Jennie Karlsson

Ligand recognition and activation of formyl peptide receptors in neutrophils

Neutrophil granulocytes, professional phagocytes of the innate immune system, can migrate in response to gradients of chemoattractants, soluble molecules serving as “danger signals.” The chemotactic behavior of these cells is of great importance for the outcome of the continuously ongoing combat with invading microorganisms. In a number of inflammatory disorders, the chemoattractant-guided accumulation of neutrophils and their subsequent release of reactive oxygen species (ROS) and proteolytic enzymes are responsible for the tissue damage associated with such disease conditions. Research about the structure and function of neutrophil chemoattractants and their receptors is therefore of direct clinical importance and relevance. Although chemotaxis was defined already as an important part of active immune reactivity already by Elie Metchnikoff in the late 19th century [1], the modern chemotaxis research era started first, by the introduction of a filter technique in 1962 [2], which allowed quantitative determinations of neutrophil migration and a rational search for specific attractants derived from intruding microbes or activated/damaged host cells [3]. Following the discovery of bacteria-derived, formylated peptides as potent neutrophil chemoattractants in the mid-1970s [4], the list of structurally well-characterized leukocyte chemoattractants has steadily grown. Other microbial components, cleavage products from the complement system (e.g., C5a), lipid metabolites such as platelet-activating factor (PAF) and leukotriene B4 (LTB4), as well as a large group of chemokines are examples of such molecules [5] (Table 1). During the last two decades, a broad application of molecular biology techniques has also led to identification of the chemoattractant receptors. Despite the fact that these receptors recognize different chemoattractants specifically, they exhibit some sequence homologies and share structural features, all belonging to a pertussis toxin (PTX)-sensitive subfamily within the G protein-coupled receptor (GPCR) superfamily. The reader is referred to several excellent review articles, which in more detail, discuss general aspects of the chemoattractant receptor family and neutrophil activation in inflammation [5 –7, 16]. We intend to summarize the current knowledge about structure and function of two closely related neutrophil G proteincoupled chemoattractant receptors: the FPR, which was the first characterized member in the FPR family, and the closely related FPRL1, also called LXA4 receptor, as this eicosanoid was the first specific agonist described for the receptor. A large number of agonists for these receptors have now been identified, and the same basic neutrophil functional responses are triggered by ligation of these receptors [16]—chemotaxis, receptor mobilization, secretion of proteolytic enzymes and inflammatory mediators, and production of ROS. Ligand recognition by the two neutrophil FPR, linked to activation and signaling, is the subject of this review.

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‐α 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.

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

BackgroundThe 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.ResultsWe 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.ConclusionsThis 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.

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.

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.

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 (LTB4R). Inhibition of the FPR as well as desensitization of the receptor rendered eosinophils anergic to activation by the allergens. Blockade of CCR3 or LTB4R 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.

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.

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 β-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.

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.