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In order to avoid a prolonged pro-inflammatory neutrophil response, signaling downstream of an agonist-activated G protein-coupled receptor (GPCR) has to be rapidly terminated. Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, GRK2, which is highly expressed by immune cells, plays an important role. The medium chain fatty acid receptor GPR84 as well as formyl peptide receptor 2 (FPR2), receptors expressed in neutrophils, play a key role in regulating inflammation. In this study, we investigated the effects of GRK2 inhibitors on neutrophil functions induced by GPR84 and FPR2 agonists. GRK2 was shown to be expressed in human neutrophils and analysis of subcellular fractions revealed a cytosolic localization. The GRK2 inhibitors enhanced and prolonged neutrophil production of reactive oxygen species (ROS) induced by GPR84- but not FPR2-agonists, suggesting a receptor selective function of GRK2. This suggestion was supported by β-arrestin recruitment data. The ROS production induced by a non β-arrestin recruiting GPR84 agonist was not affected by the GRK2 inhibitor. Termination of this β-arrestin independent response relied, similar to the response induced by FPR2 agonists, primarily on the actin cytoskeleton. In summary, we show that GPR84 utilizes GRK2 in concert with β-arrestin and actin cytoskeleton dependent processes to fine-tune the activity of the ROS generating NADPH-oxidase in neutrophils., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFAR2), transfers the orthosteric FFAR2 agonists propionate and the P2Y 2 R specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFAR2 modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist. The activation signals down-stream of FFAR2 when stimulated by the two interdependent allosteric modulators were biased in that, unlike for orthosteric agonists, the two complementary modulators together triggered an activation of the NADPH-oxidase, but not any transient rise in the cytosolic concentration of free calcium ions (Ca 2+ ). Furthermore, following AZ1729/Cmp58 activation, the signaling by the desensitized FFAR2s was functionally selective in that the orthosteric agonist propionate could still induce a transient rise in intracellular Ca 2+ . The novel neutrophil activation and receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFAR2 modulators represent a new regulatory mechanism that controls receptor signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo. In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The study of human leukocytes is almost exclusively conducted using cells isolated from peripheral blood. This is especially true for neutrophils, despite the fact that these cells are of main (pathological) importance in extravascular tissues upon e.g., infection and/or tissue damage. The journey from circulation to tissue is typically associated with a number of cellular changes, making the cells primed, or hyper-responsive, and in many aspects distinct from the cells present in circulation. Models to obtain in vivo transmigrated leukocytes from human tissue are available, but not widely used. We describe here an easy-to-use model for the study of local inflammation, stemming from limited tissue damage, which can be used to isolate viable and functional leukocytes. The model is based on the generation of aseptic skin blisters, formed by the application of negative pressure, and allows for investigations of the cellular infiltrate as well as of soluble mediators present in the exudate. We believe that this method, combined with modern analysis equipment suitable for small volumes and cell numbers, could be of great use for increasing our understanding of the nature and function of leukocytes that have left circulation and transmigrated to inflamed tissues., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Neutrophil accumulation at an inflammatory site or an infected tissue is dependent on the recognition of chemotactic peptides that bind to G-protein coupled receptors (GPCRs) exposed on the surface of the inflammatory cells. A GPCR activated by a chemoattractant quickly becomes refractory to further stimulation by ligands using the same receptor. This desensitization phenomenon has been used frequently to characterize new receptor agonists and to determine receptor hierarchies. In this study we show that desensitization patterns differ depending on what read out systems are used to follow neutrophil activity. When monitoring release of superoxide, neutrophils were readily desensitized against repeated stimulations with the prototypical agonist formylmethionyl-leucyl-phenylalanine (fMLF). In contrast, neutrophils were not desensitized for fMLF when cell activity was determined by intracellular calcium ([Ca(2+)](i)). The difference observed was dependent on inactivation of the agonist in one read out system but not in the other, and we suggest several different solutions to the problem. Agonist inactivation occurs through a myeloperoxidase (MPO)/hydrogen peroxide catalyzed reaction, and the problem could be avoided by using a FACS based technique to measure the change in [Ca(2+)](i), by the use of an agonist insensitive to the MPO/hydrogen peroxide-system or, by adding an MPO inhibitor or a scavenger that removes either superoxide/hydrogen peroxide or the MPO-derived metabolites., (Copyright 2009 Elsevier B.V. All rights reserved.)

Neutrophils express the G protein-coupled N-formyl peptide receptor (FPR) as well as its closely related homologue, formyl peptide like receptor 1 (FPRL1), and activation of these receptors induce a release of superoxide anions. The magnitude of the responses induced by the two peptide agonists fMLF and WKYMVM, specific for FPR and FPRL1, respectively, was found to be very variable in different neutrophil populations. The ratio between the FPR and FPRL1 triggered respiratory burst was, however, very constant and close to 1. The ratio was changed in neutrophils that were desensitized as well as when the signaling through either of the receptors was inhibited by receptor specific antagonists or by a PIP(2) binding peptide. The FPR/FPRL1 ratio was not changed in primed neutrophils or in differentiated HL-60 cells. We show that the change in the ratio, calculated from the amount of radical release in neutrophils triggered with FPR and FPRL1 specific agonists can be used as a valuable tool to find/identify receptor specific/selective changes mediated by peptides/proteins/drugs, as well as to identify cells from patients or groups of patients that diverge from normal cells in their FPR/FPRL1 triggered functions.