A3 adenosine receptor-mediated suppression of pro-inflammatory functions of mouse bone marrow neutrophils through inhibition of Rac.

摘要

The goal of this study was to test the possibility that the A3AR subtype participates in mediating the suppressive effects of adenosine on neutrophils. Specific aims were: (1) to comprehensively characterize the expression profile of the A3AR in mouse bone marrow neutrophils and human promyelocytic HL60 cells, (2) to determine whether the A3AR suppresses the pro-inflammatory actions of neutrophils, and (3) to determine the signaling mechanisms by which the A3AR regulates neutrophil functions. The general experimental approach involved the use of the highly selective A3AR agonist CP-532,903 and bone marrow neutrophils obtained from wild-type and AR gene knock-out mice.;Using quantitative real-time RT-PCR and radioligand binding analysis, the A3AR was abundantly expressed in murine neutrophils at levels that were equal to or even greater than the A2AAR. Expression of the A3AR increased more than 25-fold in HL60 cells cultured under conditions that promote neutrophil differentiation. Thus, these initial findings provided compelling evidence in support of the theory that the A3AR plays an important regulatory role in neutrophils. Expression of the A 3AR was not increased in bone marrow neutrophils obtained from mice pre-treated with a large dose of lipopolysaccharide, whereas mRNA and protein expression of the A2AAR was increased 10- and 3-fold, respectively. These studies suggest that, unlike the A2AAR, the A3AR appears to be stably expressed in murine neutrophils and is not transcriptionally induced in response to pro-inflammatory stimuli.;Subsequent work focused on examining the potential role of the A 3AR in regulating stimulated superoxide production and chemotaxis. Pre-treating murine neutrophils with CP-532,903 inhibits neutrophil superoxide production induced by the bacterial tripeptide fMLP, platelet-activating factor, and complement component 5a with a potency and efficacy similar to that of the A2AAR agonist CGS 21680. The selectivity of CP-532,903 for the A3AR was confirmed in assays using bone marrow neutrophils obtained from A2A and A3AR gene knock-out mice. These results demonstrated, for the first time, that stimulating the A3AR suppresses neutrophil superoxide production, suggesting that the A3AR participates along with the A2AAR in mediating the suppressive actions of adenosine on neutrophils. Subsequent studies using a trans-well migration assay system using isolated neutrophils as well as an in vivo model of thioglycollate-induced peritonitis demonstrated that activating the A 3AR also inhibits neutrophil chemotaxis.;Using an ELISA-based assay, it was found that pre-treating neutrophils with CP-532,903 reduced activation of the monomeric GTPase Rac, a central regulator of chemoattractant-induced neutrophil chemotaxis and superoxide production. CP-532,903 inhibited Rac activation via specific interaction with the A3AR, since it was ineffective in neutrophils obtained from A3AR gene knock-out mice. CP-532,903 also inhibited fMLP-induced F-actin formation, a downstream effector function of Rac relevant to neutrophil migration, but it did not alter activation of p38 or ERK1/2. These studies therefore provide strong evidence that the A3AR signals to suppress neutrophil functions by interfering with Rac. Subsequent studies showed that CP-532,903 did not alter fMLP-induced calcium transients or membrane association of P-Rex1, implicating that A3AR stimulation does not inhibit Rac activation by inducing heterologous desensitization of chemoattractant receptors, suppressing calcium signaling, or interfering with translocation of an important guanine nucleotide exchange factor of Rac in neutrophils, P-Rex1.;In conclusion, this study provides evidence supporting the theory that the A3AR, which is abundantly expressed in murine neutrophils, signals to inhibit stimulated superoxide production and chemotaxis by interfering with activation of the monomeric GTPase Rac, thus contributing to the anti-inflammatory actions of adenosine. Several previous studies have demonstrated that selective A3AR agonists provide benefit in experimental animal models of inflammation, including models of sepsis, asthma, arthritis, inflammatory bowel disease, and ischemia/reperfusion injury. However, the precise mechanism of action of these agents remained uncertain. This work provides evidence that these agents are likely to be effective by inhibiting the pro-inflammatory actions of neutrophils. (Abstract shortened by UMI.)