A3 adenosine receptor signaling influences pulmonary inflammation and fibrosis

Abstract

Adenosine is a signaling molecule produced during conditions that cause cellular stress or damage. This signaling pathway is implicated in the regulation of pulmonary disorders through the selective engagement of adenosine receptors. The goal of this study was to examine the involvement of the A(3) adenosine receptor (A(3)R) in a bleomycin model of pulmonary inflammation and fibrosis. Results demonstrated that A(3)R-deficient mice exhibit enhanced pulmonary inflammation that included an increase in eosinophils. Accordingly, there was a selective up-regulation of eosinophil-related chemokines and cytokines in the lungs of A(3)R-deficient mice exposed to bleomycin. This increase in eosinophil numbers was accompanied by a decrease in the amount of extracellular eosinophil peroxidase activity in lavage fluid from A(3)R-deficient mice exposed to bleomycin, an observation suggesting that the A(3)R is necessary for eosinophil degranulation in this model. Despite an increase in inflammatory metrics associated with A(3)R-deficient mice treated with bleomycin, there was little difference in the degree of pulmonary fibrosis. Examination of fibrotic mediators demonstrated enhanced transforming growth factor (TGF)-beta1 expression, but not a concomitant increase in TGF-beta1 activity. This was associated with the loss of expression of matrix metalloprotease 9, an activator of TGF-beta1, in alveolar macrophages and airway mast cells in the lungs of A(3)R-deficient mice. Together, these results suggest that the A(3)R serves antiinflammatory functions in the bleomycin model, and is also involved in regulating the production of mediators that can impact fibrosis.

Figure 1.

Pulmonary histopathology and inflammation. Mice were killed 14 days after saline or bleomycin exposure, and the lungs were lavaged and processed for hematoxylin and eosin staining. (A) Lung section from an A₃R^+/+ mouse exposed to saline. (B) Lung section from an A₃R^−/− mouse exposed to saline. (C) Lung section from an A₃R^+/+ mouse exposed to bleomycin. (D) Lung section from an A₃R^−/− mouse exposed to bleomycin. Photographs are representative of six to eight mice from each condition; scale bars = 100 μm. (E) Total bronchoalveolar lavage (BAL) cell counts presented as mean cell counts × 10⁴ ± SEM. *Significant difference compared with saline treatment, ^#significant difference compared with bleomycin-treated A₃R^+/+ mice (P < 0.05, n = 6–8).

Figure 2.

Pulmonary eosinophil recruitment. (A) BAL was collected on Days 1, 3, 7, 14, and 21 after bleomycin exposure, and cells were cytospun onto slides, stained with Diff-Quick, and eosinophils counted using a hemocytometer. Triangles depict eosinophils from A₃R^+/+ mice exposed to bleomycin, while squares depict eosinophils from A₃R^−/− mice exposed to bleomycin. Data are presented as mean cell counts × 10⁴ ± SEM. *Significant difference compared with A₃R^+/+ mice (P < 0.05, n = 3–8). (B) Lung sections from A₃R^+/+ and A₃R^−/− mice 14 days after exposure to bleomycin were stained with mMBP-1 to mark eosinophils; scale bar = 100 μm. (C) The number of mMBP-1–positive cells per mm² was quantified in lung sections using ImagePro software. Data are presented as mean eosinophils/mm² ± SEM. (D) Total cellular RNA was isolated from the lungs of wild-type (A₃R +) or A₃R^−/− (A₃R −) mice 14 days after exposure to saline (open bars) or bleomycin (solid bars). Quantitative rtPCR was used to quantify the levels of key eosinophil related cytokines and chemokines. Values were normalized to 18S ribosomal RNA and are presented as mean normalized transcript levels (Δ^−Ct) ± SEM. *Significant difference from saline-treated A₃R^+/+ mice, ^#significant difference from bleomycin-treated A₃R^+/+ mice (P < 0.05, n = 6–8).

Figure 3.

Eosinophil activity. (A) Eosinophil peroxidase activity was measured in the BAL fluid of mice 14 days after exposure to saline or bleomycin. (B) Eosinophil peroxidase activity was measured in the BAL cell pellets from mice 14 days after exposure to saline or bleomycin. Eosinophil peroxidase activity is presented as mean optical density (OD) at 490 nm/ml ± SEM. *Significant difference from saline-treated A₃R^+/+ mice, ^#significant difference from bleomycin-treated A₃R^+/+ mice (P < 0.05, n = 6–8).

Figure 4.

Pulmonary fibrosis in A₃R^+/+ and A₃R^−/− mice. (A) A₃R^+/+ and A₃R^−/− mice were killed 14 days after saline or bleomycin exposure, and the lungs were processed for Masson's trichrome staining. Photographs are representative of six to eight mice from each condition; scale bars = 100 μm. (B) Ashcroft scores were determined by examining multiple sections from lungs of mice from each condition. Data are presented as mean Ashcroft score ± SEM (n = 6–8 lungs per condition). (C) RNA was extracted from the lungs of mice 14 days after saline or bleomycin exposure, and α1 procollagen levels were quantified using quantitative rtPCR. Values were normalized to 18S ribosomal RNA and are presented as mean normalized transcript levels (Δ^−Ct) ± SEM (n = 6–8). (D) Collagen protein levels were measured in whole lungs 14 days after saline or bleomycin exposure using the Sircol assay. Data are presented as mean μg collagen per lung ± SEM (n = 6–8). *Significant difference from saline-treated A₃R^+/+ mice (P < 0.05).

Figure 5.

TGF-β1 expression and activity. Total cellular RNA was isolated from the lungs (A) or BAL cell pellets (B) of wild-type (A₃R +) or A₃R^−/− (A₃R −) mice 14 days after exposure to saline (open bars) or bleomycin (solid bars). Quantitative rtPCR was used to measure TGF-β1 transcript levels. Values were normalized to 18S ribosomal RNA and are presented as mean normalized transcript levels (Δ^−Ct) ± SEM (n = 6–8). (C) Active TGF-β1 protein levels were determined in BAL fluid from mice treated the same as in A and B. Values are presented as mean pg/ml TGF-β1 ± SEM (n = 4–6). *Significant difference from saline-treated A₃R^+/+ mice, ^#significant difference from bleomycin-treated A₃R^+/+ mice (P < 0.05).

Figure 6.

Matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 expression and activity. (A) Total cellular RNA was isolated from the lungs of wild-type (A₃R +) or A₃R^−/− (A₃R −) mice 14 days after exposure to saline (open bars) or bleomycin (solid bars). Quantitative rtPCR was used to quantify the levels MMP-9 transcripts. Values were normalized to 18S ribosomal RNA and are presented as mean normalized transcript levels (Δ^−Ct) ± SEM (n = 6–8). (B) BAL fluid (20 μl from saline-, and 10 and 5 μl from bleomycin-treated samples) were subjected to 10% SDS-PAGE in gels containing 0.02% gelatin. Gels were fixed and stained with 50% methanol and 10% acetic acid that contained 0.3% wt/vol Coomassie blue. MMP-9 and MMP-2 bands were identified by their molecular weight. MMP-9 (C) and TIMP-1 (D) activities were determined in BAL fluid using specific immunoassays. (E) MMP-9 and TIMP-1 BAL fluid activities were used to determine a ratio of MMP-9 to TIMP-1. Data are presented as means ± SEM. *Significant difference from saline-treated A₃R^+/+ mice, ^#significant difference from bleomycin-treated A₃R^+/+ mice (P < 0.05, n = 6–8).

Figure 7.

MMP-9 immunolocalization. Mice were killed 14 days after saline or bleomycin exposure, and the lungs were lavaged and processed for MMP-9 immunolocalization. Immunoperoxidase detection using a MMP-9 antibody was used to localize MMP-9 (brown stain) to inflammatory cells in the lungs of (A) A₃R^+/+ mice exposed to bleomycin and (B) A₃R^−/− mice exposed to bleomycin. The same antibody was used in an immunofluorecence assay to examine MMP-9 expression (green) in BAL cells from (C) A₃R^+/+ mice exposed to bleomycin and (D) A₃R^−/− mice exposed to bleomycin. Blue staining denotes DAPI-stained nuclei. Mast cells were localized in bronchi of (E) A₃R^+/+ mice exposed to bleomycin and (G) A₃R^−/− mice exposed to bleomycin using tolidine blue staining. MMP-9 immunoperoxidase detection (brown stain) was used to localize MMP-9 to mast cells in the bronchi of (F) A₃R^+/+ mice exposed to bleomycin and (G) A₃R^−/− mice exposed to bleomycin. n, neutrophils; Φ, macrophages; arrows denote mast cells. Scale bars: A and B, 50 μm; C and D, 10 μm; H, 10 μm (also applies to E–G).