Activation of the A(3) adenosine receptor suppresses superoxide production and chemotaxis of mouse bone marrow neutrophils

Abstract

Adenosine is formed in injured/ischemic tissues, where it suppresses the actions of essentially all cells of the immune system. Most of the anti-inflammatory actions of adenosine have been attributed to signaling through the G(s) protein-coupled A(2A) adenosine receptor (AR). Here, we report that the A(3)AR is highly expressed in murine neutrophils isolated from bone marrow. Selective activation of the A(3)AR with (2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-532,903) potently inhibited mouse bone marrow neutrophil superoxide generation and chemotaxis induced by various activating agents. The selectivity of CP-532,903 was confirmed in assays using neutrophils obtained from A(2A)AR and A(3)AR gene "knockout" mice. In a model of thioglycollate-induced inflammation, treating mice with CP-532,903 inhibited recruitment of leukocytes into the peritoneum by specifically activating the A(3)AR. Collectively, our findings support the theory that the A(3)AR contributes to the anti-inflammatory actions of adenosine on neutrophils and provide a potential mechanistic explanation for the efficacy of A(3)AR agonists in animal models of inflammation (i.e., inhibition of neutrophil-mediated tissue injury).

Figure 1

Purity of mouse bone marrow neutrophil preparations obtained by Percoll gradient separation (A) coupled with immunomagnetic selection using the anti-Gr-1 antibody (B). Shown are representative cytospin images and results of flow cytometry analysis using Gr-1 and CD11b as markers. The percentage of neutrophils in the entire cell population is indicated. n=3.

Figure 2

Expression of AR subtypes in mouse bone marrow neutrophils. (A) mRNA levels of AR subtypes in mouse bone marrow neutrophils quantified by real time RT-PCR, n = 5–18. (B and C) Protein levels of AR receptor subtypes in Percoll gradient purified mouse bone marrow neutrophils quantified using radioligand binding analysis. Total binding (fmols/mg of total membrane protein) of the A_2AAR antagonist radioligand ¹²⁵I-ZM241385 ( 0.4 nM; B) or the A₁/A₃AR agonist radioligand [¹²⁵I]I-AB-MECA ( 0.4 nM; C) to neutrophil membranes in the presence of vehicle or various competitors at the concentrations indicated. Results are presented as the mean ± SEM. *, p < 0.05 versus the vehicle-treated group by one-way ANOVA and Dunnett’s t test, n = 3.

Figure 3

Changes in AR mRNA (A) and protein (B) expression in bone marrow neutrophils from LPS-treated mice. Mice were injected ip with either vehicle or 10 mg/kg LPS. Four hours later, bone marrow neutrophils were obtained by Percoll gradient separation. AR mRNA levels were determined by real time RT-PCR normalized to 18S RNA. A_2A and A₃AR protein levels were determined by radioligand binding analysis with crude membrane preparations using ¹²⁵I-ZM 241385 (~0.4 nM) or [¹²⁵I]I-AB-MECA (~0.4 nM). Specific binding was defined by including 1 μM of the respective non-radiolabeled ligand in the assays. The data (mean ± SEM; n = 3) are presented as the fold increase in mRNA or protein expression in neutrophils from LPS-treated mice compared to vehicle-treated mice.

Figure 4

Superoxide production by Percoll gradient purified mouse bone marrow neutrophils in response to fMLP (1 μM). Studies were conducted with naïve neutrophils, neutrophils primed with TNF-α (30 min incubation with 100 ng/mL rm TNF-α), or treated with superoxide dismutase (SOD). Superoxide production was measured using the chemiluminescent probe MCLA. Relative light units (RLUs) were obtained for 3 minutes at 10 second intervals. The inset depicts the cumulative relative light units generated over 3 minutes after subtracting baseline values. Results are presented as the mean ± SEM. *, p < 0.05 versus the unprimed fMLP-induced group by one-way ANOVA followed by a Student’s t test, n = 3.

Figure 5

Effect of pretreating with AR agonists on superoxide production by Percoll gradient purified mouse bone marrow neutrophils in response to fMLP (A), C5a (B), PAF (C), or PMA (D). Neutrophils were pretreated with either vehicle or the AR agonists for 30 min in the presence of ADA (1 unit/mL), and then stimulated with the activating agents while measuring superoxide production using the chemiluminescent probe MCLA. In studies with fMLP, the effect of the agonists on superoxide production was tested with both unprimed and TNF-α-primed (100 ng/mL) neutrophils. The data (mean ± SEM) are presented as the percentage of superoxide produced compared to the vehicle-treated group. *, p < 0.05 versus the vehicle-treated group by one-way ANOVA and Dunnett’s t test, n = 4 - 6.

Figure 6

Inhibition of fMLP-induced (1 μM) superoxide production by Percoll gradient purified bone marrow neutrophils from WT, A_2AKO, or A₃KO mice by increasing concentrations of CGS 21680 (A) or CP-532,903 (B). Results (mean ± SEM; n = 3) are displayed as percentage of superoxide produced by the vehicle-treated group.

Figure 7

Effect of duration of pretreatment with CGS 21680 or CP-532,903 on superoxide production by Percoll gradient purified bone marrow neutrophils in response to fMLP. Neutrophils were incubated (37° C) with vehicle, CGS 21680 (100 nM), or CP-532,903 (100 nM) in the presence of ADA (1 unit/mL) for the times indicated prior to stimulation with fMLP (1 μM). Superoxide produced was measured using the chemiluminescent probe MCLA (0.5 μM). Results (mean ± SEM, n = 4) are presented as the percentage of superoxide produced compared to the corresponding vehicle-treated group.

Figure 8

Effect of CP-532,903 on neutrophil chemotaxis in trans-well migration assays using Percoll gradient purified mouse bone marrow neutrophils. Fluorescently labeled (Calcein-AM) neutrophils were added into upper wells, with fMLP and/or CP-532,903 in the lower wells in the presence of ADA (1 unit/mL), separated by a polycarbonate membrane. After incubating at 37° C for 35 min, migrated cells were quantified and chemotaxis was calculated as described in Methods. (A) Effect of adding increasing concentrations of CP-532,903 into lower wells. (B) Effect of adding increasing concentration of CP-532,903 into bottom wells on fMLP-induced (1 μM) chemotaxis of unprimed and TNF-αprimed neutrophils. Results are presented as the mean ± SEM. n = 3–5.

Figure 9

Effect of pretreating with CP-532,903 or CGS 21680 on fMLP-induced chemotaxis of unprimed WT (A), TNF-αprimed WT (B), TNF-α primed A_2AKO (C) or TNF-αprimed A₃KO (D) Percoll gradient purified mouse bone marrow neutrophils. Neutrophils were incubated (37° C) with vehicle, CP-532,903 (100 nM), or CGS 21680 (100 nM; A and B) for 30 min in the presence of ADA (1 unit/mL) with (B, C and D) or without rm TNF-α (100 ng/mL; A), prior to addition to the upper wells of trans-well assays containing increasing concentrations of fMLP in lower wells. The chemotaxis index was calculated as described in Methods after allowing the cells to migrate for 35 min. Results are presented as the mean ± SEM. *, p < 0.05 versus the vehicle-treated group by two-way ANOVA, n = 5–16.

Figure 10

Effect of pretreating with CP-532,903 on chemotaxis of Percoll gradient purified mouse bone marrow neutrophils towards various chemoattractants. Neutrophils were incubated (37° C) with vehicle or CP-532,903 (100 nM) for 30 min in the presence of ADA (1 unit/mL) with or without rm TNF-α(100 ng/mL), prior to addition to the upper wells of trans-well assays containing increasing concentrations of C5a (A and B), IL-8 (C and D), or PAF (E and F) in lower wells. The chemotaxis index was calculated as described in Methods after allowing the cells to migrate for 35 min. Results are presented as the mean ± SEM. p < 0.05 versus the vehicle-treated group by two-way ANOVA, n = 3–6.

Figure 11

Effect of pretreating with CP-532,903 on fMLP-induced chemokinesis of Percoll gradient purified mouse bone marrow neutrophils. Neutrophils were incubated (37° C) with vehicle or CP-532,903 (100 nM) for 30 min in the presence of ADA (1 unit/mL) with or without rm TNF-α(100 ng/mL), prior to addition to the upper wells of trans-well assays containing increasing concentrations of fMLP in both the upper and lower wells. The chemokinesis index was calculated as described in Methods after allowing the cells to migrate for 35 min. Results are presented as the mean ± SEM. *, p < 0.05 versus the vehicle-treated group by two-way ANOVA, n = 5.

Figure 12

Effect of CP-532,903 on leukocyte accumulation during thioglycollate-induced peritonitis. Mice were injected i.v. with either vehicle or CP-532,903 (100 μg/kg) immediately prior to a single intraperitoneal injection of thioglycollate (2 ml of a 3% solution). Four hours later, the number of leukocytes within peritoneal exudates was quantitated, as described in Methods. Results are presented as the mean ± SEM. *, p < 0.05 versus the vehicle-treated group by Student’s t test, n = 6.