Female 6- to 8-week old C57BL/6 mice were obtained from the Center for Animal Experiment of Wuhan University (Wuhan, China) and maintained in the Animal Biosafety Level 3 Laboratory of the university. All animals were bred at the animal facilities under specific pathogen-free conditions. Animal experiments were conducted under the approval of the Institutional Animal Care and Use Committee of Wuhan University.

The mice were immunized intraperitoneally with 20 μg OVA (Sigma-Aldrich, St. Louis, MO, USA) adsorbed to 2 mg aluminum hydroxide (Thermo Fisher Scientific Inc., Rockford, IL, USA) in 200 μl PBS on days 0 and 14 and challenged with 100 μg OVA in 50 μl PBS by intranasal (i.n.) administration for over 3 consecutive days (days 25–27). Age- and gender-matched control mice were treated in the same manner with PBS as a substitute for OVA. Apyrase (diluted with PBS, 0.2 IU/g body weight; Sigma-Aldrich) or PBS was intraperitoneally administered to the OVA-sensitized mice prior to each OVA challenge.

At 48 h after the final challenge, the mice were sacrificed and the left lungs were excised. The lungs were fixed in 4% paraformaldehyde (PFA) buffer, dehydrated, embedded and sectioned. Paraffin-embedded 5-μm sections were stained with hematoxylin and eosin (H&E) to evaluate airway inflammation and periodic acid-Schiff (PAS) to evaluate goblet cell hyperplasia and mucus secretion. A total of 4–5 sections were evaluated per lung under a magnification of ×200. The inflammation degree of peribronchial and perivascular regions was assessed on a subjective scale of 0–3, as previously described (18). A value of 0 was regarded as undetectable inflammation, a value of 1 as occasional cuffing with inflammatory cells, a value of 2 as bronchi or vessels surrounded by a thin layer of inflammatory cells (1–5 layer cells), and a value of 3 was regarded as bronchi or vessels surrounded by a thick layer of inflammatory cells (>5 layers of cells).

The location of CD39 in the lungs obtained from the mice was detected by immunofluorescence. Briefly, 5-μm-thick sections were incubated with a rabbit anti-mouse CD39 antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. For detection, Rhodamine-labeled goat anti-rabbit secondary antibody (Santa Cruz Biotechnology, Inc.) was used. The nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Sigma-Aldrich).

At 48 h after the final challenge, the mice were sacrificed and bronchoalveolar lavage was performed 3 times with 0.5-ml aliquots of PBS containing 1 mM sodium EDTA. Cells in the BALF were collected by centrifugation and were counted with a hemocytometer. Smears of cells were prepared by cytospin and stained with Wright-Giemsa in order to differentiate the inflammatory cells (eosinophils, neutrophils and lymphocytes). Approximately 400 cells were counted in each random location. The supernatants in the BALF were collected and stored at −70°C until use.

The right upper lung lobes were measured for gene transcript levels. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo, Tokyo, Japan) according to the manufacturer’s instructions. qPCR was conducted in triplicate using SYBR Premix Ex Taq™ (Takara Bio, Inc., Otsu, Japan). The primer sequences are presented in Table I. The thermal cycling conditions were as follows: denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 61°C for 10 sec and extension at 72°C for 10 sec. The data were normalized to the internal reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative expression of the target gene was calculated using the comparative 2^−ΔΔCt method.

Protein was extracted from the right median lobes using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Total protein (60 μg) was separated by electrophoresis on SDS-PAGE gels, transferred onto PVDF membranes (Millipore Corp., Billerica, MA, USA), blocked with TBST-containing 5% non-fat dried milk at room temperature for 1 h and probed with rabbit anti-mouse CD39 or GAPDH (1:200, Santa Cruz Biotechnology, Inc.). The membranes were incubated with primary antibody overnight at 4°C and then with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:5,000; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Chemoluminescence images were captured with ECL (Beyotime Institute of Biotechnology) using the Fusion Fx7 image acquisition system (Vilber Lourmat, Marne La Vallée, France). The quantification of the bands was performed by densitometry using ImageJ software. The CD39 protein expression level was calculated by densitometry relative to GAPDH.

The levels of cytokines were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. The lower limit of detection was 4 pg/ml for interleukin (IL)-4 and IL-5. All ELISA kits for cytokines were purchased from eBioscience, Inc. (San Diego, CA, USA).

Immature DCs were prepared from bone marrow progenitors. Bone marrow mononuclear cells were prepared from femur bone marrow suspensions of C57BL/6 male mice (5–6 weeks old) and then cultured at a density of 2×10⁶ cells/ml in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 10 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng/ml recombinant murine IL-4 (both from PeproTech, Rocky Hill, NJ, USA). Non-adherent cells were gently washed out on the 3rd day of culture. On days 5 and 7, the medium was refreshed. The purity of the DCs was at least 90%. On day 8, the BMDCs were incubated with 1 μg/ml lipopolysaccharide (LPS; Sigma-Aldrich) overnight and collected for subsequent experiments.

A migration assay was performed in 24-well Transwell chambers with 5 μm pore size polycarbonate filters (Corning Life Sciences, Tewksbury, MA, USA). Various concentrations of ATP diluted in RPMI-1640 containing 0.5% bovine serum albumin (BSA) were added to the lower chambers in a volume of 0.6 ml; 0.1 ml RPMI-1640 containing DCs (2×10⁵ cells/well) was added to the upper chambers followed by culture at 37°C for 4 h. In some experiments, apyrase (1 IU) was added to the medium containing ATP in the lower chambers. The number of migrated DCs to the lower chambers was calculated using a hemocytometer. The results are presented as the chemotactic index and calculated as the number of cells in the lower chamber containing the different stimuli divided by the number of cells in the chamber containing medium alone.

The data are expressed as the means ± SD, representing at least 2 independent experiments with consistent results (n=4–6 mice per group). The data from 1 representative experiment of 3 are shown. The differences were compared using the unpaired Student’s t-test for 2 groups or one-way ANOVA analysis for 3 groups. Statistical analysis was performed using SPSS 17.0 software (IBM SPSS, Chicago, IL, USA). A value of P<0.05 was considered to indicate a statistically significant difference.

We investigated the distribution of CD39 in the lungs obtained from mice by immunofluorescence assay. As shown in Fig. 1A, CD39 was located at the cytomembrane and cytoplasm of bronchial epithelial cells (red area indicates the positive location). The fluorescence intensity was reduced in the lungs from OVA-sensitized and challenged (asthma) mice compared to that in the lungs from PBS-sensitized and challenged (control) mice. We further assessed the expression levels of CD39 in the lungs from the control and asthmatic mice. At the mRNA level, CD39 expression was decreased in the asthmatic mice, as shown by qPCR (Fig. 1B). A marked decrease in CD39 protein expression was also observed in the asthmatic mice, as shown by western blot analysis (Fig. 1C). These data indicate that a reduction in CD39 expression is associated with OVA-induced allergic airway inflammation.

Having shown that a reduced CD39 expression is associated with OVA-induced allergic airway inflammation, we exogenously supplemented apyrase, an eATP scavenger, to investigate its effects on airway inflammation. Lung tissues were collected 48 h after the final OVA challenge. In histological analysis, the OVA-exposed mice showed cardinal pathological features of asthma-like inflammation. In contrast to the PBS-sensitized and challenged controls (Fig. 2A), OVA-sensitized and challenged mice displayed numerous inflammatory cells in the peribronchiolar and perivascular zones (Fig. 2B). Compared to treatment with PBS, treatment with apyrase markedly decreased the number of eosinophil-rich inflammatory cells infiltrating the peribronchiolar and perivascular regions (Fig. 2C). As shown in Fig. 2D–F, PAS staining was used to detect goblet cells. In contrast to the controls (Fig. 2D), the OVA-exposed mice (Fig. 2E) showed goblet cell hyperplasia in the airways, which was significantly reduced by treatment with apyrase (Fig. 2F). OVA exposure markedly increased the inflammation scores of the peribronchial and perivascular regions (Fig. 3), compared to PBS sensitization and challenge. The increased lung inflammation following exposure to OVA was reduced by approximately 40% following treatment with apyrase. Taken together, these results reveal that apyrase significantly reduces OVA-induced inflammatory cell infiltration into the lungs, goblet cell hyperplasia in the airways and airway inflammation.

BALF was collected 48 h after the final OVA challenge and the number of the total cells and the different inflammatory cells were counted. Exposure to OVA markedly increased the number of eosinophils, lymphocytes and neutrophils, compared to PBS sensitization and challenge (Fig. 4). The administration of apyrase markedly reduced the number of eosinophils, lymphocytes and neutrophils in BALF, compared to the PBS-treated asthmatic mice. In addition, a reduction in the number of total cells was observed in the apyrase-treated mice.

Given the essential role of Th2 cytokines in triggering the allergic inflammatory response, we detected the levels of IL-4 and IL-5 in BALF. OVA sensitization and challenge markedly increased the concentration of cytokines in BALF, as shown by ELISA (Fig. 5A) compared to PBS sensitization and challenge. The increased levels of cytokines in BALF were significantly reduced by apyrase treatment.

GATA3, the key transcription factor, controls the differentiation of Th2 cells. We further determined whether apyrase decreases GATA3 mRNA expression in the lungs obtained from asthmatic mice. In the OVA-exposed mice, the mRNA level of GATA3 was increased by 4-fold in the lung tissues compared with that in the PBS-sensitized and challenged control mice (Fig. 5B). The administration of apyrase effectively reduced the expression of GATA3 in the lung tissues compared with the PBS-treated asthmatic mice (Fig. 5B).

Our results revealed that apyrase attenuated airway inflammation. ATP is a chemoattractant for DCs in vitro (8,19). Therefore, we explored whether apyrase decreases the chemotactic migration of DCs towards ATP. Various concentrations of ATP were added to the medium in the lower chamber. As shown in Fig. 6A, the DCs showed the strongest chemotactic activity with 10 μM ATP. Apyrase was then added to the medium containing 10 μM ATP. We found that upon the administration of apyrase, the chemotactic migration of DCs towards ATP was markedly decreased compared to the cells treated with ATP alone (Fig. 6B).