Chemical Composition and Immunomodulatory Activity of Essential Oils from Rhododendron albiflorum.

Rhododendron (Ericaceae) extracts contain flavonoids, chromones, terpenoids, steroids, and essential oils and are used in traditional ethnobotanical medicine. However, little is known about the immunomodulatory activity of essential oils isolated from these plants. Thus, we isolated essential oils from the flowers and leaves of R. albiflorum (cascade azalea) and analyzed their chemical composition and innate immunomodulatory activity. Compositional analysis of flower (REOFl) versus leaf (REOLv) essential oils revealed significant differences. REOFl was comprised mainly of monoterpenes (92%), whereas sesquiterpenes were found in relatively low amounts. In contrast, REOLv was primarily composed of sesquiterpenes (90.9%), with a small number of monoterpenes. REOLv and its primary sesquiterpenes (viridiflorol, spathulenol, curzerene, and germacrone) induced intracellular Ca2+ mobilization in human neutrophils, C20 microglial cells, and HL60 cells transfected with N-formyl peptide receptor 1 (FPR1) or FPR2. On the other hand, pretreatment with these essential oils or component compounds inhibited agonist-induced Ca2+ mobilization and chemotaxis in human neutrophils and agonist-induced Ca2+ mobilization in microglial cells and FPR-transfected HL60 cells, indicating that the direct effect of these compounds on [Ca2+]i desensitized the cells to subsequent agonist activation. Reverse pharmacophore mapping suggested several potential kinase targets for these compounds; however, these targets were not supported by kinase binding assays. Our results provide a cellular and molecular basis to explain at least part of the beneficial immunotherapeutic properties of the R. albiflorum essential oils and suggest that essential oils from leaves of this plant may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration.

Anti-glioma Activity of Dapsone and Its Enhancement by Synthetic Chemical Modification.

The sulfone dapsone is an old antibiotic used for the treatment of mycobacterial and protozoal infections. We postulated before that dapsone might possess biological activity exceeding its anti-infectious properties and that it could potentially be repurposed for the treatment of glioma. To test this hypothesis, we treated established and primary cultured glioma cells with dapsone or several dapsone analogues which we previously synthesized (D2-D5) and determined effects on proliferation, anchorage-independent growth and migration. While dapsone and its synthetic analogues D2-D5 displayed only modest anti-proliferative activity, important neoplastic features such as anchorage-independent growth, clonogenic survival and directed migration were significantly inhibited by dapsone treatment. Moreover, dapsone analogues D3, D4 and D5 yielded even enhanced anti-glioma activity against different pro-neoplastic features. Overall these data suggest that dapsone provides activity against glioma which can be further enhanced by molecular modifications. These compounds could potentially serve as a therapeutic adjunct to the treatment of gliomas in a repurposing approach.

Smoking-induced expression of the GPR15 gene indicates its potential role in chronic inflammatory pathologies.

Despite the described clear epigenetic effects of smoking, the effect of smoking on genome-wide gene expression in the blood is obscure. We therefore studied the smoking-induced changes in the gene-expression profile of the peripheral blood. RNA was extracted from the whole blood of 48 individuals with a detailed smoking history (24 never-smokers, 16 smokers, and 8 ex-smokers). Gene-expression profiles were evaluated with RNA sequencing, and results were analyzed separately in 24 men and 24 women. In the male smokers, 13 genes were statistically significantly (false-discovery rate <0.1) differentially expressed; in female smokers, 5 genes. Although most of the differentially expressed genes were different between the male and female smokers, the G-protein-coupled receptor 15 gene (GPR15) was differentially expressed in both male and female smokers compared with never-smokers. Analysis of GPR15 methylation identified significantly greater hypomethylation in smokers compared with that in never-smokers. GPR15 is the chemoattractant receptor that regulates T-cell migration and immunity. Up-regulation of GPR15 could explain to some extent the health hazards of smoking with regard to chronic inflammatory diseases.

Propofol inhibits superoxide production, elastase release, and chemotaxis in formyl peptide-activated human neutrophils by blocking formyl peptide receptor 1.

Neutrophils play a critical role in acute and chronic inflammatory processes, including myocardial ischemia/reperfusion injury, sepsis, and adult respiratory distress syndrome. Binding of formyl peptide receptor 1 (FPR1) by N-formyl peptides can activate neutrophils and may represent a new therapeutic target in either sterile or septic inflammation. Propofol, a widely used i.v. anesthetic, has been shown to modulate immunoinflammatory responses. However, the mechanism of propofol remains to be established. In this study, we showed that propofol significantly reduced superoxide generation, elastase release, and chemotaxis in human neutrophils activated by fMLF. Propofol did not alter superoxide generation or elastase release in a cell-free system. Neither inhibitors of γ-aminobutyric acid receptors nor an inhibitor of protein kinase A reversed the inhibitory effects of propofol. In addition, propofol showed less inhibitory effects in non-FPR1-induced cell responses. The signaling pathways downstream from FPR1, involving calcium, AKT, and ERK1/2, were also competitively inhibited by propofol. These results show that propofol selectively and competitively inhibits the FPR1-induced human neutrophil activation. Consistent with the hypothesis, propofol inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analog of fMLF, to FPR1 in human neutrophils, differentiated THP-1 cells, and FPR1-transfected human embryonic kidney-293 cells. To our knowledge, our results identify, for the first time, a novel anti-inflammatory mechanism of propofol by competitively blocking FPR1 in human neutrophils. Considering the importance of N-formyl peptides in inflammatory processes, our data indicate that propofol may have therapeutic potential to attenuate neutrophil-mediated inflammatory diseases by blocking FPR1.

Stimulation of human formyl peptide receptors by calpain inhibitors: homology modeling of receptors and ligand docking simulation.

Calpain inhibitors, including peptide aldehydes (N-acetyl-Leu-Leu-Nle-CHO and N-acetyl-Leu-Leu-Met-CHO) and α-mercapto-acrylic acid derivatives (PD150606 and PD151746), have been shown to stimulate phagocyte functions via activation of human formyl peptide receptor (hFPR) and/or hFPR-like 1 (hFPRL1). Using the homology modeling of the receptors and the ligand docking simulation, here we show that these calpain inhibitors could bind to the putative N-formyl-Met-Leu-Phe (fMLF) binding site on hFPR and/or hFPRL1. The studies with HEK-293 cells stably expressing hFPR or hFPRL1 showed that the concentrations of calpain inhibitors required to induce an increase in cytoplasmic free Ca(2+) ([Ca(2+)](i)) was much higher (>100 folds) than those of fMLF and Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm). HEK-293 cells expressing hFPR or hFPRL1 with the mutated fMLF binding site never exhibited the [Ca(2+)](i) response to calpain inhibitors. When the optimal concentrations of each stimulus were used, pretreatment of cells with fMLF or WKYMVm abolished an increase in [Ca(2+)](i) induced by calpain inhibitors as well as the same stimulus, whereas pretreatment of cells with calpain inhibitors significantly suppressed, but never abolished, the [Ca(2+)](i) response induced by fMLF or WKYMVm, suggesting that the binding affinity of the inhibitors to the putative fMLF binding site may be lower than that of fMLF or WKYMVm.

Antiallergic cromones inhibit neutrophil recruitment onto vascular endothelium via annexin-A1 mobilization.

OBJECTIVE: To determine whether the inhibitory action of the antiallergic cromone "mast cell stabilizing" drugs on polymorphonuclear leukocyte (PMN) trafficking is mediated through an annexin-A1 (Anx-A1) dependent mechanism. METHODS AND RESULTS: Intravital microscopy was used to monitor the actions of cromones in the inflamed microcirculation. Reperfusion injury provoked a dramatic increase in adherent and emigrated leukocytes in the mesenteric vascular bed, associated with augmented tissue levels of myeloperoxidase. Nedocromil, 2 to 20 mg/kg, significantly (P<0.05) inhibited cell adhesion and emigration, as well as myeloperoxidase release, in wild-type but not Anx-A1(-/-) mice. Short pretreatment of human PMNs with nedocromil, 10 nmol/L, inhibited cell adhesion (P<0.05) in the flow chamber assay, and this effect was reversed by specific anti-AnxA1 or a combination of antiformyl peptide receptors 1 and 2, but not irrelevant control, antibodies. Western blotting experiments revealed that cromones stimulate protein kinase C-dependent phosphorylation and release Anx-A1 in human PMNs. CONCLUSIONS: We propose a novel mechanism to explain the antiinflammatory actions of cromones on PMN trafficking, an effect that has long puzzled investigators.

Functional and physical interactions between formyl-peptide-receptors and scavenger receptor MARCO and their involvement in amyloid beta 1-42-induced signal transduction in glial cells.

Recent studies suggest that the chemotactic G protein-coupled receptor formyl-peptide-receptor-like-1 (FPRL1) or the scavenger receptor MARCO (macrophage receptor with collagenous structure) plays an essential role in the inflammatory response of host defense mechanisms and neurodegenerative disorders such as Alzheimer's disease. We therefore analyzed the involvement of FPRL1 and MARCO in amyloid beta1-42 (Abeta1-42)-induced signalling by extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia) and in transfected HEK293 cells. Receptors were inhibited by small interference RNA and the consequences in Abeta1-42- and MARCO agonist fucoidan-induced signal transduction were determined. Receptor deactivation by antagonists or small interference RNA verified the importance of FPRL1 for Abeta1-42-mediated signal transduction by ERK1/2 phosphorylation and cAMP level measurement in glial cells. Furthermore, for the first time, we have demonstrated a functional interaction between FPRL1 and scavenger receptors in fucoidan-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition, co-immunoprecipitation data and fluorescence microscopy measurements revealed a physical interaction between FPR, FPRL1 and MARCO. These results suggest that FPRL1 plays a pivotal role for Abeta1-42-induced signal transduction in glial cells and the interaction with MARCO could explain the broad ligand spectrum of formyl peptide receptors.

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.

Activated N-formyl peptide receptor and high-affinity IgE receptor occupy common domains for signaling and internalization.

Immune cells display multiple cell surface receptors that integrate signals for survival, proliferation, migration, and degranulation. Here, immunogold labeling is used to map the plasma membrane distributions of two separate receptors, the N-formyl peptide receptor (FPR) and the high-affinity IgE receptor (FepsilonRI). We show that the FPR forms signaling clusters in response to monovalent ligand. These domains recruit Gi, followed by the negative regulatory molecule arrestin2. There are low levels of colocalization of FPR with FcepsilonRI in unstimulated cells, shown by computer simulation to be a consequence of receptor density. Remarkably, there is a large increase in receptor coclustering when cells are simultaneously treated with N-formyl-methionyl-leucyl-phenylalanine and IgE plus polyvalent antigen. The proximity of two active receptors may promote localized cross-talk, leading to enhanced inositol-(3,4,5)-trisphosphate production and secretion. Some cointernalization and trafficking of the two receptors can be detected by live cell imaging, but the bulk of FPR and FcepsilonRI segregates over time. This segregation is associated with more efficient internalization of cross-linked FcepsilonRI than of arrestin-desensitized FPR. The observation of receptors in lightly coated membrane invaginations suggests that, despite the lack of caveolin, hematopoietic cells harbor caveolae-like structures that are candidates for nonclathrin-mediated endocytosis.

Targeting AnxA1/Formyl Peptide Receptor 2 Pathway Affords Protection against Pathological Thrombo-Inflammation.

Stroke is a leading cause of death and disability globally and is associated with a number of co-morbidities including sepsis and sickle cell disease (SCD). Despite thrombo-inflammation underlying these co-morbidities, its pathogenesis remains complicated and drug discovery programs aimed at reducing and resolving the detrimental effects remain a major therapeutic challenge. The objective of this study was to assess whether the anti-inflammatory pro-resolving protein Annexin A1 (AnxA1) was able to reduce inflammation-induced thrombosis and suppress platelet activation and thrombus formation in the cerebral microvasculature. Using two distinct models of pathological thrombo-inflammation (lipopolysaccharide (LPS) and sickle transgenic mice (STM)), thrombosis was induced in the murine brain using photoactivation (light/dye) coupled with intravital microscopy. The heightened inflammation-induced microvascular thrombosis present in these two distinct thrombo-inflammatory models was inhibited significantly by the administration of AnxA1 mimetic peptide AnxA1Ac2-26 (an effect more pronounced in the SCD model vs. the endotoxin model) and mediated by the key resolution receptor, Fpr2/ALX. Furthermore, AnxA1Ac2-26 treatment was able to hamper platelet aggregation by reducing platelet stimulation and aggregation (by moderating αIIbß3 and P-selectin). These findings suggest that targeting the AnxA1/Fpr2/ALX pathway represents an attractive novel treatment strategy for resolving thrombo-inflammation, counteracting e.g., stroke in high-risk patient cohorts.

Effect of treatment on mononuclear cell migration in cervical cancer patients.

AIMS AND BACKGROUND: Our aim was to evaluate the effect of treatment on the in vitro migration of circulating mononuclear cells in cervical cancer patients at different stages. METHODS: We prospectively investigated 24 patients with cervical neoplasia, without prior treatment, submitted to surgery or chemotherapy as therapeutic conduct. Controls were healthy volunteer women (n = 23). Mononuclear cells were isolated from peripheral venous blood before and after treatment, and their migration capacity was evaluated in a microchemotaxis chamber assay towards the chemotactic stimuli fMLP, MCP-1 and RANTES, compared to basal migration. Serum levels of nitric oxide metabolites were assayed by the Griess reaction. RESULTS: Increased mononuclear cell migration in response to the chemotactic stimuli, compared to basal migration, was observed in controls and patients, without differences between them. After treatment (n = 14), mononuclear cell migration in response to MCP-1 and RANTES was increased compared to pre-treatment. Serum levels of nitric oxide metabolites were more elevated in patients (n = 19) than in controls (n = 17), but decreased after treatment (n = 15). CONCLUSIONS: The results suggest that the production of soluble circulating factors by tumor cells could interfere with the functional activity of blood mononuclear cells.

Role of Formyl Peptide Receptors in Gastrointestinal Healing.

The wound healing and the barrier restoration of the gastrointestinal (GI) mucosa must be continuously ensured to allow homeostasis of the gastrointestinal tract and of all the surrounding tissues. Several lines of the evidence report a key role of innate immunity, and in particular of Pattern Recognition Receptors (PRRs), in controlling the homeostasis of GI tract by sensing commensal and pathogen bacteria, activating the immune response and regulating epithelial repair, thus guaranteeing the morphological and functional recovery of the injured tissue. We will discuss the role of a particular class of PRRs - the Formyl Peptide Receptors - in the homeostasis of GI mucosa. We here report the results of studies that strongly suggest the possibility that the activation of FPRs is crucial in the maintenance of homeostasis of the GI tract and provide indications of the potential clinical relevance of new treatment regimens involving FPR modulation for several GI disorders.

Formyl Peptide Receptors in Mice and Men: Similarities and Differences in Recognition of Conventional Ligands and Modulating Lipopeptides.

The pattern recognition formyl peptide receptors (FPRs) belong to the class of G-protein-coupled receptors (GPCRs), the largest group of cell surface receptors involved in a range of physiological processes and pathologies. The FPRs have regulatory function in the initiation as well as resolution of inflammatory reactions, making them highly interesting as targets for drug development. Recent research in the GPCR/FPR fields has uncovered novel receptor biology concepts, including biased signalling/functional selectivity, allosteric modulation, receptor reactivation and receptor cross-talk. When it comes to allosteric modulators, 'tailor-made' lipopeptides (pepducins and lipopeptoids) represent a novel concept of GPCR/FPR regulation. This MiniReview is focused on the basis for recognition of conventional ligands and immunomodulating lipopeptides, novel allosteric modulators for the FPRs, receptors that are highly expressed by both human and mouse neutrophils. The FPRs play key roles in host defence against microbial infections, tissue homeostasis and the initiation as well as resolution of inflammation but there are both similarities and differences in ligand recognition between mice and men. Thus, identification and functional characterization of activating and inhibiting ligands should provide insights into future design of FPR-based animal models of human diseases and development of therapeutics for treating inflammatory diseases.

Inhibition of platelet-activating factor biosynthesis by adenosine and histamine in human neutrophils: involvement of cPLA2alpha and reversal by lyso-PAF.

Leukotrienes (LT) and platelet-activating factor (PAF) are important lipid mediators of inflammation. We and others reported previously that autacoids such as adenosine, histamine, prostaglandin E2, and beta-adrenergic agents inhibit LT biosynthesis in activated human polymorphonuclear leukocytes (PMN). In this study, we demonstrate that CGS-21680 (a selective agonist of the adenosine A2A receptor) and histamine also potently inhibit PAF biosynthesis in agonist [formyl Met-Leu-Phe (fMLP)]- and thapsigargin-activated human PMN. The observed inhibitions of PAF biosynthesis were reversed effectively by exogenous 1-O-alkyl-lyso-sn-glyceryl-3-phosphocholine (lyso-PAF), suggesting that these effects of CGS-21680 and histamine implicate the blockade of cytosolic phospholipase A2alpha (cPLA2alpha) activity and lyso-PAF release and that the acetyl-coenzyme A/lyso-PAF acetyl transferase is not inhibited by the autacoids. Accordingly, the cPLA2alpha inhibitor pyrrophenone completely blocked PAF formation, and lyso-PAF similarly prevented this effect of pyrrophenone. The inhibitory effects of CGS-21680 and histamine on PAF biosynthesis were prevented by the protein kinase A inhibitor H-89, supporting roles for the Gs -coupled receptors A2A and H2, respectively, and cyclic adenosine monophosphate in the inhibitory mechanism. The fMLP-induced phosphorylations of p38 and extracellular signal-regulated kinase 1/2 were not altered significantly by the CGS-21680, indicating that inhibition of these kinases is not involved in the inhibitory effect of the adenosine A2A receptor ligand on LT and PAF biosynthesis. These data further emphasize the multiple and potent inhibitory effects of adenosine and histamine on leukocyte functions, in particular, on the biosynthesis of two classes of important lipid mediators and their putative regulatory roles in immune processes in health and diseases.

Antiflammin-2 activates the human formyl-peptide receptor like 1.

The anti-inflammatory actions of the nonapeptide antiflammin-2, identified by homology with uteroglobin and annexin-A1 sequences, have been described in some detail, yet its mechanisms of action remain elusive. Since recent data indicate an involvement of the formyl peptide receptor (FPR)-like 1 (or FPRL-1) in the effects of annexin-A1, we have tested here the effect of antiflammin-2 with respect to this receptor family. Using HEK-293 cells expressing either human FPR and FPRL-1, and an annexin-A1 peptide as tracer ([125I-Tyr]-Ac2-26), we found that antiflammin-2 competed for binding only at FPRL-1, and not FPR, with an approximate EC50 of 1 mM. In line with data produced for the full-length protein, genuine receptor activation by antiflammin-2 was confirmed by rapid phosphorylation of extracellular-regulated kinase 1 and 2. Finally, study of the neutrophil interaction with activated endothelium under flow demonstrated an inhibitory effect of antiflammin-2, thus providing functional support to a role for the antiflammin-2/FPRL-1 anti-inflammatory axis.

Anti-alopecia mechanisms of soymetide-4, an immunostimulating peptide derived from soy beta-conglycinin.

Previously, we found that orally administered soymetide-4 (MITL), an immunostimulating peptide derived from soybean beta-conglycinin alpha' subunit, suppressed alopecia induced by the anti-cancer drug etoposide in neonatal rats. Soymetide-4 has weak affinity for N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor. fMLP showed an anti-alopecia effect after intraperitoneal administration, though it was inactive after oral administration. Anti-alopecia effect of fMLP was blocked by pyrilamine or cimetidine, antagonists for histamine H1 or H2 receptor, respectively. However, the anti-alopecia effect of soymetide-4 was not inhibited by the histamine antagonists but by indomethacin, an inhibitor of cyclooxygenase (COX), or AH-23848B, an antagonist of the EP4 receptor for PGE2. Anti-alopecia effect of soymetide-4 was also blocked by pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-kappaB (NF-kappaB). These results suggest that PGE2, which is produced after activation of COX by soymetide-4, might suppress apoptosis of hair matrix cells and etoposide-induced alopecia by activating NF-kappaB.

Peptide length and folding state govern the capacity of staphylococcal ß-type phenol-soluble modulins to activate human formyl-peptide receptors 1 or 2.

Most staphylococci produce short α-type PSMs and about twice as long ß-type PSMs that are potent leukocyte attractants and toxins. PSMs are usually secreted with the N-terminal formyl group but are only weak agonists for the leukocyte FPR1. Instead, the FPR1-related FPR2 senses PSMs efficiently and is crucial for leukocyte recruitment in infection. Which structural features distinguish FPR1 from FPR2 ligands has remained elusive. To analyze which peptide properties may govern the capacities of ß-type PSMs to activate FPRs, full-length and truncated variants of such peptides from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis were synthesized. FPR2 activation was observed even for short N- or C-terminal ß-type PSM variants once they were longer than 18 aa, and this activity increased with length. In contrast, the shortest tested peptides were potent FPR1 agonists, and this property declined with increasing peptide length. Whereas full-length ß-type PSMs formed α-helices and exhibited no FPR1-specific activity, the truncated peptides had less-stable secondary structures, were weak agonists for FPR1, and required N-terminal formyl-methionine residues to be FPR2 agonists. Together, these data suggest that FPR1 and FPR2 have opposed ligand preferences. Short, flexible PSM structures may favor FPR1 but not FPR2 activation, whereas longer peptides with α-helical, amphipathic properties are strong FPR2 but only weak FPR1 agonists. These findings should help to unravel the ligand specificities of 2 critical human PRRs, and they may be important for new, anti-infective and anti-inflammatory strategies.

A truncated form of CKbeta8-1 is a potent agonist for human formyl peptide-receptor-like 1 receptor.

1. Human formyl peptide-receptor-like-1 (FPRL-1) is a promiscuous G protein-coupled receptor (GPCR), and belongs to a chemoattractant receptor family protein. This receptor has been reported to interact with various host-derived peptides and lipids involved in inflammatory responses. We described here, a novel role for FPRL-1 as a high-affinity beta-chemokine receptor for an N-terminally truncated form of the CKbeta8 (CCL23/MPIF-1) splice variant CKbeta8-1 (22-137 aa). 2. RT-PCR analysis of mRNA derived from human tissues and cells revealed a predominant expression of FPRL-1 in inflammatory cells, particularly in neutrophils. 3. Intracellular calcium mobilisation assay, used as screening tool, in recombinant Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293s) cells coexpressing FPRL-1 and Galpha(16), demonstrated FPRL-1 is a functional high-affinity receptor for CKbeta8-1 (46-137 aa, sCKbeta8-1), with pEC(50) values of 9.13 and 8.85, respectively. 4. The FPRL-1 activation in CHO-K1 cells is mediated by Galpha(i)/Galpha(o) proteins, as assessed by pertussis toxin sensitivity and inhibition of forskolin-induced cyclic AMP accumulation. 5. Binding experiments were performed with a radio-iodinated synthetic peptide, [(125-)I]-WKYMVm, a known potent FPRL-1 agonist. CHO-K1 cell membranes expressing FPRL-1 bound [(125-)I]-WKYMVm with a K(d) value of 9.34. Many known FPRL-1 agonists were tested and sCKbeta8-1 was the most effective nonsynthetic ligand in displacing the radiolabelled agonist, with a pIC(50) of 7.97. 6. The functional significance of sCKbeta8-1 interaction with FPRL-1 was further demonstrated by the activation of polymorphonuclear leukocytes (PMNs) calcium mobilisation and chemotaxis. These interactions were shown to be via FPRL-1 by specific blockade of PMNs activation in the presence of an FPRL-1 antibody.

Opposing regulation of interleukin-8 and NF-kappaB responses by lipoxin A4 and serum amyloid A via the common lipoxin A receptor.

Lipoxin A4 (LXA4) is a potent eicosanoid that inhibits IL-1beta-induced activation of human fibroblast like synoviocytes (FLS) via the ALX4 receptor. Serum amyloid A (SAA) is an acute phase reactant with cytokine-like properties. SAA has been shown to bind the same seven transmembrane G protein-coupled receptor ligated by LXA4. Here we compared the inflammatory responses of lipid (LXA4) and peptide (SAA) ligands in human FLS via the shared ALX, and characterized their downstream signaling. LXA4 induced stimulation of tissue inhibitors of metalloproteinase-2, whereas SAA induced interleukin-8 and matrix metalloproteinase-3 production. SAA up-regulated NF-kappaB and AP-1 DNA binding activity, while LXA4 markedly inhibited these responses after IL-1beta stimulation. A human IL-8 promoter luciferase construct was transfected into CHO cells stably expressing ALXR in order to determine the role of NF-kappaB and/or AP-1 in the regulation of IL-8 gene expression. The NF-kappaB pathway proved to be the preeminent for the biological responses elicited by both ligands. These findings suggest that two endogenous molecules, targeting a common receptor, could participate in the pathogenesis of inflammatory arthritis by differentially regulating inflammatory responses in tissues expressing the ALXR.