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Neutrophils express several G protein-coupled receptors (GPCRs) connected to intracellular Gα i or Gα q containing G proteins for down-stream signaling. To dampen GPCR mediated inflammatory processes, several inhibitors targeting the receptors and/or their down-stream signals, have been developed. Potent and selective inhibitors for Gα q containing G proteins are available, but potent and specific inhibitors of Gα i containing G proteins are lacking. Recently, Larixol, a compound extracted from the root of Euphorbia formosana , was shown to abolish human neutrophil functions induced by N -formyl-methionyl-leucyl-phenylalanine (fMLF), an agonist recognized by formyl peptide receptor 1 (FPR1) which couple to Gα i containing G proteins. The inhibitory effect was suggested to be due to interference with/inhibition of signals transmitted by βγ complexes of the Gα i containing G proteins coupled to FPR1. In this study, we applied Larixol, obtained from two different commercial sources, to determine the receptor- and G protein- selectivity of this compound in human neutrophils. However, our data show that Larixol not only lacks inhibitory effect on neutrophil responses mediated through FPR1, but also on responses mediated through FPR2, a Gα i coupled GPCR closely related to FPR1. Furthermore, Larixol did not display any features as a selective inhibitor of neutrophil responses mediated through the Gα q coupled GPCRs for platelet activating factor and ATP. Hence, our results imply that the inhibitory effects described for the root extract of Euphorbia formosana are not mediated by Larixol and that the search for a selective inhibitor of G protein dependent signals generated by Gα i coupled neutrophil GPCRs must continue. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: 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 PIP2 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. [Copyright &y& Elsevier]

The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin's ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core 2 O-linked glycans mediate this lubricin-galectin-3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core 2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary cells with the core 2 GlcNAc transferase acting on a mucin-type O-glycoprotein displayed increased galectin-3 binding. Both the level of galectin-3 and the galectin-3 interactions with synovial lubricin were found to be decreased in late-stage OA patients, coinciding with an increase in unsialylated core 1 O-glycans (T-antigens) and Tn-antigens. These data suggest a defect in crosslinking of surface-active molecules in OA and provide novel insights into OA molecular pathology. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

[Display omitted] Formyl peptide receptor 1 (FPR1), a G protein-coupled receptor expressed in phagocytes, recognizes short N-formylated peptides originating from proteins synthesized by bacteria and mitochondria. Such FPR1 agonists are important regulators of neutrophil functions and by that, determinants of inflammatory reactions. As FPR1 is implicated in promoting both pro-inflammatory and pro-resolving responses associated with inflammatory diseases, characterization of ligands that potently and selectively modulate FPR1 induced functions might be of high relevance. Accordingly, a number of FPR1 specific antagonists have been identified and shown to inhibit agonist binding or receptor down-stream signaling as well as neutrophil functions such as granule secretion and NADPH oxidase activity. The inhibitory effect on neutrophil chemotaxis induced by FPR1 agonists has generally not been part of basic antagonist characterization. In this study we show that the inhibitory effects on neutrophil chemotaxis of established FPR1 antagonists (i.e., cyclosporin H, BOC1 and BOC2) are limited. Our data demonstrate that the recently described small molecule AZ2158 is a potent and selective FPR1 antagonist in human neutrophils. In contrast to the already established FPR1 antagonists, AZ2158 also potently inhibits chemotaxis. Whereas the cyclosporin H inhibition was agonist selective, AZ2158 inhibited the FPR1 response induced by both a balanced and a biased FPR1 agonist equally well. In accordance with the species specificity described for many FPR1 ligands, AZ2158 was not recognized by the mouse orthologue of FPR1. Our data demonstrate that AZ2158 may serve as an excellent tool compound for further mechanistic studies of human FPR1 mediated activities. [ABSTRACT FROM AUTHOR]

Lubricin (or proteoglycan 4 (PRG4)) is an abundant mucin-like glycoprotein in synovial fluid (SF) and a major component responsible for joint lubrication. In this study, it was shown thatO-linked core 2 oligosaccharides (Galβ1-3(GlcNAcβ1-6)GalNAcα1- Thr/Ser) on lubricin isolated from rheumatoid arthritis SF contained both sulfate and fucose residues, and SF lubricin was capable of binding to recombinant L-selectin in a glycosylationdependent manner. Using resting human polymorphonuclear granulocytes (PMN) from peripheral blood, confocal microscopy showed that lubricin coated circulating PMN and that it partly co-localized with L-selectin expressed by these cells. In agreement with this, activation-induced shedding of L-selectin also mediated decreased lubricin binding to PMN. It was also found thatPMNrecruited to inflamed synovial area and fluid in rheumatoid arthritis patients kept a coat of lubricin. These observations suggest that lubricin is able to bind to PMN via an L-selectin-dependent and -independent manner and may play a role in PMN-mediated inflammation. [ABSTRACT FROM AUTHOR]

Abstract: 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 ([Ca2+]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 [Ca2+]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 &y& Elsevier]

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 (Ca2+). 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 Ca2+. 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. • The agonist occupied ATP receptor P2Y 2 R activates allosterically modulated FFAR2s. • Activation of FFAR2s by allosteric modulators without any orthosteric agonist • Biased receptor signaling induced by two interdependent allosteric modulators. • New regulatory mechanisms for control of GPCR-signaling [ABSTRACT FROM AUTHOR]