To characterize the basic functions of GPR in phagocytes we

To characterize the basic functions of GPR84 in phagocytes, we have utilized the receptor selective antagonist GLPG1205 (a small selective antagonist of GPR84 developed by Galapagos NV [14]) together with ZQ16 (2-(hexylthio) pyrimidine-4,6-diol), a potent GPR84 selective agonist identified in a high throughput screening study [15]. We show that GPR84 is functionally expressed by human neutrophils, monocytes and monocyte-derived macrophages (MDMs). The GPR84 selective agonist triggered the G-protein coupled PLC-IP3-Ca2+ signaling pathway, mobilization of intracellular granules, chemotactic migration, as well as an assembly of ROS generating NADPH-oxidase. The receptor selectivity was confirmed as the ZQ16 induced responses were inhibited by the GPR84 selective antagonist GLPG1205. The very low release of ROS induced by the GPR84 agonist in naïve neutrophils was significantly increased by pre-treatment of neutrophils with the pro-inflammatory priming agent tumour necrosis factor α (TNFα). Although the ZQ16 induced response was augmented in neutrophils with a disrupted MPI-0479605 cytoskeleton, GPR84 signaling was terminated by a cytoskeleton independent mechanism. In summary, our results demonstrate that the GPR84 antagonist GLPG1205 selectively inhibits the function of human professional phagocytes activated with the GPR84 agonist ZQ16. We also provide data revealing some mechanistic insights into GPR84-mediated immune modulation related to activation and termination of phagocyte functions, which shows similarities as well as differences to those of FFA2R.
Materials and methods
Results
Discussion GPR84 (gene name EX33) was cloned in 2001 as a novel GPCR from human blood leukocytes and the expression pattern revealed high levels primarily in neutrophils [36]. When later de-orphanized GPR84 was found to be a specific receptor for medium chain fatty acids containing 9–14 carbons [7,11]. The mechanism that regulates GPR84 activities and its biological role in inflammation has however not been much explored. The background to this is that ligands well suited for experimental research have been missing and potent and selective molecular tools are in highly demand in order to perform basic characterization studies with GPR84. Despite the lack of proper ligands, attempts were made almost ten years ago to determine the function of GPR84 by generation of a GPR84 deficient mice strain, however no distinct phenotype was observed in these animals [37]. The recent identification of new synthetic GPR84 agonistic-, as well as antagonistic-tools has created new research opportunities, and using the two recently developed GPR84 targeting ligands, ZQ16 (agonist) and GLPG1205 (antagonist), we have in this study performed a basic characterization of the receptor regarding priming, activation and desensitization, and provide some new molecular insights into GPR84-mediated pro-inflammatory response in peripheral blood neutrophils and tissue recruited neutrophils following in vivo transmigration (summary Fig. 9). Firstly, Compared to the neutrophil-specific expression profile of the short chain fatty acid receptor FFA2R, we show that GPR84 is functionally expressed by monocytes/macrophages in addition to neutrophils, indicating that GPR84 may have a broader functional spectrum in modulating inflammatory processes where both neutrophils and monocytes are involved. We show that activation of GPR84 triggers pro-inflammatory responses such as a modest chemoattraction and a modest ability of inducing granule-localized receptor mobilization in neutrophils, as well as an assembly and activation of the superoxide generating phagocyte NADPH-oxidase in phagocytes. Furthermore, through phenotypic characterization of neutrophils following tissue recruitment in vivo, we provide some molecular insights into GPR84 regulation in peripheral blood neutrophils and transmigration of these cells to an aseptic inflammatory site. ZQ16 was the first potent and selective GPR84 agonist that was identified almost five years ago in a high through-put-screening project involving a library of 160,000 small molecules [15]. Two other small GPR84 agonists (diindolylmethane and 6-OAU) were also described, but when compared to ZQ16, they were both less potent and not highly selective for GPR84 [38,39]. Using ZQ16 together with the GPR84 specific antagonist (GLPG1205, Galapagos NV; [14]), we show that GPR84 is functionally expressed by both neutrophils and monocytes. Further, the functional expression of GPR84 is retained during differentiation from monocytes to macrophages (MDMs) with regards to production and release of ROS. Interestingly, the functional expression pattern of GPR84 is different from that of the short chain fatty acid receptor FFA2R which is functionally expressed by neutrophils but not by monocytes or macrophages. Although it has been shown that FFA2R is highly expressed at the mRNA level in both human neutrophils and monocytes, only neutrophils respond to FFA2R agonists, when measured as a receptor mediated rise in intracellular Ca2+ and activation of the superoxide generating oxidase [40]. These data are in line with earlier studies showing that FFA2R has a neutrophil-specific expression profile [4]. Whether the mRNA/functional discrepancy is due to purity of the cells from which the mRNA was isolated or a lack of correlation between the generation of mRNA and expression of a functional protein is not yet known.