Huamei Fu

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.

The mechanism for activation of the neutrophil NADPH-oxidase by the peptides formyl-Met-Leu-Phe and Trp-Lys-Tyr-Met-Val-Met differs from that for interleukin-8

Neutrophil chemotaxis has been shown to be regulated by two different signalling pathways that allow strong chemoattractants, such as bacterial‐derived formylated peptides, to dominate over endogenous attractants, such as interleukin‐8 (IL‐8). Here we show that triggering of the formyl peptide receptor (FPR) with f‐Met‐Leu‐Phe (fMLF) substantially reduced the neutrophil superoxide production induced by activation of the CXC receptors with IL‐8. When the order of agonists was reversed, the cells were primed in their response to fMLF, suggesting that the signalling hierarchy between strong, so‐called end‐type (i.e. fMLF) and weak or intermediate‐type (i.e. IL‐8) chemoattractants, is also operating during activation of the NADPH‐oxidase. The same result was obtained when fMLF was replaced with the hexapeptide, WKYMVM, specific for the formyl peptide‐like receptor 1 (FPRL1). There were additional differences between the agonist receptor pairs fMLF/FPR, WKYMVM/FPRL1 and IL‐8/CXCR. In contrast to FPR and FPRL1, no reserve pool of CXCR was present in subcellular granules and it was impossible to prime the oxidative response transduced through CXCR by the addition of priming agents such as tumour necrosis factor‐α and platelet‐activating factor. Moreover, the cytoskeleton‐disrupting substance, cytochalasin B, had no effect either on IL‐8‐triggered oxidase activation or on CXCR reactivation. A pertussis toxin‐sensitive G‐protein is involved in signalling mediated through both FPR and CXCR, and the signalling cascades include a transient intracellular calcium increase, as well as downstream p38 MAPK and phosphoinositide 3‐kinase activation. The data presented in this study provide support for two different signalling pathways to the neutrophil NADPH‐oxidase, used by ligand binding to FPR/FPRL1 or CXCR, respectively.

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.

Interleukin-8-Derived Peptide Has Antibacterial Activity

ABSTRACT Chemokines are inflammatory mediators with effects on diverse processes associated with the immune response. Some of the proteins belonging to the CXC chemokine subfamily, one of four groups in the family, possess inherent antibacterial activity against a wide range of bacteria. The CXC chemokine interleukin-8 (IL-8) has not been ascribed any direct antibacterial activity, but the fact that several of the amino acids in the carboxy-terminal part of the protein are identical or similar to those in a bactericidal cecropin-like peptide [Hp(2-20)] from Helicobacter pylori suggests that processing of the cytokine might generate peptide fragments with antibacterial properties. Synthetic peptides representing the carboxy-terminal part of IL-8 were investigated for antibacterial activities. These fragments possessed an antibacterial activity absent in the full-length IL-8. The antibacterial effects were reduced at increasing salt concentrations whereas the activity was increased when the pH was lowered. The IL-8-derived peptide shared structural similarity with and was also functionally additive to the Hp(2-20) peptide. The IL-8-derived peptide lacked the proinflammatory effects of the full-length protein. We also showed that acid hydrolysis of IL-8 generated a major peptide fragment corresponding to the antibacterial carboxyl terminus of the protein. The results presented are of special interest when put in the context of the suggested importance of antimicrobial peptides for microbial colonization of the gastric mucosa.

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.

Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils

BackgroundCytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncoupling results in respiratory burst activity when signals generated by reactivated formyl peptide receptors trigger the NADPH-oxidase to produce superoxide anions.ResultsTumor necrosis factor alpha (TNF-alpha) primes neutrophils for subsequent activation by cytochalasin B. Pretreatment with TNF-alpha induced mobilization of receptor-storing neutrophil organelles, suggesting that receptor up-regulation significantly contributes to the response, but the receptor mobilization was not sufficient for induction of the cytochalasin B sensitive state. The TNF-alpha primed state resembled that of the desensitized non-signaling state of agonist-occupied neutrophil formyl peptide receptors. The fact that the TNF-alpha primed, cytochalasin B-triggered activation process was pertussis toxin sensitive suggests that the activation process involves a GPCR. Based on desensitization experiments the unidentified receptor was found to be distinct from the C5a receptor as well as the formyl peptide receptor family members FPR and FPRL1. Based on the fact the occupied and desensitized receptors for interleukin-8 and platelet activating factor could not be reactivated by cytochalasin B, also these could be excluded as receptor candidates involved in the TNF-alpha primed state.ConclusionsThe TNF-alpha-induced priming signals could possibly trigger a release of an endogenous GPCR-agonist, amplifying the response to the receptor-uncoupling effect of cytochalasin B. However, no such substance could be found, suggesting that TNF-alpha can transfer G-protein coupled receptors to a signaling state independently of agonist binding.

Subinhibitory Concentrations of the Deformylase Inhibitor Actinonin Increase Bacterial Release of Neutrophil-Activating Peptides: a New Approach to Antimicrobial Chemotherapy

ABSTRACT Bacterial protein synthesis starts with a formylated methionine residue, and this residue is sequentially cleaved away by a unique peptide deformylase (PDF) and a methionine aminopeptidase to generate mature proteins. The formylation-deformylation of proteins is a unique hallmark of bacterial metabolism and has recently become an attractive target for the development of antimicrobial agents. The innate immune system uses the formylation of bacterial proteins as a target, and professional phagocytes, e.g., neutrophils, express specific receptors for bacterium-derived formylated peptides. Activation of formyl peptide receptors (FPR) mediates neutrophil migration and the release of oxygen radicals and other antimicrobial substances from these cells. We hypothesize that the use of a PDF inhibitor would increase the production of proinflammatory peptides from the bacteria and thus trigger a more pronounced innate immune response. We tested this hypothesis by exposing Escherichia coli to subinhibitory doses of the PDF inhibitor actinonin and show that actinonin indeed increases the production and secretion of neutrophil-activating peptides that activate human neutrophils through FPR. These findings could be potentially used as a new approach to antibacterial chemotherapy.

Outer membrane protein A deficient Escherichia coli activates neutrophils to produce superoxide and shows increased susceptibility to antibacterial peptides.

The outer membrane protein A (OmpA) of Gram-negative bacteria has been ascribed multiple functions including maintenance of structural membrane integrity and porin activity. OmpA has also been implicated in various host defense processes in that it contributes to bacterial serum resistance and activates certain immune cells. Recently, OmpA was shown to be the molecular target for neutrophil elastase (NE), and Escherichia coli mutants lacking OmpA were resistant to the bactericidal effects of NE. In addition to NE, neutrophils use a variety of other antibacterial effector molecules such as oxygen radicals and bactericidal peptides or proteins. The aim of this study was to investigate the role of E. coli OmpA regarding susceptibility to other neutrophil-derived defense systems. We found that OmpA-deficient (OmpA(-)), but not wild-type isogenic, E. coli activated human neutrophils to produce oxygen radicals intracellularly. This activation was found to require an intact neutrophil cytoskeleton but was independent of bacterial phagocytosis. Furthermore, we found that the OmpA(-) strain was more susceptible to membrane-acting bactericidal peptides than the wild-type strain, although the susceptibility to different oxygen radicals was independent of the presence of OmpA. Taken together, these data suggest an important role for OmpA in the context of bacteria vs. host interactions.

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.