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. 2006 Aug;116(8):2173-2182.
doi: 10.1172/JCI27303.

Role of A2B adenosine receptor signaling in adenosine-dependent pulmonary inflammation and injury

Chun-Xiao Sun  1 , Hongyan Zhong, Amir Mohsenin, Eva Morschl, Janci L Chunn, Jose G Molina, Luiz Belardinelli, Dewan Zeng, Michael R Blackburn
Affiliations

Role of A2B adenosine receptor signaling in adenosine-dependent pulmonary inflammation and injury

Chun-Xiao Sun et al. J Clin Invest. 2006 Aug.

Abstract

Adenosine has been implicated in the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease. In vitro studies suggest that activation of the A2B adenosine receptor (A2BAR) results in proinflammatory and profibrotic effects relevant to the progression of lung diseases; however, in vivo data supporting these observations are lacking. Adenosine deaminase-deficient (ADA-deficient) mice develop pulmonary inflammation and injury that are dependent on increased lung adenosine levels. To investigate the role of the A2BAR in vivo, ADA-deficient mice were treated with the selective A2BAR antagonist CVT-6883, and pulmonary inflammation, fibrosis, and airspace integrity were assessed. Untreated and vehicle-treated ADA-deficient mice developed pulmonary inflammation, fibrosis, and enlargement of alveolar airspaces; conversely, CVT-6883-treated ADA-deficient mice showed less pulmonary inflammation, fibrosis, and alveolar airspace enlargement. A2BAR antagonism significantly reduced elevations in proinflammatory cytokines and chemokines as well as mediators of fibrosis and airway destruction. In addition, treatment with CVT-6883 attenuated pulmonary inflammation and fibrosis in wild-type mice subjected to bleomycin-induced lung injury. These findings suggest that A2BAR signaling influences pathways critical for pulmonary inflammation and injury in vivo. Thus in chronic lung diseases associated with increased adenosine, antagonism of A2BAR-mediated responses may prove to be a beneficial therapy.

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Figures

Figure 1
Figure 1. Pharmacological characterization of CVT-6883 as an A2B AR antagonist.
(A) Structure of CVT-6883. (B) Competition of CVT-6883 for specific radioligand binding to membranes prepared from CHO cells overexpressing the A1AR or A3AR or from HEK cells over-expressing the A2AAR or A2BAR. Increasing concentrations of CVT-6883 were used to displace the specific binding of selective receptor radioligands: 3H-CPX (A1AR antagonist; 0.45 nM), 3H-ZM241385 (A2AAR antagonist; 1.6 nM), 3H-ZM241385 (A2BAR antagonist; 9.3 nM), or 3H-MRE3008F20 (A3AR antagonist; 0.63 nM). Data are presented as mean ± SEM percent specific binding of control from 6 determinations. (C) Concentration response curves of NECA-induced increases in cAMP in the absence or presence of increasing concentrations of CVT-6883. Mouse NIH/3T3 cells were treated with NECA (1 nM to 100 μM) in the absence or presence of increasing concentrations of CVT-6883, and cAMP accumulation was measured. Data are mean ± SEM from 3 experiments performed in duplicate. Inset is the Schild plot. DR – 1, concentration ratio minus 1.
Figure 2
Figure 2. Histopathology of the lungs of mice treated with CVT-6883.
Lungs were collected from postnatal day 38 mice and prepared routinely for sectioning and H&E staining. (A) Lung from an ADA+ mouse treated with vehicle. (B) Lung from an ADA+ mouse treated with CVT-6883. (C) Lung from an ADA–/– mouse treated with vehicle. (D) Lung from an ADA–/– mouse treated with CVT-6883. Sections are representative of 6–8 different mice from each treatment group. Scale bars: 100 μm.
Figure 3
Figure 3. Airway inflammatory cells in mice treated with CVT-6883.
(A) BAL fluid was collected from postnatal day 38 mice, and total cell numbers were counted. (B and C) BAL cells were cytospun and stained with Diff-Quick, allowing for determination of cellular differentials. Data are mean cell counts ± SEM. *P ≤ 0.05 versus vehicle-treated ADA+ mice; #P ≤ 0.05 versus vehicle-treated ADA–/– mice. n = 8 (ADA+), 6–8 (ADA–/–). (D) Increased numbers of foam cells were found by directly examining cytospun cells stained with Diff-Quick. Scale bars: 10 μm.
Figure 4
Figure 4. Production of proinflammatory cytokines.
Transcript levels of various proinflammatory cytokines were measured in whole-lung extracts from postnatal day 38 mice using quantitative RT-PCR. Shown are levels of (A) TNF-α, (B) IL-6, (C) CXCL2, (D) CCL11, (E) CCL17, and (F) CXCL1. Results are presented as mean pg transcript/μg RNA ± SEM. *P ≤ 0.05 versus vehicle-treated ADA+ mice; #P ≤ 0.05 versus vehicle-treated ADA–/– mice. n = 4 (ADA+), 8 (ADA–/–). ND, not detected.
Figure 5
Figure 5. CVT-6883 treatment inhibits alveolar airway enlargement inADA–/– mice.
Lungs from postnatal day 38 mice were infused with fixative under constant pressure (25 cm H2O) and processed for H&E staining. (A) Lung from an ADA+ mouse treated with vehicle. (B) Lung from an ADA+ mouse treated with CVT-6883. (C) Lung from an ADA–/– mouse treated with vehicle. (D) Lung from an ADA–/– mouse treated with CVT-6883. Images are representative of 8 animals from each group. Scale bars: 100 μm. (E) Alveolar airspace size was calculated using ImagePro analysis software; data are mean chord length ± SEM. *P ≤ 0.05 versus vehicle-treated ADA+ mice; #P ≤ 0.05 versus vehicle-treated ADA–/– mice. n = 5 per group.
Figure 6
Figure 6. CVT-6883 treatment inhibits the expression of genes associated with alveolar airway destruction.
Transcript levels of TIMP-1 (A), MMP-9 (B), and MMP-12 (C) were measured in whole-lung RNA extracts from postnatal day 38 mice using quantitative RT-PCR. Data are mean pg transcript/μg RNA ± SEM. *P ≤ 0.05 versus vehicle-treated ADA+ mice; #P ≤ 0.05 versus vehicle-treated ADA–/–mice. n = 4 (ADA+), 8 (ADA–/–).
Figure 7
Figure 7. Pulmonary fibrosis inADA–/– mice treated with CVT-6883.
(A–C) Lung sections from postnatal day 38 mice were stained with an antibody against α-SMA to visualize myofibroblast (brown stain). (A) Lung from an ADA+ mouse treated with vehicle. (B) Lung from an ADA–/– mouse treated with vehicle. (C) Lung from an ADA–/– mouse treated with CVT-6883. (D–F) Lung sections were stained with Masson’s trichrome to visualize collagen deposition (blue stain). (D) Lung from an ADA+ mouse treated with vehicle. (E) Lung from an ADA+ mouse treated with CVT-6883. (F) Lung from an ADA–/– mouse treated with vehicle. (G) Lung from an ADA–/– mouse treated with CVT-6883. Sections are representative of 6 different mice from each treatment. Scale bars: 100 μm. (H) Whole-lung α1-procollagen transcript levels. Data are mean pg transcript/μg RNA ± SEM. (I) Soluble collagen protein levels. Data are mean μg collagen/ml BAL fluid ± SEM. *P ≤ 0.05 versus vehicle-treated ADA+; #P ≤ 0.05 versus vehicle-treated ADA–/–. n = 4 (ADA+), 8 (ADA–/–).
Figure 8
Figure 8. CVT-6883–treatedADA–/– mice had decreased expression of TGF-β1 and OPN in alveolar macrophages.
RNA was extracted from whole lungs of postnatal day 38 mice for analysis using quantitative RT-PCR for various fibrosis-associated transcripts. Results demonstrate that lungs from CVT-6883–treated ADA–/– mice had lower levels of transcripts for TGF-β1 (A) and OPN (B) compared with that seen in the lungs of vehicle-treated ADA–/–mice. *P ≤ 0.05 versus ADA+; #P ≤ 0.05 versus vehicle-treated ADA–/–. n = 4 (ADA+), 8 (ADA+). (C–E) Immunolocalization of TGF-β1. (F–H) Immunolocalization of OPN. Lung sections from vehicle-treated ADA+ mice (C and F), vehicle-treated ADA–/– mice (D and G), and CVT-6883–treated ADA–/– mice (E and H) are shown. Arrows denote macrophages. Scale bars: 100 μm.
Figure 9
Figure 9. A2B AR transcript and adenosine levels in the lungs.
(A) RNA was extracted from whole lungs of postnatal day 38 mice for analysis of A2BAR expression using quantitative RT-PCR. Data are mean transcript levels ± SEM. *P ≤ 0.05 versus vehicle-treated ADA+; #P ≤ 0.05 versus vehicle-treated ADA–/–. n = 4 (ADA+), 8 (ADA–/–). (B) Adenine nucleotides were extracted from whole lungs of postnatal day 38 mice, and adenosine levels were quantified by reverse-phase HPLC. Data are mean fold increase ± SEM. *P ≤ 0.05 versus ADA+. n = 4 per group.
Figure 10
Figure 10. CVT-6883 treatment in a model of bleomycin-induced pulmonary injury.
(A–C) Lung sections were stained with an antibody against α-SMA to visualize myofibroblast (brown). (D–F) Lung sections were stained with Masson’s trichrome to visualize collagen (blue). Shown are lungs from saline-treated mice 14 days after treatment (A and D) and bleomycin-treated mice 14 days after treatment that were treated with vehicle (B and E) or CVT-6883 (C and F) beginning on day 5 of the protocol. Sections are representative of 7 different mice from each treatment. Scale bars: 100 μm. (G) Total BAL cells. Data are mean ± SEM. (H) Whole-lung collagen protein levels. Data are mean μg collagen/lung ± SEM. *P ≤ 0.05 versus saline-treated mice; #P ≤ 0.05 versus vehicle-treated bleomycin-exposed mice. n = 7 per group.
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