Structural characterization and inhibitory profile of formyl peptide receptor 2 selective peptides descending from a PIP2-binding domain of gelsolin.

The neutrophil formyl peptide receptors, FPR1 and FPR2, play critical roles for inflammatory reactions, and receptor-specific antagonists/inhibitors can possibly be used to facilitate the resolution of pathological inflammatory reactions. A 10-aa-long rhodamine-linked and membrane-permeable peptide inhibitor (PBP10) has such a potential. This FPR2 selective inhibitor adopts a phosphatidylinositol 4,5-bisphosphate-binding sequence in the cytoskeletal protein gelsolin. A core peptide, RhB-QRLFQV, is identified that displays inhibitory effects as potent as the full-length molecule. The phosphatidylinositol 4,5-bisphosphate-binding capacity of PBP10 was not in its own sufficient for inhibition. A receptor in which the presumed cytoplasmic signaling C-terminal tail of FPR2 was replaced with that of FPR1 retained the PBP10 sensitivity, suggesting that the tail of FPR2 was not on its own critical for inhibition. This gains support from the fact that the effect of cell-penetrating lipopeptide (a pepducin), suggested to act primarily through the third intracellular loop of FPR2, was significantly inhibited by PBP10. The third intracellular loops of FPR1 and FPR2 differ in only two amino acids, but an FPR2 mutant in which these two amino acids were replaced by those present in FPR1 retained the PBP10 sensitivity. In summary, we conclude that the inhibitory activity on neutrophil function of PBP10 is preserved in the core sequence RhB-QRLFQV and that neither the third intracellular loop of FPR2 nor the cytoplasmic tail of the receptor alone is responsible for the specific inhibition.

In vivo-transmigrated human neutrophils are resistant to antiapoptotic stimulation.

Neutrophils respond to microbial invasion or injury by transmigration from blood to tissue. Transmigration involves cellular activation and degranulation, resulting in altered levels of surface receptors and changed responsiveness to certain stimuli. Thus, fundamental functional changes are associated with neutrophil transmigration from blood to tissue. Neutrophils isolated from peripheral blood spontaneously enter apoptosis, a process that can be accelerated or delayed by different pro- or antiapoptotic factors. How tissue neutrophils that have transmigrated in vivo regulate cell death is poorly understood. In this study, in vivo-transmigrated neutrophils (tissue neutrophils) were collected using a skin chamber technique and compared with blood neutrophils from the same donors with respect to regulation of cell death. Skin chamber fluid contained a variety of cytokines known to activate neutrophils and regulate their lifespan. Freshly prepared tissue neutrophils had elevated activity of caspase 3/7 but were fully viable; spontaneous cell death after in vitro culture was also similar between blood and tissue neutrophils. Whereas apoptosis of cultured blood neutrophils was delayed by soluble antiapoptotic factors (e.g., TLR ligands), tissue neutrophils were completely resistant to antiapoptotic stimulation, even though receptors were present and functional. In vitro transmigration of blood neutrophils into skin chamber fluid did not fully confer resistance to antiapoptotic stimulation, indicating that a block of antiapoptotic signaling occurs specifically during in vivo transmigration. We describe a novel, functional alteration that takes place during in vivo transmigration and highlights the fact that life and death of neutrophils may be regulated differently in blood and tissue.

The anionic amphiphile SDS is an antagonist for the human neutrophil formyl peptide receptor 1.

The anionic amphiphil sodium dodecyl sulfate (SDS) is commonly used to activate the superoxide-generating NADPH-oxidase complex in cell-free systems, but very little is known about the effects of SDS on intact cells. It was, however, recently shown that SDS causes a translocation and an activation of Rac (a small G-protein) in intact cells, but this signal is not in its own sufficient to activate the oxidase (Nigorikawa et al. (2004) [1]). We found that SDS acted as an antagonist for FPR1, one of the neutrophil members of the formyl peptide receptor family. Accordingly, SDS reduced superoxide anion production induced by the chemoattractant formylmethionyl-leucyl-phenylalanine (fMLF). The receptor specificity of SDS was fairly high, but the concentration range in which it worked was narrow. The length of the carbohydrate chain as well as the charge of the molecule was of importance for the antagonistic effects. Signaling through FPR2, a closely related receptor also expressed in neutrophils, was not inhibited by SDS. On the contrary, the response induced by the FPR2-specific agonist WKYMVM was primed by SDS. The precise mechanism behind the primed state is not known, but might be related to the effects earlier described for SDS on the small G-protein Rac, that is of importance for a proper transduction of the down-stream signals from the occupied receptor.

A methodological approach to studies of desensitization of the formyl peptide receptor: Role of the read out system, reactive oxygen species and the specific agonist used to trigger neutrophils.

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.

The FPR2-specific ligand MMK-1 activates the neutrophil NADPH-oxidase, but triggers no unique pathway for opening of plasma membrane calcium channels.

Human neutrophils express formyl peptide receptor 1 and 2 (FPR1 and FPR2), two highly homologous G-protein-coupled cell surface receptors important for the cellular recognition of chemotactic peptides. They share many functional as well as signal transduction features, but some fundamental differences have been described. One such difference was recently presented when the FPR2-specific ligand MMK-1 was shown to trigger a unique signal in neutrophils [S. Partida-Sanchez, P. Iribarren, M.E. Moreno-Garcia, et al., Chemotaxis and calcium responses of phagocytes to formyl peptide receptor ligands is differentially regulated by cyclic ADP ribose, J. Immunol. 172 (2004) 1896-1906]. This signal bypassed the emptying of the intracellular calcium stores, a route normally used to open the store-operated calcium channels present in the plasma membrane of neutrophils. Instead, the binding of MMK-1 to FPR2 was shown to trigger a direct opening of the plasma membrane channels. In this report, we add MMK-1 to a large number of FPR2 ligands that activate the neutrophil superoxide-generating NADPH-oxidase. In contrast to earlier findings we show that the transient rise in intracellular free calcium induced by MMK-1 involves both a release of calcium from intracellular stores and an opening of channels in the plasma membrane. The same pattern was obtained with another characterized FPR2 ligand, WKYMVM, and it is also obvious that the two formyl peptide receptor family members trigger the same type of calcium response in human neutrophils.

The beta-galactoside binding immunomodulatory lectin galectin-3 reverses the desensitized state induced in neutrophils by the chemotactic peptide f-Met-Leu-Phe: role of reactive oxygen species generated by the NADPH-oxidase and inactivation of the agonist.

Neutrophils interacting with a chemoattractant gradually become nonresponsive to further stimulation by the same agonist, a process known as desensitization. Receptor desensitization is a highly regulated process that involves different mechanisms depending on which receptor-ligand pair that is studied. Galectin-3, a member of a large family of beta-galactoside-binding lectins, has been suggested to be a regulator of the inflammatory process, augmenting or directly triggering the neutrophil functional repertoire. We show here that the desensitized state of neutrophils interacting with the chemotactic peptide fMLF is broken by galectin-3 and that this is achieved through an oxygen radical-mediated inactivation of the chemoattractant. The effect was inhibited by the competitor lactose and required the affinity of galectin-3 for N-acetyllactosamine, a saccharide typically found on cell surface glycoproteins. The latter was shown using a galectin-3 mutant that lacked N-acetyllactosamine binding activity, and this protein was not active. The mechanism behind the inactivation of the chemoattractant was found to depend on the ability of galectin-3 to induce a neutrophil generation/secretion of reactive oxygen species which in combined action with myeloperoxidase inactivated the peptides.

Changes in the ratio between FPR and FPRL1 triggered superoxide production in human neutrophils-a tool in analysing receptor specific events.

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.

Serum amyloid A mediates human neutrophil production of reactive oxygen species through a receptor independent of formyl peptide receptor like-1.

Serum amyloid A (SAA) is one of the acute-phase reactants, a group of plasma proteins that increases immensely in concentration during microbial infections and inflammatory conditions, and a close relationship between SAA levels and disease activity in rheumatoid arthritis (RA) has been observed. RA is an inflammatory disease, where neutrophils play important roles, and SAA is thought to participate in the inflammatory reaction by being a neutrophil chemoattractant and inducer of proinflammatory cytokines. The biological effects of SAA are reportedly mediated mainly through formyl peptide receptor like-1 (FPRL1), a G protein-coupled receptor (GPCR) belonging to the formyl peptide receptor family. Here, we confirmed the affinity of SAA for FPRL1 by showing that stably transfected HL-60 cells expressing FPRL1 were activated by SAA and that the response was inhibited by the use of the FPRL1-specific antagonist WRWWWW (WRW4). We also show that SAA activates the neutrophil NADPH-oxidase and that a reserve pool of receptors is present in storage organelles mobilized by priming agents such as TNF-alpha and LPS from Gram-negative bacteria. The induced activity was inhibited by pertussis toxin, indicating the involvement of a GPCR. However, based on FPRL1-specific desensitization and use of FPRL1 antagonist WRW4, we found the SAA-mediated effects in neutrophils to be independent of FPRL1. Based on these findings, we conclude that SAA signaling in neutrophils is mediated through a GPCR, distinct from FPRL1. Future identification and characterization of the SAA receptor could lead to development of novel, therapeutic targets for treatment of RA.

A monocyte-specific peptide from herpes simplex virus type 2 glycoprotein G activates the NADPH-oxidase but not chemotaxis through a G-protein-coupled receptor distinct from the members of the formyl peptide receptor family.

We have recently identified a peptide derived from the secreted portion of the HSV-2 glycoprotein G, gG-2p20, to be proinflammatory. Based on its ability to activate neutrophils and monocytes via the formyl peptide receptor (FPR) to produce reactive oxygen species (ROS) that down-regulate NK cell function, we suggested it to be of importance in HSV-2 pathogenesis. We now describe the effects of an overlapping peptide, gG-2p19, derived from the same HSV-2 protein. Also, this peptide activated the ROS-generating NADPH-oxidase, however, only in monocytes and not in neutrophils. Surprisingly, gG-2p19 did not induce a chemotactic response in the affected monocytes despite using a pertussis toxin-sensitive, supposedly G-protein-coupled receptor. The specificity for monocytes suggested that FPR and its homologue FPR like-1 (FPRL1) did not function as receptors for gG-2p19, and this was also experimentally confirmed. Surprisingly, the monocyte-specific FPR homologue FPRL2 was not involved either, and the responsible receptor thus remains unknown so far. However, the receptor shares some basic signaling properties with FPRL1 in that the gG-2p19-induced response was inhibited by PBP10, a peptide that has earlier been shown to selectively inhibit FPRL1-triggered responses. We conclude that secretion and subsequent degradation of the HSV-2 glycoprotein G can generate several peptides that activate phagocytes through different receptors, and with different cellular specificities, to generate ROS with immunomodulatory properties.

Cyclosporin H, Boc-MLF and Boc-FLFLF are antagonists that preferentially inhibit activity triggered through the formyl peptide receptor.

In order to properly interpret receptor inhibition experiments, the precise receptor specificities of the employed antagonists are of crucial importance. Lately, a great number of agonists for various formyl peptide receptors have been identified using a selection of antagonists. However, some confusion exists as to the precise receptor specificities of many of these antagonists. We have investigated the effects of formyl peptide receptor family antagonists on the neutrophil response induced by agonists for the formyl peptide receptor (FPR) and the formyl peptide receptor like 1 (FPRL1). To determine FPR- and FPRL1-specific interactions, these antagonists should not be used at used at concentrations above 10 microM. Signaling through FPR was inhibited by low concentrations of the antagonists cyclosporin H, Boc-MLF (also termed Boc-1), and Boc-FLFLFL (also termed Boc-2), while higher concentrations also partly inhibited the signaling through FPRL1. The antagonist WRWWWW (WRW(4)) specifically inhibited the signaling through FPRL1 at low concentrations but at high concentrations also partly the signaling through FPR. Based on the difference in potency of cyclosporin H and the two Boc-peptides, we suggest using cyclosporin H as a specific inhibitor for FPR. To specifically inhibit the FPRL1 response the antagonist WRW(4) should be used.

House dust mite allergen activates human eosinophils via formyl peptide receptor and formyl peptide receptor-like 1.

The objective was to evaluate which receptors house dust mite (HDM) and birch pollen extracts engage to activate human eosinophils. Chemotaxis and degranulation were studied in eosinophils pretreated with pertussis toxin and other antagonists of G protein-coupled receptors, e.g. the formyl peptide receptor (FPR), CC chemokine receptor 3 (CCR3) and leukotriene receptor B4 (LTB(4)R). Inhibition of the FPR as well as desensitization of the receptor rendered eosinophils anergic to activation by the allergens. Blockade of CCR3 or LTB(4)R did not affect eosinophilic reactivity. It was determined by PCR that human eosinophils express the FPR family members FPR and FPR-like 1 (FPRL1). HDM, unlike birch pollen, evoked calcium fluxes in HL-60 cells transfected with FPR or FPRL1. Although both allergens gave rise to calcium transients in neutrophils, which also express FPR and FPRL1, only the HDM response was decreased by the FPR antagonist. Moreover, neutrophils migrated toward HDM but not to birch pollen. Eosinophils pretreated with inhibitors of MAPK p38, ERK1/2 or protein kinase C exhibited diminished responsiveness to the aeroallergens. This study indicates that FPR and FPRL1 mediate the activation of eosinophils by HDM, whereas birch pollen employs other pathways shared with FPR to activate human eosinophils.

Ability of monocyte-derived dendritic cells to secrete oxygen radicals in response to formyl peptide receptor family agonists compared to that of myeloid and plasmacytoid dendritic cells.

We show that human monocyte-derived dendritic cells (DC) differ considerably from freshly isolated blood-derived myeloid and plasmacytoid DC in their abilities to produce reactive oxygen species in response to different agonists to the formyl peptide receptor family and are thus poor representatives of blood DC in this field of research.

The peptide Trp-Lys-Tyr-Met-Val-D-Met activates neutrophils through the formyl peptide receptor only when signaling through the formylpeptide receptor like 1 is blocked. A receptor switch with implications for signal transduction studies with inhibitors and receptor antagonists.

Neutrophils express the G protein-coupled N-formyl peptide receptor (FPR) and its homologue FPRL1. The hexapeptide Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm) activates HL-60 cells transfected either with FPRL1 or with FPR. The signaling through the stably expressed receptors was inhibited by specific receptor antagonists, cyclosporine H and WRWWWW (WRW4) for FPR and FPRL1, respectively. The neutrophil release of superoxide was used to determine receptor preference, when these cells were triggered with WKYMVm. The response was not affected by the FPR specific antagonist suggesting that no signals are transduced through this receptor. The response was only partly inhibited by WRW4, but this antagonist induced a receptor switch, perceptible as a change in sensitivity to the FPR antagonist. The activity remaining in the presence of WRW4 was inhibited by cyclosporine H. A cell permeable peptide (PBP10) corresponding to the phosphatidyl-inositol-bisphosphate binding region of gelsolin, inhibited the FPRL1-, but not the FPR-induced cellular response and induced the same type of receptor switch. We show that an agonist that has the potential to bind and activate neutrophils through FPRL1 as well as through FPR, uses the latter receptor and its signaling route, only when the activating signal generated through FPRL1 is blocked. The receptor switch is achieved when signaling through FPRL1 is inhibited both by a receptor antagonist, and by an inhibitor operating from the inside of the plasma membrane. The phenomenon described is of general importance for proper interpretation of results generated through the use of different "silencing technologies" in receptor operated signaling transduction research.

Neutrophil NADPH-oxidase activation by an annexin AI peptide is transduced by the formyl peptide receptor (FPR), whereas an inhibitory signal is generated independently of the FPR family receptors.

Truncation of the N-terminal part of the calcium-regulated and phospholipid-binding protein annexin AI has been shown to change the functional properties of the protein and to generate immunoregulatory peptides. Proinflammatory as well as anti-inflammatory signals are triggered by these peptides, and the two formyl peptide receptor (FPR) family members expressed in neutrophils, FPR and FPR-like 1 (FPRL1), have been suggested to transduce these signals. We now report that an annexin AI peptide (Ac9-25) activates, as well as inhibits, the neutrophil release of superoxide anions. Results obtained from experiments with receptor antagonists/inhibitors, desensitized cells, and transfected cells reveal that the Ac9-25 peptide activates the neutrophil reduced nicotinamide adenine dinucleotide phosphate oxidase through FPR but not through FPRL1. The Ac9-25 peptide also inhibits the oxidase activity in neutrophils triggered, not only by the FPR-specific agonist N-formyl-Met-Leu-Phe but also by several other agonists operating through different G protein-coupled receptors. Our data show that the two signals generated by the Ac9-25 peptide are transmitted through different receptors, the inhibitory signal being transduced by a not-yet identified receptor distinct from FPR and FPRL1.

The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide.

BACKGROUND: The formylpeptide receptor family members FPR and FPRL1, expressed in myeloid phagocytes, belong to the G-protein coupled seven transmembrane receptor family (GPCRs). They share a high degree of sequence similarity, particularly in the cytoplasmic domains involved in intracellular signaling. The established model of cell activation through GPCRs states that the receptors isomerize from an inactive to an active state upon ligand binding, and this receptor transformation subsequently activates the signal transducing G-protein. Accordingly, the activation of human neutrophil FPR and FPRL1 induces identical, pertussis toxin-sensitive functional responses and a transient increase in intracellular calcium is followed by a secretory response leading to mobilization of receptors from intracellular stores, as well as a release of reactive oxygen metabolites. RESULTS: We report that a cell permeable ten amino acid peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate (PIP2) binding region of gelsolin (an uncapper of actin filaments) blocks granule mobilization as well as secretion of oxygen radicals. The inhibitory effect of PBP10 is, however, receptor specific and affects the FPRL1-, but not the FPR-, induced cellular response. The transient rise in intracellular calcium induced by the active receptors is not affected by PBP10, suggesting that the blockage occurs in a parallel, novel signaling pathway used by FPRL1 to induce oxygen radical production and secretion. Also the FPR can activate neutrophils through a PBP10-sensitive signaling pathway, but this signal is normally blocked by the cytoskeleton. CONCLUSIONS: This study demonstrates that the two very closely related chemoattractant receptors, FPR and FPRL1, use distinct signaling pathways in activation of human neutrophils. The PIP2-binding peptide PBP10 selectively inhibits FPRL1-mediated superoxide production and granule mobilization. Furthermore, the activity of this novel PBP10 sensitive pathway in neutrophils is modulated by the actin cytoskeleton network.