/JCI22656.

A protective role for the A1 adenosine receptor in adenosine-dependent pulmonary injury

Chun-Xiao Sun  1 , Hays W Young, Jose G Molina, Jonathan B Volmer, Jurgen Schnermann, Michael R Blackburn
Affiliations

A protective role for the A1 adenosine receptor in adenosine-dependent pulmonary injury

Chun-Xiao Sun et al. J Clin Invest. 2005 Jan.

Abstract

Adenosine is a signaling nucleoside that has been implicated in the regulation of asthma and chronic obstructive pulmonary disease. Adenosine signaling can serve both pro- and anti-inflammatory functions in tissues and cells. In this study we examined the contribution of A(1) adenosine receptor (A(1)AR) signaling to the pulmonary inflammation and injury seen in adenosine deaminase-deficient (ADA-deficient) mice, which exhibit elevated adenosine levels. Experiments revealed that transcript levels for the A(1)AR were elevated in the lungs of ADA-deficient mice, in which expression was localized predominantly to alveolar macrophages. Genetic removal of the A(1)AR from ADA-deficient mice resulted in enhanced pulmonary inflammation along with increased mucus metaplasia and alveolar destruction. These changes were associated with the exaggerated expression of the Th2 cytokines IL-4 and IL-13 in the lungs, together with increased expression of chemokines and matrix metalloproteinases. These findings demonstrate that the A(1)AR plays an anti-inflammatory and/or protective role in the pulmonary phenotype seen in ADA-deficient mice, which suggests that A(1)AR signaling may serve to regulate the severity of pulmonary inflammation and remodeling seen in chronic lung diseases by controlling the levels of important mediators of pulmonary inflammation and damage.

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Figures

Figure 1
Figure 1
A1AR expression in the lungs of ADA–/– mice. (A) Transcript levels for the A1AR were measured in whole lung RNA extracts from postnatal day 18 ADA-containing (ADA+) and ADA-deficient (ADA–/–) mice, using semiquantitative RT-PCR. Findings from 2 different pairs of littermates are shown. RNA extracted from the brain of an ADA+ mouse was used as a positive control, and β-actin was used as an RNA-positive control for each sample. M, DNA size ladder. Quantitative RT-PCR was used to determine the levels of A1AR transcripts in day 18 whole-lung extracts (B) or BAL cell pellets (C) from ADA+ and ADA–/– mice. Data are presented as mean percentage of β-actin transcripts ± SEM; n = 4 for each. *P – 0.05 compared to ADA+. nd, not detectable. Images show (D) lung section from a postnatal day 18 ADA+ mouse hybridized with antisense A1AR riboprobe, (E) lung section from a postnatal day 18 ADA–/– mouse hybridized with antisense A1AR riboprobe, and (F) lung section from a postnatal day 18 ADA–/– mouse hybridized with sense A1AR riboprobe. Purple represents specific hybridization; pink shows counterstained nuclei. Arrows denote alveolar macrophages. Scale bar: 10 μm
Figure 2
Figure 2
Histological findings in the lungs of ADA/A1AR double-knockout mice. Lungs were collected on postnatal day 14 and processed for H&E staining. Images show (A) lung from an ADA+A1AR+/+ mouse, (B) lung from an ADA+A1AR–/– mouse, (C) lung from an ADA–/–A1AR+/+ mouse, and (D) lung from an ADA–/–A1AR–/– mouse. Images are representative of 10 animals from each group. Scale bar: 100 μm. Br, bronchiole.
Figure 3
Figure 3
Increased airway inflammation in ADA/A1AR double-knockout mice. BALF was collected from the lungs of postnatal day 14 mice, and total cells were determined (A). BALF cells were then cytospun and stained with Diff-Quick (Dade Behring), allowing for determination of cellular differentials. Cells examined in BALF included eosinophils, neutrophils, and lymphocytes (B) as well as alveolar macrophages (C). All data are presented as total cells ± SEM; n = 10 for each group. *P – 0.05 compared to ADA+ mice; **P – 0.05 compared to ADA–/–A1AR+/+ mice.
Figure 4
Figure 4
Increased mucus production in the lungs of ADA/A1AR double-knockout mice. Lung sections from postnatal day 14 mice were stained with PAS for the detection of mucus (arrows). Images show (A) lung from an ADA+A1AR+/+ mouse, (B) lung from an ADA+A1AR–/– mouse, (C) lung from an ADA–/–A1AR+/+ mouse, and (D) lung from an ADA–/–A1AR–/– mouse. Scale bar: 100 μm. (E) A mean mucus index ± SEM was determined as described in Methods; n = 5 for each group. (F) Levels of mucus-associated genes were determined in whole-lung extracts from postnatal day 14 mice, using quantitative RT-PCR. Data are presented as mean pg of transcripts/μg RNA ± SEM; n = 8 for each group. *P – 0.05 compared to ADA+ mice; **P – 0.05 compared to ADA–/–A1AR+/+ mice.
Figure 5
Figure 5
Cytokine transcript levels in the lungs of ADA/A1AR double-knockout mice. Levels of IL-4, IL-13, and IL-6 transcripts were determined in whole-lung extracts from postnatal day 14 mice using quantitative RT-PCR. Data are presented as mean pg of transcripts/μg RNA ± SEM; n = 8 for each. *P – 0.05 compared to ADA+ mice; **P – 0.05 compared to ADA–/–A1AR+/+ mice.
Figure 6
Figure 6
Chemokine transcript levels in the lungs of ADA/A1AR double-knockout mice. Levels of eotaxin 1, eotaxin 2, TARC, and MCP-3 transcripts were determined in whole-lung extracts from postnatal day 14 mice using quantitative RT-PCR. Data are presented as mean pg of transcripts/μg RNA ± SEM; n = 8 for each. *P – 0.05 compared to ADA+ mice; **P – 0.05 compared to ADA–/–A1AR+/+ mice.
Figure 7
Figure 7
Adenosine and AR levels in the lungs of ADA/A1AR double-knockout mice. (A) Adenosine levels were measured in the lungs of postnatal day 17 mice using reversed phase HPLC. Data are presented as mean nmol adenosine/mg protein ± SEM; n = 8 for each group. *P – 0.05 compared to ADA+ADA+/+ mice; **P – 0.05 compared to ADA+ mice; ***P – 0.05 compared to ADA–/–A1AR+/+ mice. (B) Levels of AR transcripts were determined in whole-lung extracts from postnatal day 14 mice using quantitative RT-PCR. Data are presented as mean transcripts/100 ng RNA ± SEM; n = 6 for each. *P – 0.05 compared to ADA+ mice.
Figure 8
Figure 8
Regulation of lung adenosine levels using ADA enzyme therapy. ADA–/– mice were maintained on ADA enzyme therapy until postnatal day 17 as described in Methods. Whole-lung adenosine levels were measured in mice at 3 and 14 days after the cessation of ADA enzyme therapy. Data are presented as mean nmol adenosine/mg protein ± SEM; day 3, n = 4; day 14, n = 7. *P – 0.05 compared to ADA+ mice; **P – 0.05 compared to ADA–/–A1AR+/+ mice.
Figure 9
Figure 9
Alveolar destruction in ADA/A1AR double-knockout mice. ADA–/– mice were treated with ADA enzyme therapy from birth until postnatal day 14 as described in Methods. Lungs from ADA–/– mice and age-matched ADA+ mice were collected 14 days after the cessation of ADA enzyme therapy and processed for H&E staining. Images show (A) lung from an ADA+A1AR+/+ mouse, (B) lung from an ADA+A1AR–/– mouse, (C) lung from an ADA–/–A1AR+/+ mouse, and (D) lung from an ADA–/–A1AR–/– mouse. Images are representative of 10 animals from each group. Scale bar: 100 μm. (E) Alveolar airway sizes were calculated using Image-Pro Plus (Media Cybernetics). Data are presented as mean cord length ± SEM; n = 5. (F) Levels of MMP-9 and MMP-12 transcripts were determined in whole-lung extracts from postnatal day 14 mice using quantitative RT-PCR. Data are presented as mean pg of transcripts/μg RNA ± SEM; n = 8 for each. *P – 0.05 compared to ADA+ mice; **P – 0.05 compared to ADA–/–A1AR+/+ mice.
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