Activation of the A(3) adenosine receptor inhibits fMLP-induced Rac activation in mouse bone marrow neutrophils

Abstract

Adenosine is released from injured or hypoxic tissues where it exerts numerous anti-inflammatory effects including suppression of neutrophil functions. Although most previous work has implicated the A(2A)AR, we have recently shown that selective activation of the abundantly expressed A(3)AR inhibits neutrophil superoxide production and chemotaxis providing a potential mechanistic explanation for the efficacy of A(3)AR agonists in experimental animal models of inflammation. In this study, we hypothesized that the A(3)AR suppresses neutrophil functions by inhibiting the monomeric GTPase Rac, a central regulator of chemokine-directed neutrophil migration and superoxide production. We found that pre-treating neutrophils with the highly selective A(3)AR agonist CP-532,903 reduced fMLP-induced Rac activation using an ELISA-based assay that detects all three Rac isoforms. CP-532,903 also inhibited fMLP-induced F-actin formation, a downstream effector function of Rac relevant to neutrophil migration, but not activation of ERK1/2 or p38. Pre-treating neutrophils with CP-532,903 did not stimulate cAMP production or alter fMLP-induced calcium transients, implicating that A(3)AR stimulation does not inhibit Rac activation or neutrophil activities by suppressing Ca(2+) signaling, elevating the intracellular concentration of cAMP, or by cross-desensitizing fMLP receptors. Our results suggest that activation of the A(3)AR signals to suppress neutrophil functions by interfering with the monomeric GTPase Rac, thus contributing to the ant-inflammatory actions of adenosine.

Figure 1

Effect of CP-532,903 on fMLP-induced Rac activation in mouse bone marrow neutrophils obtained from wild-type mice and from A₃ARKO mice. Rac activity in whole-cell lysates was quantified using an ELISA-based assay kit (G-LISA kit, Cytoskeleton, Inc), as described under Materials and Methods. (A) The time-course of Rac activation following stimulation with fMLP (1 μM). (B & C) Rac activity 15 sec after the addition of 1 μM fMLP to wild-type (B) and A₃KO (C) cells pretreated for 30 min with 100 nM CP-532,903 (CP). The data are presented as the percent increase over baseline activity. In control assays, cell lysates were incubated with 200 μM GTPγS. All assays were conducted in the presence of 1 unit/ml ADA. Mean ± SEM. *, p < 0.05 versus the fMLP-treated group by one-way ANOVA and Bonferroni’s t test, n = 3-7.

Figure 2

Effect of CP-532,903 on fMLP-induced F-actin formation in mouse bone marrow neutrophils. Neutrophils were pretreated with vehicle (A) or 100 nM CP-532,903 (B) for 30 min at 37°C in the presence of 1 unit/ml ADA, and then stimulated with 1 μM fMLP for 30 sec. Cells were stained with FITC-conjugated phalloidin and intracellular fluorescence was quantified by flow cytometry assessing a total of 10,000 cells per sample. Mean ± SEM. *, p < 0.05 versus the vehicle-treated group by Student’s t test, n = 8.

Figure 3

Effect of CP-532,903 on fMLP-induced activation of ERK1/2 and p38 in mouse bone marrow neutrophils. (A) Representative Western immunoblots showing phosphorylated and total levels of ERK1/2 and p38. (B) and (C) show the results of densitometric analysis of the Western immunoblots for ERK1/2 and p38, respectively. Ratios of phosphorylated to total protein were normalized to baseline levels. Mean ± SEM. n = 3-4.

Figure 4

Changes in intracellular [Ca²⁺] in mouse bone marrow neutrophils in response to fMLP. The cells were pretreated for 30 min with vehicle or CP-532,903 (100 nM) prior to measurement of intracellular [Ca²⁺] during stimulation with fMLP (1 μM). The intracellular [Ca²⁺] was measured in suspended cells loaded with FURA-2/AM in HBSS containing 1 unit/ml ADA, as described in Materials and Methods. The data shown are representative of 3-4 independent experiments.

Figure 5

CP-532,903 does not stimulate cAMP production in mouse bone marrow neutrophils. Neutrophils suspended in HBSS containing 1 unit/ml ADA and 20 μM Ro 20-1724 were stimulated with vehicle or increasing concentrations of CP-532,903 for 15 min. In control experiments, the cells were stimulated with the A_2AAR agonist CGS 21680 (1 μM) or forskolin (10 μM). The assays were terminated by adding 0.15 N HCl. cAMP in the acid extract was determined by radioimmunoassay. Mean ± SEM. *, p < 0.05 versus the vehicle-treated group by one-way ANOVA and Dunnett’s t test, n = 3.

Figure 6

Treating neutrophils with CP-532,903 (100 nM) directly produced a transient increase in ERK1/2 phosphorylation (A) but did not induce changes in the intracellular [Ca²⁺] at concentrations as high as 10 μM (B). The data shown are representative of three independent experiments.