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Department of Clinical and Experimental Medicine, Pharmacology Section, and Department of Clinical and Experimental Medicine, Respiratory Disease Section, and Department of Surgical Sciences, Thoracic Surgery Section, University of Ferrara, Ferrara, Italy
King Pharmaceuticals, Cary, North Carolina
Airway Disease Section, National Heart and Lung Institute, Imperial College London, United Kingdom
ABSTRACT
Rationale: Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of mortality worldwide. Adenosine is an inflammatory regulator that acts through four distinct receptors to mediate pro- and antiinflammatory effects.
Objectives: The primary aim of this study was to investigate the expression, affinity, and density of adenosine receptors in peripheral lung parenchyma from age-matched smokers with COPD (n = 14) and smokers with normal lung function (control group; n = 20).
Methods: Adenosine receptors were analyzed by immunohistochemistry and saturation binding assays using typical antagonist radioligands.
Results: A1, A2A, A2B, and A3 receptors were expressed in different cells in peripheral lung parenchyma. The affinity of A1, A2A, and A3 receptors was significantly decreased in patients with COPD compared with the control group (KD[A1] = 3.15 ± 0.19 vs. 1.70 ± 0.14 nM; KD[A2A] = 7.88 ± 0.68 vs. 1.87 ± 0.09 nM; KD[A3] = 9.34 ± 0.27 vs. 4.41 ± 0.25 nM; p < 0.01), whereas their density was increased (Bmax[A1] = 53 ± 4 vs. 32 ± 3 fmol/mg protein; Bmax[A2A] = 852 ± 50 vs. 302 ± 12 fmol/mg protein; Bmax[A3] = 2,078 ± 108 vs. 770 ± 34 fmol/mg protein; p < 0.01). The affinity of A2B receptors was not altered, but the density was significantly decreased in patients with COPD compared with the control group (Bmax = 66 ± 5 vs. 189 ± 16 fmol/mg protein; p < 0.01). A significant correlation was found between the affinity and density of the adenosine receptors and the FEV1/FVC ratio.
Conclusions: This is the first report showing the presence of adenosine receptors in lung parenchyma in subjects with COPD compared with control smokers. These novel findings strengthen the hypothesis of a potential role played by adenosine receptors in the pathogenesis of COPD.
Key Words: adenosine receptors chronic obstructive pulmonary disease inflammation small airways
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of mortality worldwide (1). COPD is a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lung to noxious particles or gases (1). The main cause of COPD is cigarette smoking (1). Adenosine has been suggested to play a role in the pathogenesis of COPD (2). The exact role of adenosine in the pathogenesis of COPD is unknown and probably complex because adenosine receptors in the lungs, in vitro, and in animal models have pro- and antiinflammatory effects and may also cause bronchoconstriction (3–5). It is accepted that adenosine acts on its cell targets through the interaction with specific receptors classified as A1, A2A, A2B, and A3 (3), and a better understanding of the contribution of these different adenosine receptors to the pathogenesis of COPD may make these receptors potential therapeutic targets (6). Until recently, the limited specificity of available selective agonists and antagonists has made it difficult to identify the expression of the different adenosine receptors. However, in the last few years, significant advances in A1 and A2A adenosine receptor pharmacology have been made through the use of highly potent and selective agonist and/or antagonist radioligands, such as [3H]-1,3-dipropyl-8-cyclopentyl-xanthine ([3H]-DPCPX) and [3H]-4-(2-[7-amino-2-(2-furyl)[1,2,4] triazolo[2,3-a] [1,3,5] triazin-5-yl-amino]ethyl)phenol ([3H]-ZM 241,385), respectively (7, 8). More recently, the pharmacologic characterization of the new, high-affinity, potent, and selective radioligand ([3H]-N-benzo[1,3[dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yl-oxy]-acetamide, [3H]-MRE 2029F20), which is able to bind human A2B adenosine receptors, has allowed a better characterization of this receptor in different human tissues (9). In addition, the discovery of the new, high-affinity, and selective radioligand [3H]-5N-(4-methoxyphenylcarbamoyl) amino-8-propyl-2-(2-furyl) pyrazolo [4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([3H]-MRE 3008F20) that is able to bind the human A3 adenosine receptor with high affinity has allowed its pharmacologic characterization (10, 11). Combined immunohistochemical and radioligand binding studies could be useful to clarify the specific effects determined by differential expression of A1, A2A, A2B, and A3 adenosine receptors.
This article describes, for the first time, a detailed analysis of A1, A2A, A2B, and A3 adenosine receptors expression in peripheral lung parenchyma, the major site of airflow obstruction in COPD (12), using immunohistochemistry, radioligand binding, and real-time quantitative polymerase chain reaction (QPCR). Adenosine receptors were analyzed in age-matched smokers with normal lung function (control group) and patients with COPD. Moreover, we have investigated whether changes in affinity and density of these receptors are correlated with clinical parameters, such as the FEV1/FVC ratio. We have also investigated, using the in vitro model of human lung type 2 alveolar-like cells (A549 cells), the effect of proinflammatory cytokines on adenosine receptor subsets. Some of the results of this study have been previously reported in abstract form (13, 14).
METHODS
Subjects
All subjects were recruited from the Section of Respiratory Diseases of the University Hospital of Ferrara, Italy. We recruited 34 subjects undergoing lung resection for a solitary peripheral carcinoma. For the immunohistochemistry study, six subjects were smokers with COPD, and 12 subjects were smokers with normal lung function (Table 1). In addition, eight smokers with COPD and eight smokers with normal lung function were selected for the binding and real-time QPCR experiments (Table 2). All former smokers had stopped smoking for more than 1 yr. COPD and chronic bronchitis were respectively defined, according to international guidelines, as the presence of postbronchodilator FEV1/FVC ratio of less than 70% or the presence of cough and sputum production for at least 3 mo in each of 2 consecutive yr (1). All subjects were free from preoperative chemotherapy and radiotherapy. Pulmonary function tests were performed as previously described (15) according to published guidelines. Predicted values for the different measures were calculated from the regression equations published by Quanjer (16). The study was approved by the local ethics committee of the University Hospital of Ferrara, and informed consent was obtained from each participant in accordance with the principles outlined in the Declaration of Helsinki.
Lung Tissue Processing
Two to four randomly selected tissue blocks were taken from the subpleural parenchyma of the lobe obtained at surgery; areas invaded by tumor were avoided. Tissue specimens were cut for immunohistochemical analysis and placed on charged slides as previously reported (17). Another piece of lung parenchyma was taken and used in radioligand binding and real-time QPCR experiments.
Immunostaining for A1, A2A, A2B, and A3 Adenosine Receptors in Lung Sections
Single and double immunohistochemical staining was performed as previously described (17). A detailed description of the methods used is presented in the online supplement.
Radioligand Binding Assays for A1, A2A, A2B, and A3 Adenosine Receptors
The lung parenchyma tissues were used to prepare a membrane suspension for saturation binding assays as previously described (7–9). Saturation binding experiments to A1, A2A, A2B, and A3 adenosine receptors were performed using [3H]-DPCPX, [3H]-ZM241385, [3H]-MRE2029F20, and [3H]-MRE3008F20 as radioligands, respectively. A detailed description of the methods used is presented in the online supplement.
Real-Time QPCR in Peripheral Lung Parenchyma
Real-time QPCR assay was performed using specific primers for A1, A2A, A2B, and A3 receptor mRNA (18). A description of the methods used is presented in the online supplement.
Adenosine Receptor Expression and Modulation by Proinflammatory Cytokines in Human Lung Type 2 Alveolar-like Cells
Human lung type 2 alveolar-like cells (A549 cells; ATCC CCL185) were grown as previously described and used for real-time QPCR, radioligand binding, and Western blotting assays as described in the online supplement.
Statistical Analysis
A weighted, nonlinear, least-squares curve fitting program ligand (19) was used for computer analysis of saturation experiments. Analysis of data was performed by one-way analysis of variance. Differences between control subjects and subjects with COPD were analyzed with Dunnett's test and were considered significant at p < 0.05. All data are reported as mean ± SEM.
RESULTS
Clinical parameters and pulmonary function of the patients are summarized in Tables 1 and 2. The two groups of subjects were similar with regard to age and sex, and there was no significant difference in the smoking history (pack-years) between smokers with COPD and smokers with normal lung function. No difference was found in the prevalence of chronic bronchitis between groups. As expected from the selection criteria, smokers with COPD had a significantly lower FEV1 and FEV1/FVC ratio as compared with control smokers.
Immunohistochemical Localization of A1, A2A, A2B, and A3 Adenosine Receptors in Peripheral Lung Parenchyma
Peripheral lung parenchyma is a mixture of many cell types, including bronchiolar and alveolar epithelial cells, endothelial, smooth muscle cells (localized in bronchiolar and vessel walls), fibroblasts, mast cells, neutrophils, macrophages, and lymphocytes. Immunohistochemical analysis with anti-A2A adenosine receptor antibody demonstrated staining of the bronchiolar and alveolar epithelial cells, bronchiolar smooth muscle cells, endothelial cells, and infiltrating cells with no significant difference seen between patients with COPD and the control group (Figure 1). A similar expression pattern was seen for the A3 receptors (Figure 2). In contrast, A2B receptor was expressed only in mast cells and macrophages (Figure 3), whereas A1 receptor was expressed only in a few alveolar macrophages (Figure 4). A quantitative score for the immunohistochemical staining is presented in Table 3.
Density and Affinity of A1, A2A, A2B, and A3 Adenosine Receptors in Peripheral Lung Parenchyma
The affinity and density of A1, A2A, A2B, and A3 adenosine receptors in membranes of peripheral lung from control group and subjects with COPD are shown in Figures 5 and 6.
The affinity of A1 receptors was significantly decreased in patients with COPD compared with the control group (KD 3.15 ± 0.19 vs. 1.70 ± 0.14 nM, p < 0.01; Figure 5A). However, A1 receptor density was significantly increased in patients with COPD compared with the control group (Bmax 53 ± 4 vs. 32 ± 3 fmol/mg protein, p < 0.01; Figure 5A).
Similarly, the affinity of A2A and A3 receptors was signifi- cantly decreased in patients with COPD compared with the control group, whereas their density was increased (KD 7.88 ± 0.68 vs. 1.87 ± 0.09 nM for A2A [Figure 5B] and 9.34 ± 0.27 vs. 4.41 ± 0.25 nM for A3 [Figure 6B]; Bmax 852 ± 50 vs. 302 ± 12 fmol/mg protein for A2A [Figure 5B], and 2,078 ± 108 vs. 770 ± 34 fmol/mg protein for A3 [Figure 6B]; p < 0.01).
In comparison, the affinity of A2B receptors was not significantly different between the control group and patients with COPD (KD 2.46 ± 0.45 vs. 2.10 ± 0.26 nM; Figure 6A). However, the density of A2B receptors was significantly decreased in patients with COPD compared with the control group (Bmax 66 ± 5 vs. 189 ± 16 fmol/mg protein, p < 0.01; Figure 6A).
Expression of A1, A2A, A2B, and A3 Adenosine Receptors mRNA in Peripheral Lung Parenchyma
Using real-time QPCR, we examined the expression of mRNA for all four adenosine receptors in the peripheral lung from both groups of subjects (Figure 7). GAPDH mRNA was used as an internal control for loading (20, 21). The fold increase in ratio between A2A receptor/GAPDH (2.46 ± 0.25, p < 0.01) and A3 receptor/GAPDH mRNA (1.71 ± 0.18, p < 0.01) was significantly increased in patients with COPD (Figure 7). In contrast, the A2B receptor/GAPDH mRNA ratio was significantly decreased (0.47 ± 0.05 vs. 1.03 ± 0.12, p < 0.01) in patients with COPD (Figure 7). No differences in A1 receptor mRNA expression were seen between groups (Figure 7). Similar results were obtained by using -actin mRNA/ratio as internal control (data not shown).
Effect of Inflammatory Stimuli on Adenosine Receptor Expression in A549 Cells
Saturation binding experiments on A549 membranes revealed the presence of A1, A2A, and A3 adenosine receptors. However, A2BR were not detectable on A549 membranes. Interleukin (IL)-1 (1 ng/ml) and tumor necrosis factor (TNF)- (10 ng/ml) significantly induced A2A receptor expression (Bmax) 1.5-fold without affecting binding affinity (Table 4). Up-regulation of A2A receptor expression was attenuated by pretreatment of cells with the nuclear factor (NF)-B inhibitor AS602868. Similar data were seen for the induction of A2A receptor mRNA and protein as determined by real-time QPCR and Western blotting analysis (Figure 8). In contrast, neither IL-1 nor TNF- was able to modify the affinity and the density of the A1 and A3 adenosine receptors (Table 4).
Correlation between Binding and Clinical Parameters
A direct correlation was found between the FEV1/FVC ratio and A2A (Figure 9A) and A3 (Figure 9B) receptor affinity (KD) and density (Bmax). An inverse correlation was found between the affinity and density of A2B receptors and the FEV1/FVC ratio (Figure 9C). No other significant correlation was found between the affinity or density of A1 receptors and any clinical parameters.
DISCUSSION
In this article, we report for the first time the localization, mRNA expression, affinity, and density of A1, A2A, A2B, and A3 adenosine receptors in peripheral lung parenchyma from age-matched smokers with normal lung function (control group) and patients with COPD. We have examined the peripheral lung because this is the major site of airflow obstruction in patients with COPD and because histopathologic studies have demonstrated that most of the airway inflammation in COPD is localized in the small airways and lung parenchyma (12). We have demonstrated that A2B receptors are expressed only in mast cells and macrophages and that there is reduced expression of their protein (Bmax) and mRNA in peripheral lung parenchyma from patients with COPD compared with the control group. These data suggest that there may be a compensatory feedback mechanism regulating A2B receptor expression. Such reduction could be explained as the consequence of an increased adenosine concentration in peripheral lung with secondary down-regulation of the A2B receptors. The role of mast cells in the pathogenesis of COPD is controversial (12) but, in vitro, A2B receptor stimulation has been shown to enhance the release of proinflammatory mediators from human lung mast cells (22, 23). On the contrary, the number of alveolar macrophages is highly increased in the peripheral lung of patients with COPD compared with control smokers, and they seem to represent the key inflammatory cells in the pathogenesis of COPD (4). Furthermore, there is a significant inverse correlation between binding parameters of the A2B adenosine receptor and the FEV1/FVC ratio, suggesting a potential role of this receptor in the pathogenesis of airflow obstruction in COPD.
Radioligand binding and real-time QPCR experiments have demonstrated a significant decrease of the affinity, associated with an increased density of the A2A and A3 adenosine receptor protein and mRNA in patients with COPD compared with the control group.
In vitro studies have demonstrated that the long-term exposure of target cells to adenosine causes desensitization of adenosine receptors (3). We hypothesize that high concentrations of adenosine in the peripheral lung of patients with COPD might mediate desensitization of the A2A and A3 receptors. Consequently, the up-regulation of the A2A and A3 receptors may represent a compensatory response mechanism and may contribute to the antiinflammatory effects mediated by the stimulation of these receptors. This is in keeping with the increased expression in vitro of A2A receptor after exposure of A549 cells to the proinflammatory cytokines IL-1 and TNF- through the activation of the key proinflammatory NF-B pathway. Radioligand binding experiments show the presence of A1, A2A, and A3 adenosine receptors on A549 membranes. The proinflammatory cytokines IL-1 and TNF- are able to increase the A2A density but not that of A1 and A3 receptors. On the contrary, A2B adenosine receptors are not detectable on A549 membranes.
In the lower airways of patients with COPD, compared with smokers with normal lung function, IL-1 and TNF- expression are increased (4), and the NF-B pathway is activated (17). Consistent with this hypothesis, stimulation of the A2A and A3 receptors in vitro has antiinflammatory effects (10, 24, 25). We have also found a direct correlation between KD and Bmax of A2A and A3 receptors and the FEV1/FVC ratio, indicating that these adenosine subtypes may play a role in the pathogenesis of airflow obstruction in COPD. However, as with all association studies, it is necessary to confirm the data in other groups of COPD, and until selective agonists or antagonists are used clinically in patients with COPD, we are unlikely to be able to resolve whether specific subsets of adenosine receptors have distinct roles in the pathogenesis or progression of COPD.
There is also a significant decrease of the affinity associated with an increased density of the A1 receptors in patients with COPD compared with the control group. However, the radioligand binding and immunohistochemical studies demonstrate a low A1 receptor density in the peripheral lung parenchyma, suggesting a secondary role for this receptor in the pathogenesis of COPD in contrast to the protective role of A1 receptor in the pulmonary inflammation and injury seen in adenosine deaminase–deficient mice (26).
In conclusion, our results suggest that (1) A1, A2A, A2B, and A3 adenosine receptors are differentially expressed in peripheral lung parenchyma; (2) the affinity and/or density of these receptors are altered in patients with COPD compared with control smokers with normal lung function; and (3) there is a significant correlation between the density and affinity of A2A, A2B, and A3 and the FEV1/FVC ratio, an established index of airflow obstruction. These data suggest a potential role of adenosine receptors in the pathogenesis of COPD. However, to understand the relative importance of each adenosine receptor in the pathogenesis of COPD, we need controlled clinical trials using specific agonists and antagonists, some of which are already in preclinical and early clinical development (27, 28).
FOOTNOTES
The work in our research laboratories is partially supported by GlaxoSmithKline, Associazione per la Ricerca e la Cura dell'Asma, and King Pharmaceuticals; the work reported in this article, however, has not been funded by any pharmaceutical companies.
These authors contributed equally to this article.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200506-869OC on December 1, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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