Formyl Peptide Receptor Ligands Research Articles

Selective Display of a Chemoattractant Agonist on Cancer Cells Activates the Formyl Peptide Receptor 1 on Immune Cells.

Current immunotherapeutics often work by directing components of the immune system to recognize biomarkers on the surface of cancer cells to generate an immune response. However, variable changes in biomarker distribution and expression can result in inconsistent patient response. The development of a more universal tumor-homing strategy has the potential to improve selectivity and extend therapy to cancers with decreased expression or absence of specific biomarkers. Here, we designed a bifunctional agent that exploits the inherent acidic microenvironment of most solid tumors to selectively graft the surface of cancer cells with a formyl peptide receptor ligand (FPRL). Our approach is based on the pH(Low) insertion peptide (pHLIP), a unique peptide that selectively targets tumors in vivo by anchoring to cancer cells in a pH-dependent manner. We establish that selectively remodeling cancer cells with a pHLIP-based FPRL activates formyl peptide receptors on recruited immune cells, potentially initiating an immune response towards tumors.

Functional and signaling characterization of the neutrophil FPR2 selective agonist Act-389949

Despite the steadily increased numbers of formyl peptide receptor (FPR) ligands identified over the years, few have been characterized in studies using animal disease models and even less have entered clinical trials in human subjects. A small-molecule compound, Act-389949, was however recently tested in a phase I clinical trial and found to be safe and well tolerated in healthy human subjects. The desired anti-inflammatory property of Act-389949 was proposed to be mediated through FPR2, one of the FPRs expressed in neutrophils, but no basic characterization was included in the study. To gain more insights into FPR2 recognition of this first-in-class compound for future utility of the agonist, we have in this study determined the receptor preference and down-stream signaling characteristics induced by Act-389949 in human blood neutrophils isolated from healthy donors. Our data demonstrate that Act-389949 is an agonist for FPR2 that triggers functional/signaling repertoires comparable to what has been earlier described for other FPR2 agonists, including neutrophil chemotaxis, granule mobilization and activation of the NADPH-oxidase. In fact, Act-389949 was found to be as potent as the prototype FPR2 peptide agonist WKYMVM and had the advantage of being resistant to oxidation by MPO-H2O2-halide derived oxidants, as compared to the sensitive WKYMVM. The down-stream signals generated by Act-389949 include an FPR2-dependent and Gαq-independent transient rise in intracellular Ca2+ and recruitment of β-arrestin. In summary, our data show that Act-389949 serves as an excellent tool-compound for further dissection of FPR2-regulated activities in vitro and in vivo. Potent and stable FPR ligands such as Act-389949 may therefore be used to develop the next generation of FPR signaling regulating anti-inflammatory therapeutics.

Defective formyl peptide receptor 2/3 and annexin A1 expressions associated with M2a polarization of blood immune cells in patients with chronic obstructive pulmonary disease

BackgroundControversy exists in previous studies on macrophage M1/M2 polarization in chronic obstructive pulmonary disease (COPD). We hypothesized that formyl peptide receptor (FPR), a marker of efferocytosis and mediator of M1/M2 polarization, may be involved in the development of COPD.MethodsWe examined FPR 1/2/3 expressions of blood M1/M2a monocyte, neutrophil, natural killer (NK) cell, NK T cell, T helper (Th) cell, and T cytotoxic (Tc) cell by flowcytometry method in 40 patients with cigarette smoking-related COPD and 16 healthy non-smokers. Serum levels of five FPR ligands were measured by ELISA method.ResultsThe COPD patients had lower M2a percentage and higher percentages of NK, NK T, Th, and Tc cells than the healthy non-smokers. FPR2 expressions on Th/Tc cells, FPR3 expressions of M1, M2a, NK, NK T, Th, and Tc cells, and serum annexin A1 (an endogenous FPR2 ligand) levels were all decreased in the COPD patients as compared with that in the healthy non-smokers. FPR1 expression on neutrophil was increased in the COPD patient with a high MMRC dyspnea scale, while FPR2 expression on neutrophil and annexin A1 were both decreased in the COPD patients with a history of frequent moderate exacerbation (≥ 2 events in the past 1 year). In 10 COPD patients whose blood samples were collected again after 1-year treatment, M2a percentage, FPR3 expressions of M1/NK/Th cells, FPR2 expression on Th cell, and FPR1 expression on neutrophil were all reversed to normal, in parallel with partial improvement in small airway dysfunction.ConclusionsOur findings provide evidence for defective FPR2/3 and annexin A1 expressions that, associated with decreased M2a polarization, might be involved in the development of cigarette smoking induced persistent airflow limitation in COPD.

Follicular dendritic cells modulated by formyl peptide receptor ligands are capable of enhancing the germinal center reaction in mucosal immune system

Abstract In various vaccination protocols, induction of germinal center (GC) is one of the critical factors involved in successful immune induction because GC formation results in the production of high affinity antibodies via B cell clonal expansion, somatic hypermutation, and affinity-based B cell selection. Although GC formation in Peyer’s patch (PP) is slowly and spontaneously induced by intestinal microbiota, it is unknown whether follicular dendritic cells (FDCs) in PP directly contribute to the antigen-specific immune response induction. In this study, we found the expression of formyl peptide receptor (FPR-2), a receptor for cathelicidin LL-37 which could play a role as an adjuvant in mucosal vaccine, on FDCs. The interaction between LL-37 and FPR-2 on FDCs promotes the expression of CXCL13 which is closely associated with the GC formation. Based on these findings, we conclude that the adjuvant role of LL-37 in mucosal immune induction is closely associated with the enhancement of GC reaction in PPs by FDC stimulation.

Flow cytometric analysis with a fluorescently labeled formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2 receptor.

The histamine H2 receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models, or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMP-dependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca(2+)]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression, and potencies and efficacies of fMLP-induced increases in [Ca(2+)]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive, and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression.

Formyl peptide receptor ligands exert differential effects on corticosterone secretion from the hypothalamo‐pituitary‐adrenal (HPA) axis

The stress response results in increased secretion of cortisol in man and corticosterone (CORT) in rodents. Our previous studies have identified roles for annexin A1 (ANXA1) and its receptor, a member of the formyl peptide receptor (FPR) family (possibly Fpr2) within the hypothalamus and pituitary gland in regulating CORT secretion. The FPRs are expressed in abundance in the adrenal gland but their role has not been determined. We hypothesised that FPRs contribute to the local mechanisms regulating CORT release. Adrenal glands from male Wistar rats (n = 6, 220 – 250 g) were dispersed in Earle's balanced salt solution (EBSS) and collagenase (95% O2, 5% CO2, 37 °C, 2 h) then stimulated (4 h) with a submaximal concentration of ACTH1–24 [Tetracosactide, 10−7 mol.L−1] in the presence and absence of various FPR ligands. ACTH stimulation resulted in a 60% increase in CORT release (P < 0.001). Versus basal release, fMLF (Fpr1), Ac2–26 (Fpr2/3), and 15‐epi‐LXA4 (Fpr3) were inhibitory, whereas F2L (Fpr2) was stimulatory (P < 0.001). Versus ACTH alone, ACTH‐stimulated CORT release was reduced in cells treated with fMLF [10−7], peptide Ac2–26 [10−7], and 15‐epi‐LXA4 [10−9] by 80%, but was increased in the presence F2L [10−10] by 50% (P < 0.001). Our work showed certain Fpr1/Fpr3 agonists inhibit adrenal CORT release whereas Fpr2 appears to be stimulatory.This was funded by the BBSRC.

Microfluidic devices for characterizing the agonist of formyl peptide receptor in RBL-FPR cells

The human formyl peptide receptor (FPR) plays an important role in inflammation and immunity. Finding of specific agonists and antagonists of FPR may provide potential therapeutic agents for FPR related disorders. The binding of agonist by FPR induces a cascade of G protein-mediated signaling events leading to neutrophil chemotaxis, intracellualr calcium mobilization, FPR ligand uptake and so on. This work proposed a microfluidic-based method to characterize FPR-related cellular events in response to small peptides, N-formyl-Met-Leu-Phe (fMLF), in rat basophilic leukemia cell line RBL-2H3 expressing human FPR (RBL-FPR). The results showed that fMLF triggered chemotaxis, calcium mobilization and FPR ligand uptake in RBL-FPR cells, indicating the potential role of FPR agonist. The chemotaxis index and the calcium mobilization intensity increased but the time course of calcium mobilization decreased, as the rising of fMLF concentration. The basic agreement between the microfluidic results and the previous studies demonstrated good feasibility of the microfluidic method for characterization of FPR agonist. Microfluidic technology displays significant advantages over traditional methods in terms of sample consumption and assay time. It also facilitates experimental process and real-time observation of cellular responses at single cell resolution.

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.

Flow and image cytometry side by side for the new frontiers in quantitative single‐cell analysis

THE role of regulatory (CD4CD25) T-cells (Tregs) as therapeutics to replace common anti-inflammatory drugs is of increasing interest since cell therapy seems to be within reach (1). Practical aspects of purification and propagation of Tregs is now summarized by Trzonkowski et al. in the present issue (2). The authors demonstrate that novel advances in flow cytometry and also cellular immunology opens the door for the clinical application of Tregs. Affordable diagnostic tools for HIV and AIDS are of imminent importance in resource poor countries (3). To this end, PCR (4) and cell-based analysis (i.e. CD4 counting) by flow cytometry (5,6) or automated microscopic cytometry (7) is often the method of choice. The current PCR assays, however, often fail to detect virus variants that are prevalent in the third world. Thereby, these patients, despite clinical symptoms, may be diagnosed as virus negative and will not receive therapy. Now, Greve et al. present a new microparticle-based PCR assay that enables detection and quantification of HIV-1 viral load for most of the common variants including outlier, nonmajor, and circulating recombinant forms (8, commentary in 9). Malaria is also one of the great killers that needs appropriate diagnostic tools (7). Jimenez-Diaz et al. (10) developed an assay to quantify malaria-infected erythrocytes using the cell-permeable nuclear dye SYTO-16. The authors show the applicability of their assay for infected normal mice and human erythrocyte engrafted NOD mice. Because flow cytometers are now available in many resource poor settings, both assays can be introduced, relatively inexpensively. Finally, the research group of Edwards et al. applied a flow-cytometry-based high-throughput screening system (11) to identify novel formylpeptide receptor ligand probes. The authors screened more than 24,000 small molecules and finally came up with several candidates of potential future therapeutic or diagnostic interest (12,13). The aforementioned scientific articles of this issue illustrate that flow cytometry is still going strong with innovative technologies for versatile applications presented in our journal. However, every scientific journal has to evolve and adjust its focus to present and adjust for future requirements of the scientific community. In view of the increasing technological advances in imaging (14), our goal was to become a front journal for image cytometry and quantitative image analysis. As one can see from the citations in 2008 to Cytometry Part A, the readers find an increasing appreciation in our publications in the technological field of quantitative imaging. As one good example, Abella et al. (15) present an assay for automated quantification of cell infiltrates in histological sections based on microscopic images from allergic murine models. After extraction of texture parameters by a stepwise procedure the authors propose an algorithm that appears to be superior to manual cell counting. Yet another application is automated laser scanning cytometry (LSC) for mast cells tracking in skin as described by Zoog et al. (16). Rapid, accurate evaluation of mast cells present around wounds in a wound-healing model by automated LSC may serve as an important tool for tracking pharmacodynamic effects of MC-directed therapies. Without doubt we enter the era of automated cell detection in tissues (17). New labeling fluorescent tools for life-cell in vivo imaging are introduced by Robers et al. (18). High-affinity and selective ligand–receptor binding of 50-fluorescein-tagged synthetic ligand for FKBP12 (immunophilin) and mutated receptor

Duplex high‐throughput flow cytometry screen identifies two novel formylpeptide receptor family probes

Of recent, clinical interest have been two related human G-protein coupled receptors: formylpeptide receptor (FPR), linked to antibacterial inflammation and malignant glioma cell metastasis; and FPR like-1 (FPRL1), linked to chronic inflammation in systemic amyloidosis, Alzheimer's disease, and prion diseases. In association with the National Institutes of Health (NIH) Molecular Library Screening Network, we implemented a flow-cytometry-based high-throughput screening (HTS) approach for identifying selective small molecule FPR and FPRL1 ligands. The screening assay measured the ability of test compounds to competitively displace a high-affinity, fluorescein- labeled peptide ligand from FPR, FPRL1, or both. U937 cells expressing FPR and rat basophil leukemia (RBL) cells expressing FPRL1 were tested together in a "duplex" format. The U937 cells were color coded with red-fluorescent dye allowing their distinction during analysis. Compounds, cells, and fluorescent ligand were sequentially combined (no wash) in 15 microl assay volumes in 384-well plates. Throughput averaged approximately 11 min per plate to analyze approximately 4,000 cells ( approximately 2,000/receptor) in a 2 microl aspirate from each well. In primary single concentration HTS of 24,304 NIH Small Molecule Repository compounds, 253 resulted in inhibition >30% (181 for FPR, 72 for FPRL1) of which 40 had selective binding inhibition constants (K(i)) < or = 4 microM (34 for FPR and 6 for FPRL1). An additional 1,446 candidate compounds were selected by structure-activity-relationship analysis of the hits and screened to identify novel ligands for FPR (3570-0208, K(i) = 95 +/- 10 nM) and FPRL1 (BB-V-115, K(i) = 270 +/- 51 nM). Each was a selective antagonist in calcium response assays and the most potent small molecule antagonist reported for its respective receptor to date. The duplex assay format reduced assay time, minimized reagent requirements, and provided selectivity information at every screening stage, thus proving to be an efficient means to screen for selective receptor ligand probes.

Efficient Calculation of Molecular Properties from Simulation Using Kernel Molecular Dynamics

Understanding the relationship between chemical structure and function is a ubiquitous problem within the fields of chemistry and biology. Simulation approaches attack the problem utilizing physics to understand a given process at the particle level. Unfortunately, these approaches are often too expensive for many problems of interest. Informatics approaches attack the problem with empirical analysis of descriptions of chemical structure. The issue in these methods is how to describe molecules in a manner that facilitates accurate and general calculation of molecular properties. Here, we present a novel approach that utilizes aspects of simulation and informatics in order to formulate structure-property relationships. We show how supervised learning can be utilized to overcome the sampling problem in simulation approaches. Likewise, we show how learning can be achieved based on molecular descriptions that are rooted in the physics of dynamic intermolecular forces. We apply the approach to three problems including the analysis of corticosteroid binding globulin ligand binding affinity, identification of formylpeptide receptor ligands, and identification of resveratrol analogues capable of inhibiting activation of transcription factor nuclear factor kappaB.

Biologically Active Peptides Interacting with the G Protein-Coupled Formylpeptide Receptors

Leucocytes accumulate at sites of inflammation and microbial infection in response to locally produced chemotactic factors. N-formylpeptides produced by Gram negative bacteria were among the first chemotactic factors structurally defined which signal through G protein-coupled formylpeptide receptor (FPR) and FPR-like 1 (FPRL1) expressed by phagocytic leukocytes in human and in mouse homogogues mFPR and mFPR2. During the past few years, a number of pathogen- and host-derived agonists/antagonists for FPR, FPRL1 and another FPR variant FPR-like 2 (FPRL2) have been identified. Activation of formylpeptide receptors (FPRs) in phagocytic leukocytes by agonists results in increased cell chemotaxis, phagocytosis, and release of pro-inflammatory mediators. Peptide agonists for FPRs have also been shown to possess immune adjuvant activity when injected in mice. In addition, FPR aberrantly expressed on highly malignant human glioblastoma cells promotes tumor cell migration, proliferation and production of vascular endothelial growth factor in response to agonists released by necrotic tumor cells. Therefore, formylpeptide receptor ligands, by interacting with FPRs, play important roles in host defense and in the rapid progression of human glioblastoma.

The non-steroidal anti-inflammatory drug piroxicam blocks ligand binding to the formyl peptide receptor but not the formyl peptide receptor like 1

The anti-inflammatory drug piroxicam has been reported to affect the production of reactive oxygen species in phagocytes. This anti-inflammatory effect is thought to be mediated through inhibition of cyclooxygenase (COX), an enzyme important for prostaglandin synthesis. We have compared the effects of piroxicam on superoxide production mediated by two closely related G-protein coupled receptors expressed on neutrophils, the formyl peptide receptor (FPR) and the formyl peptide receptor like 1 (FPRL1). Neutrophils were stimulated with agonists that bind specifically to FPR (the peptide ligand N-formyl-Met-Leu-Phe, fMLF) or FPRL1 (the peptide ligand Trp-Lys-Tyr-Met-Val-L-Met-NH2, WKYMVM) or both of these receptors (the peptide ligand Trp-Lys-Tyr-Met-Val-D-Met-NH2, WKYMVm). Piroxicam reduced the neutrophil superoxide production induced by the FPR agonist but had no significant effect on the FPRL1 induced response. Neutrophil intracellular calcium changes induced by the agonist WKYMVm (that triggers both FPR and FPRL1) were only inhibited by piroxicam when the drug was combined with the FPRL1 specific antagonist, Trp-Arg-Trp-Trp-Trp-Trp (WRW4), and this was true also for the inhibition of superoxide anion release. Receptor-binding analysis showed that the fluorescently labelled FPR specific ligand N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (fNLFNYK), was competed for in a dose-dependent manner, by the FPR ligand fMLF and as well as by piroxicam.We show that piroxicam inhibits the neutrophil responses triggered through FPR, but not through FPRL1 and this inhibition is due to a reduced binding of the activating ligand to its cell surface receptor.

Biomolecular screening of formylpeptide receptor ligands with a sensitive, quantitative, high-throughput flow cytometry platform

The formylpeptide receptor (FPR) family of G protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here we describe a high-throughput flow cytometry screening approach that has successfully identified multiple families of previously unknown FPR ligands. The assay detects active structures that block the binding of a fluorescent ligand to membrane FPR of intact cells, thus detecting both agonists and antagonists. It is homogeneous in that assay reagents are added in sequence and the wells are subsequently analyzed without intervening wash steps. Microplate wells are routinely processed at a rate of 40 wells per minute, requiring a volume of only 2 microl to be sampled from each. This screening approach has recently been extended to identify a high-affinity, selective agonist for the intracellular estrogen-binding G protein-coupled receptor GPR30. With the development of appropriate assay reagents, it may be generally adaptable to a wide range of receptors. The total time required for the assay ranges between 1.5 and 2.5 h. The time required for flow cytometry analysis of a 96-well plate at the end of the procedure is less than 2.5 min. By comparison, manual processing of 96 samples will typically require 40-50 min, and a fast commercial automated sampler processes 96-well plates in less than 15 min, requiring the aspiration of 22 microl per sample for an analysis volume of 2 microl.

Integration of Virtual Screening with High-Throughput Flow Cytometry to Identify Novel Small Molecule Formylpeptide Receptor Antagonists

The formylpeptide receptor (FPR) family of G-protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. We developed a FPR homology model and pharmacophore (based on the bovine rhodopsin crystal structure and known FPR ligands, respectively) for in silico screening of approximately 480,000 drug-like small molecules. A subset of 4324 compounds that matched the pharmacophore was then physically screened with the HyperCyt flow cytometry platform in high-throughput, no-wash assays that directly measure human FPR binding, with samples (each approximately 2500 cells in 2 microl) analyzed at 40/min. From 52 confirmed hits (1.2% hit rate), we identified 30 potential lead compounds (inhibition constant, Ki= 1-32 microM) representing nine distinct chemical families. Four compounds in one family were weak partial agonists. All others were antagonists. This virtual screening approach improved the physical screening hit rate by 12-fold (versus 0.1% hit-rate in a random compound collection), providing an efficient process for identifying small molecule antagonists.

The Synthetic Peptide Trp-Lys-Tyr-Met-Val-D-Met Inhibits Human Monocyte-Derived Dendritic Cell Maturation via Formyl Peptide Receptor and Formyl Peptide Receptor-Like 2

Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) has been reported to stimulate monocytes, neutrophils, and dendritic cells (DCs). However, although WKYMVm has been reported to function as a DC chemoattractant, its role on DC maturation has not been examined. In this study, we investigated the effects of WKYMVm on human DC maturation. The costimulation of DCs with WKYMVm and LPS dramatically inhibited LPS-induced IL-12 production, CD86 and HLA-DR surface expression, and DC-mediated T cell proliferation. However, DC phagocytic activity was increased by WKYMVm stimulation. These findings demonstrate that WKYMVm inhibits DC maturation by LPS. In terms of the mechanism underlying DC maturation inhibition by WKYMVm, we found that LPS-induced DC maturation was negatively regulated by WKYMVm-stimulated ERK activity. Moreover, the costimulation of DCs with WKYMVm and LPS dramatically inhibited the LPS-induced accumulations of IL-12 mRNA, thus suggesting that WKYMVm inhibits LPS-induced IL-12 production at the transcriptional level. We also found that DCs express two WKYMVm receptors, formyl peptide receptor (FPR) and FPR-like 2 (FPRL2). In addition, formyl-Met-Leu-Phe (a FPR ligand), Trp-Lys-Tyr-Met-Val-Met, Hp(2-20) peptide, and F2L (three FPRL2 ligands) inhibited LPS-induced IL-12 production in DCs. Taken together, our findings indicate that the activations of FPR and FPRL2 inhibit LPS-induced DC maturation, and suggest that these two receptors should be regarded as important potential therapeutic targets for the modulation of DC maturation.

Chemotaxis and Calcium Responses of Phagocytes to Formyl Peptide Receptor Ligands Is Differentially Regulated by Cyclic ADP Ribose

Cyclic ADP ribose (cADPR) is a calcium-mobilizing metabolite that regulates intracellular calcium release and extracellular calcium influx. Although the role of cADPR in modulating calcium mobilization has been extensively examined, its potential role in regulating immunologic responses is less well understood. We previously reported that cADPR, produced by the ADP-ribosyl cyclase, CD38, controls calcium influx and chemotaxis of murine neutrophils responding to fMLF, a peptide agonist for two chemoattractant receptor subtypes, formyl peptide receptor and formyl peptide receptor-like 1. In this study, we examine whether cADPR is required for chemotaxis of human monocytes and neutrophils to a diverse array of chemoattractants. We found that a cADPR antagonist and a CD38 substrate analogue inhibited the chemotaxis of human phagocytic cells to a number of formyl peptide receptor-like 1-specific ligands but had no effect on the chemotactic response of these cells to ligands selective for formyl peptide receptor. In addition, we show that the cADPR antagonist blocks the chemotaxis of human monocytes to CXCR4, CCR1, and CCR5 ligands. In all cases, we found that cADPR modulates intracellular free calcium levels in cells activated by chemokines that induce extracellular calcium influx in the apparent absence of significant intracellular calcium release. Thus, cADPR regulates calcium signaling of a discrete subset of chemoattractant receptors expressed by human leukocytes. Since many of the chemoattractant receptors regulated by cADPR bind to ligands that are associated with clinical pathology, cADPR and CD38 represent novel drug targets with potential application in chronic inflammatory and neurodegenerative disease.

Functional activation of the formyl peptide receptor by a new endogenous ligand in human lung A549 cells.

The formyl peptide receptor (FPR), a heptahelical G protein-coupled receptor on phagocytic leukocytes, can be triggered by bacterially derived oligopeptides of the prototype fMLP. Although FPR expression and activation have been associated with cells of myeloid origin and bacterial inflammation, the receptor has recently been identified in nonmyeloid cells, thus suggesting additional physiological functions and the existence of an endogenous agonist. In this study, we demonstrate the presence and functional activation of the FPR in the human lung cell line A549, which represents an extrahepatic model for the regulation of acute-phase proteins. Activation of the FPR in A549 cells cannot only be triggered by fMLP, but also by an agonistic peptide of the recently identified endogenous FPR ligand, annexin 1. In addition to inducing changes in the F-actin content, annexin 1-mediated triggering of the FPR results in an increased expression of acute-phase proteins. Hence, activation of nonmyeloid FPR by its endogenous ligand annexin 1 could participate in the regulation of acute-phase responses, e.g., during inflammation and/or wound healing.

Same Receptor, Different Effects

The anti-inflammatory action of annexin I, a secreted calcium- and phospholipid-binding protein, includes inhibiting neutrophil transmigration from the blood into tissue. Walther et al. report that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. In contrast, bacterial formyl peptides, the classical FPR ligands, are proinflammatory. When bound to the receptor, annexin I-derived peptides and formyl peptides elicit a similar elevation in intracellular calcium in a G-protein-dependent manner. Competition assays indicate that the two peptides likely bind to different regions of FPR. The authors propose that the two different ligands activate different receptor-initiated signaling cascades that result in opposing inflammatory responses.Walther, A., Riehemann, K., and Gerke, V. (2000) A novel ligand of the formyl peptide receptor: Annexin I regulates neutrophil extravasation by interacting with the FPR. Mol. Cell 5: 831-840. [Online Journal]

Same Receptor, Different Effects

The anti-inflammatory action of annexin I, a secreted calcium- and phospholipid-binding protein, includes inhibiting neutrophil transmigration from the blood into tissue. Walther et al . report that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. In contrast, bacterial formyl peptides, the classical FPR ligands, are proinflammatory. When bound to the receptor, annexin I-derived peptides and formyl peptides elicit a similar elevation in intracellular calcium in a G-protein-dependent manner. Competition assays indicate that the two peptides likely bind to different regions of FPR. The authors propose that the two different ligands activate different receptor-initiated signaling cascades that result in opposing inflammatory responses. Walther, A., Riehemann, K., and Gerke, V. (2000) A novel ligand of the formyl peptide receptor: Annexin I regulates neutrophil extravasation by interacting with the FPR. Mol. Cell 5 : 831-840. [Online Journal]