Yingying Le

Formyl-peptide receptors revisited

Leukocytes accumulate at sites of inflammation and microbial infection in direct response to locally produced chemotactic factors, which signal through specific G protein-coupled receptors. The first chemotactic factors to be structurally defined were the N-formyl peptides. Unlike other leukocyte chemoattractants, N-formyl peptides could originate from either an endogenous source, such as the mitochondrial proteins of ruptured host cells, or an exogenous source, such as the proteins of invading pathogens. This suggests that the formyl-peptide receptor (FPR) and its variant FPRL1 (FPR-like 1) are involved in host defense against bacterial infection and in the clearance of damaged cells. Recently, additional, more complex, roles for these receptors have been proposed because FPR, and to a greater extent FPRL1, have been found to interact with a menagerie of structurally diverse pro- and anti-inflammatory ligands associated with different diseases, including amyloidosis, Alzheimers disease, prion disease and HIV. How these receptors recognize such diverse ligands, which are the most important in vivo, and how they contribute to disease pathogenesis and host defense are basic questions currently under investigation that could lead to new therapeutic targets.

Pleiotropic roles of formyl peptide receptors

FPR and FPRL1 belong to the seven-transmembrane, G protein-coupled chemoattractant receptor superfamily. Because of their capacity to interact with bacterial chemotactic formylated peptides, these receptors are thought to play a role in host defense against microbial infection. Recently, a variety of novel agonists have been identified for these receptors, including several host-derived endogenous molecules that are involved in proinflammatory responses. Most notably is the use of FPRL1 by at least three amyloidogenic protein and peptide ligands, the serum amyloid A (SAA), the 42 amino acid form of beta amyloid (Abeta(42)), and the prion peptide PrP106-126, to chemoattract and activate human phagocytic leukocytes. These new findings have greatly expanded the functional scope of the formyl peptide receptors and call for more in-depth investigation of the role of these receptors in pathophysiological conditions.

β Amyloid peptide (Aβ42) is internalized via the G-protein-coupled receptor FPRL1 and forms fibrillar aggregates in macrophages

The 42 amino acid form of β amyloid (Aβ42) plays a pivotal role in neurotoxicity and the activation of mononuclear phagocytes in Alzheimers disease (AD). Our recent study revealed that FPRL1, a G‐protein‐coupled receptor, mediates the chemotactic and activating effect of Aβ42 on mononuclear phagocytes (monocytes and microglia), suggesting that FPRL1 may be involved in the proinflammatory responses in AD. We investigated the role of FPRL1 in cellular uptake and the subsequent fibrillar formation of Aβ42 by using fluorescence confocal microscopy. We found that upon incubation with macrophages or HEK293 cells genetically engineered to express FPRL1, Aβ42 associated with FPRL1 and the Aβ42/FPRL1 complexes were rapidly internalized into the cytoplasmic compartment. The maximal internalization of Aβ42/ FPRL1 complexes occurred by 30 min after incubation. Removal of free Aβ42 from culture supernatants at 30 min resulted in a progressive recycling of FPRL1 to the cell surface and degradation of the internalized Aβ42. However, persistent exposure of the cells to Aβ42 over 24 h resulted in retention of Aβ42/FPRL1 complexes in the cytoplasmic compartment and the formation of Congo red positive fibrils in macrophages but not in HEK 293 cell transfected with FPRL 1. These results suggest that besides mediating the proinflammatory activity of Aβ42, FPRL1 is also involved in the internalization of Aβ42, which culminates in the formation of fibrils only in macrophages.—Yazawa, H., Yu, Z.‐X., Takeda, K., Le, Y., Gong, W., Ferrans, V. J., Oppenheim, J. J., Li, C. C. H., Wang, J. M. β Amyloid peptide (Aβ42) is internalized via the G‐protein‐coupled receptor FPRL1 and forms fibrillar aggregates in macrophages. FASEB J. 15, 2454–2462 (2001)

Cutting Edge: The Neurotoxic Prion Peptide Fragment PrP106–126 Is a Chemotactic Agonist for the G Protein-Coupled Receptor Formyl Peptide Receptor-Like 1

Prion diseases are transmissible and fatal neurodegenerative disorders which involve infiltration and activation of mononuclear phagocytes at the brain lesions. A 20-aa acid fragment of the human cellular prion protein, PrP106–126, was reported to mimic the biological activity of the pathologic isoform of prion and activates mononuclear phagocytes. The cell surface receptor(s) mediating the activity of PrP106–126 is unknown. In this study, we show that PrP106–126 is chemotactic for human monocytes through the use of a G protein-coupled receptor formyl peptide receptor-like 1 (FPRL1), which has been reported to interact with a diverse array of exogenous or endogenous ligands. Upon stimulation by PrP106–126, FPRL1 underwent a rapid internalization and, furthermore, PrP106–126 enhanced monocyte production of proinflammatory cytokines, which was inhibited by pertussis toxin. Thus, FPRL1 may act as a “pattern recognition” receptor that interacts with multiple pathologic agents and may be involved in the proinflammatory process of prion diseases.

Resveratrol differentially modulates inflammatory responses of microglia and astrocytes

BackgroundInflammatory responses in the CNS mediated by activated glial cells play an important role in host-defense but are also involved in the development of neurodegenerative diseases. Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect microglia and astrocyte from inflammatory insults and explored mechanisms underlying different inhibitory effects of resveratrol on microglia and astrocytes.MethodsA murine microglia cell line (N9), primary microglia, or astrocytes were stimulated by LPS with or without different concentrations of resveratrol. The expression and release of proinflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1) and iNOS/NO by the cells were measured by PCR/real-time PCR and ELISA, respectively. The phosphorylation of the MAP kinase superfamily was analyzed by western blotting, and activation of NF-κB and AP-1 was measured by luciferase reporter assay and/or electrophoretic mobility shift assay.ResultsWe found that LPS stimulated the expression of TNF-α, IL-1β, IL-6, MCP-1 and iNOS in murine microglia and astrocytes in which MAP kinases, NF-κB and AP-1 were differentially involved. Resveratrol inhibited LPS-induced expression and release of TNF-α, IL-6, MCP-1, and iNOS/NO in both cell types with more potency in microglia, and inhibited LPS-induced expression of IL-1β in microglia but not astrocytes. Resveratrol had no effect on LPS-stimulated phosphorylation of ERK1/2 and p38 in microglia and astrocytes, but slightly inhibited LPS-stimulated phosphorylation of JNK in astrocytes. Resveratrol inhibited LPS-induced NF-κB activation in both cell types, but inhibited AP-1 activation only in microglia.ConclusionThese results suggest that murine microglia and astrocytes produce proinflammatory cytokines and NO in response to LPS in a similar pattern with some differences in signaling molecules involved, and further suggest that resveratrol exerts anti-inflammatory effects in microglia and astrocytes by inhibiting different proinflammatory cytokines and key signaling molecules.

Bacterial lipopolysaccharide selectively up-regulates the function of the chemotactic peptide receptor formyl peptide receptor 2 in murine microglial cells.

Receptors for the bacterial chemotactic peptide fMLP are implicated in inflammation and host defense against microbial infection. We investigated the expression and function of fMLPR in microglial cells, which share characteristics of mononuclear phagocytes and play an important role in proinflammatory responses in the CNS. The expression of the genes encoding formyl peptide receptor (FPR)1 and FPR2, the high- and low-affinity fMLPR, was detected in a murine microglial cell line N9, but these cells did not respond to chemotactic agonists known for these receptors. N9 cells incubated with bacterial LPS increased the expression of fMLPR genes and developed a species of specific, but low-affinity, binding sites for fMLP, in association with marked calcium mobilization and chemotaxis responses to fMLP in a concentration range that typically activated the low-affinity receptor FPR2. In addition, LPS-treated N9 cells were chemoattracted by two FPR2-specific agonists, the HIV-1 envelope-derived V3 peptide, and the 42 aa form of the amyloid β peptide which is a pathogenic agent in Alzheimer’s disease. Primary murine microglial cells also expressed FPR1 and FPR2 genes, but similar to N9 cells, exhibited FPR2-mediated activation only after LPS treatment. In contrast to its effect on the function of FPR2, LPS reduced N9 cell binding and biological responses to the chemokine stromal cell-derived factor-1α. Thus, LPS selectively modulates the function of chemoattractant receptors in microglia and may promote host response in inflammatory diseases in the CNS.

Receptors for chemotactic formyl peptides as pharmacological targets.

Leukocytes accumulate at sites of inflammation and immunological reaction in response to locally existing chemotactic mediators. N-formyl peptides, such as fMet-Leu-Phe (fMLF), are some of the first identified and most potent chemoattractants for phagocytic leukocytes. In addition to the bacterial peptide fMLF and the putative endogenously produced formylated peptides, a number of novel peptide agonists have recently been identified that selectively activate the high-affinity fMLF receptor FPR and/or its low-affinity variant FPRL1, both of which belong to the seven-transmembrane (STM), G protein-coupled receptor (GPCR) superfamily. These agonists include peptide domains derived from the envelope proteins of human immunodeficiency virus type 1 (HIV-1) and at least three amyloidogenic polypeptides, the human acute phase protein serum amyloid A, the 42 amino acid form of beta amyloid peptide and a 21 amino acid fragment of human prion. Furthermore, a cleavage fragment of neutrophil granule-derived bactericidal cathelicidin, LL-37, is also a chemotactic agonist for FPRL1. Activation of formyl peptide receptors results in increased cell migration, phagocytosis, release of proinflammatory mediators, and the signaling cascade culminates in heterologous desensitization of other STM receptors including chemokine receptors CCR5 and CXCR4, two coreceptors for HIV-1. Thus, by interacting with a variety of exogenous and host-derived agonists, formyl peptide receptors may play important roles in proinflammatory and immunological diseases and constitute a novel group of pharmacological targets.

Synthetic peptide MMK‐1 is a highly specific chemotactic agonist for leukocyte FPRL1

Human phagocytic leukocytes express the seven‐transmembraneG‐protein‐coupled receptors formyl peptide receptor (FPR) and FPR‐like1 (FPRL1). MMK‐1, a synthetic peptide derived from a random peptidelibrary, is reported to induce calcium mobilization specifically inhuman FPRL1 gene‐transfected cells. However, its actions onhuman phagocytic leukocytes remain poorly defined. We found that MMK‐1is a potent chemotactic and calcium‐mobilizing agonist for humanmonocytes, neutrophils, and FPRL1‐transfected human embryonic kidney(HEK) 293 cells but is inactive in cells transfected with FPR. MMK‐1also activated HEK 293 cells transfected with FPR2, a mousecounterpart of human FPRL1. Furthermore, MMK‐1 increasedpertussis toxin‐sensitive production of inflammatory cytokines in humanmonocytes. MMK‐1 signaling in human phagocytes was completelydesensitized by a well‐defined FPRL1 agonist, suggesting thatFPRL1 is likely a receptor that mediates the action of MMK‐1 in primarycells. Since MMK‐1 is one of the most potent FPRL1‐specific agonistsidentified so far, it can serve as a modulator of the hostdefense and a useful agent for further studying the signaling andfunction of FPRL1.

Expression of functional formyl peptide receptors by human astrocytoma cell lines.

Activation of astrocytes is important in the pathogenesis of a variety of diseases in the central nervous system, such as infection and neurodegeneration. We found that the bacterial chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLF) induced potent migration and Ca(2+) mobilization in human astrocytoma cell lines. The effect of fMLF was pertussis toxin-sensitive, suggesting the involvement of seven transmembrane, G protein-coupled receptor(s) for fMLF. Scatchard analyses revealed that astrocytoma cell lines express both high- and low-affinity binding sites for [3H]fMLF. RT-PCR confirmed the expression of transcripts of fMLF receptors, the high-affinity FPR and the low-affinity FPRL1 by these cells. Both fMLF and F peptide, a synthetic peptide domain of HIV-1 envelope protein which specifically activates FPRL1, increased secretion of IL-6 by astrocytoma cells. Our study demonstrates for the first time that FPR and FPRL1 expressed by astrocytoma cell lines are functional, and suggests a molecular basis for the involvement of these receptors in host defense in the brain.

Toll-like receptors in inflammation of the central nervous system

Toll-like receptors (TLRs) belong to pattern-recognition receptor family that could recognize exogenous pathogen-associated molecular patterns and endogenous damage-associated molecular patterns. TLRs play pivotal roles in innate and adaptive immune responses. In this review we summarize the ligands and signal transduction pathways of TLRs and highlight recent progress of the involvement of TLRs in neuroinflammation related disorders, including cerebral ischemia/stroke, brain trauma and hemorrhage, pathogen infection and autoimmune diseases, and explore the potential of TLR signaling as therapeutic targets against these disorders.