Formyl Methionyl Leucyl Phenylalanine Receptor Research Articles

Role of sphingosine kinase and sphingosine-1-phosphate in inflammatory arthritis

The importance of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) in inflammation has been extensively demonstrated. As an intracellular second messenger, S1P plays an important role in calcium signaling and mobilization, and cell proliferation and survival. Activation of various plasma membrane receptors, such as the formyl methionyl leucyl phenylalanine receptor, C5a receptor, and tumor necrosis factor α receptor, leads to a rapid increase in intracellular S1P level via SphK stimulation. SphK and S1P are implicated in various chronic autoimmune conditions such as rheumatoid arthritis, primary Sjögren's syndrome, and inflammatory bowel disease. Recent studies have demonstrated the important role of SphK and S1P in the development of arthritis by regulating the pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets.

Inappropriate neutrophil activation in venous disease.

Neutrophil oxygen radical production was studied in 18 limbs with class 2 or 3 venous disease and compared with that of nine normal limbs. Neutrophils were isolated from arm and leg venous samples. Free radical production was determined using chemiluminescence after stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) or the ester phorbol myristate acetate (PMA). The ratio of leg to arm luminescence was greater after FMLP stimulation in patients with venous disease (median 1.52 (95 per cent confidence interval (c.i.) 1.27-2.60)) than in controls (median 0.97 (95 per cent c.i. 0.70-1.12); P < 0.01). These changes were not observed with PMA (venous disease 1.16 (95 per cent c.i. 1.05-1.40); controls 0.95 (95 per cent c.i. 0.78-1.24)). There were fewer FMLP receptors on activated leg neutrophils (median 20.19 (95 per cent c.i. 3.58-51.42) fluorescence units) than arm neutrophils (median 36.03 (95 per cent c.i. 13.00-65.28) fluorescence units; P < 0.05), indicating an amplification of signal transduction. Intracellular calcium imaging demonstrated a larger release of calcium after stimulation of leg neutrophils (median 25.0 per cent (95 per cent c.i. 15.7-43.9 per cent)) compared with neutrophils from the arm (median 8.0 per cent (95 per cent c.i. 5.6-16.1 per cent); P = 0.04), demonstrating calcium-dependent activation. Neutrophils in patients with chronic venous disease inappropriately produce more oxygen free radical as a result of amplification of a calcium-dependent signal pathway.

Cigarette Smoking Increases Neutrophil Formyl Methionyl Leucyl Phenylalanine Receptor Numbers

The purpose of this study was to explore the relationship between cigarette smoking and COPD on the number of formyl methionyl leucyl phenylalanine (FMLP) receptors on peripheral neutrophils. Three groups of subjects were studied: subjects with COPD (n = 13), healthy smokers (n = 6), and healthy nonsmokers (n = 6). Fifty milliliters of venous blood were collected from each subject, and neutrophils were isolated. Neutrophil FMLP receptor numbers were determined by incubating with tritiated FMLP at six doubling concentrations from 1.4 to 45 pmol. Three of the subjects from group 1 (the COPD group) were current smokers, and we elected to analyze these subjects as a separate group. The analysis of variance looking at the three factors-FMLP, COPD and smoking-showed significant differences among levels of FMLP (p = 0.0001), as would be expected, and also overall smoking vs nonsmoking (p = 0001) and COPD vs non-COPD (p = 0.02). Within each level of FMLP, there was only one instance of a significant difference between COPD nonsmokers and normal nonsmokers, and no instance of a significant difference between COPD smokers and normal smokers. At five of the six concentrations of tritiated FMLP, smoking was a significant factor. This study suggests that the overriding influence on peripheral neutrophil FMLP receptor numbers is current smoking rather than the presence of COPD.

Mucosal subepithelial binding sites for the bacterial chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP)

Background—The bacterial chemotactic peptideN-formyl-methionine-leucine-phenylalanine (FMLP) is produced by enteric flora and is one of the factors implicated in contributing to inflammatory bowel disease. Expression of receptors for FMLP on human...

Co-operation of phosphatidylinositol transfer protein with phosphoinositide 3-kinase gamma in the formylmethionyl-leucylphenylalanine-dependent production of phosphatidylinositol 3,4,5-trisphosphate in human neutrophils.

Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3) play an essential role in the regulation of neutrophil functions by the chemoattractant formylmethionyl-leucylphenylalanine (FMLP). Here we show that permeabilization of human neutrophils leads to loss of cytosolic components, including PI3Kgamma, and causes the loss of FMLP-dependent production of PIP3. FMLP-sensitive synthesis of PIP3 could be restored by reconstitution of permeabilized neutrophils with recombinant PI3Kgamma. Admixture of recombinant phosphatidylinositol transfer protein (PITP) to the reconstitution cocktail produced a further increase of PIP3 synthesis, whereas pertussis toxin suppressed the FMLP-dependent production of PIP3. We conclude that FMLP-sensitive PIP3 formation in human neutrophils involves the FMLP receptor, heterotrimeric G-proteins of the Gi type, PI3Kgamma and PITP.

Effect of age on second messenger generation in neutrophils

Neutrophils from healthy elderly donors generate significantly less diacylglycerol (DAG) and inositol triphosphate (IP3) than neutrophils from young donors, following stimulation by the chemotactic peptide, formyl-methionyl-leucylphenylalanine (FMLP). The defect in signal transduction occurred at a point proximal to the generation of IP3 and DAG, since the reduction in FMLP-induced superoxide generation was corrected if the intervening signal transduction steps were bypassed, either by priming with a substimulatory dose (1.62 nmol/L) of phorbol myristate acetate (PMA), by ionophore elevation of cytosolic calcium, or by using a stimulatory dose of PMA (1.62 mumol/L). FMLP receptor number and affinity were unaffected by aging. On FMLP activation, neutrophils from old, as compared with young, volunteers showed significantly greater and more long-lasting decreases in the concentrations of phosphatidylinositol (PI), phosphatidylinositol 4- monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). This indicates a reduction with age in the metabolically active precursor pools responsible for the generation of IP3 and DAG. In contrast, aging had little effect on the production of phosphatidic acid (PA), which has recently been suggested to serve as a major activator of the NADPH oxidase. This may explain why the decrease in IP3 and DAG production was not accompanied by a comparable decrement in superoxide generation, which was only 17% lower in the old than in young donor neutrophils. Thus, aging is associated with reductions in the concentration of critically important phosphoinositides, resulting in diminution in the ability to produce key second messengers. Although the aged neutrophil is largely able to compensate for the decrements in signal transduction, its reserve capacity is compromised, making it particularly vulnerable to external insults that also impair function.

Effect of Age on Second Messenger Generation in Neutrophils

AbstractNeutrophils from healthy elderly donors generate significantly less diacylglycerol (DAG) and inositol triphosphate (IP3) than neutrophils from young donors, following stimulation by the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). The defect in signal transduction occurred at a point proximal to the generation of IP3 and DAG, since the reduction in FMLP-induced superoxide generation was corrected if the intervening signal transduction steps were bypassed, either by priming with a substimulatory dose (1.62 nmol/L) of phorbol myristate acetate (PMA), by ionophore elevation of cytosolic calcium, or by using a stimulatory dose of PMA (1.62 pmol/L). FMLP receptor number and affinity were unaffected by aging. On FMLP activation, neutrophils from old, as compared with young, volunteers showed significantly greater and more long-lasting decreases in the concentrations of phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). This indicates a reduction with age in the metabolically active precursor pools responsible for the generation of IP3 and DAG. In contrast, aging had little effect on the production of phosphatidic acid (PA), which has recently been suggested to serve as a major activator of the NADPH oxidase. This may explain why the decrease in IP3 and DAG production was not accompanied by a comparable decrement in superoxide generation, which was only 17% lower in the old than in young donor neutrophils. Thus, aging is associated with reductions in the concentration of critically important phosphoinositides, resulting in diminution in the ability to produce key second messengers. Although the aged neutrophil is largely able to compensate for the decrements in signal transduction, its reserve capacity is compromised, making it particularly vulnerable to external insults that also impair function.

Subcellular distribution and characterization of GTP-binding proteins in human neutrophils

The subcellular distribution of GTP binding proteins in human neutrophils and their functional coupling to the N- formylmethionylleucylphenylalanine (FMLP) receptor was characterized to provide insight into mechanisms of cellular activation. Human neutrophils were nitrogen cavitated and fractionated on discontinuous Percoll gradients. Four subcellular fractions were obtained: cytosol, light membranes enriched for plasma membranes, specific granules and azurophilic granules. ADP-ribosylation catalyzed by pertussis toxin (PT) revealed a major substrate of 40 kDa only in plasma membrane and cytosol, and antiserum specific for Giα confirmed the presence of neutrophil Giα in plasma membrane and cytosol and its absence from specific granules. The cytosolic PT substrate was shown to be mostly in monomeric form by molecular sieve chromatography. The rate of the ribosyltransferase reaction was several-fold lower in cytosol compared to plasma membranes, and the extent of ADP-ribosylation was greatly augmented by supplementation with βγ subunits in cytosol. ADP-ribosylation catalyzed by cholera toxin (CT) revealed substrates of 52, 43 and 40 kDa in plasma membrane alone. FMLP receptors in plasma membrane were shown to be coupled to the 40 kDa substrate for CT by ligand-modulation of ADP-ribosylation, while FMLP added to specific granules did not induce ribosylation of this substrate even though FMLP receptors were found in high density in this compartment. Both 24 and 26 kDa [ 32P]GTP binding proteins were found to codistribute with FMLP receptors in specific granules and plasma membranes. Functional evidence for the coupling of GTP binding proteins to the FMLP receptor in specific granules was obtained by modulating [ 3H]FMLP binding with GTPγS, and by accelerating [ 35S]GTPγS binding with FMLP.

Human neutrophil chemiluminescence and f-Meth-Leu-Phe receptor exposure in bacterial infections.

Polymorphonuclear leukocytes were isolated from 12 patients with acute bacterial infections and the ability of the chemoattractant formylmethionyl-leucyl-phenylalanine (FMLP) to bind and induce a metabolic response in these cells was investigated. Cells isolated from the patients showed a significantly increased metabolic response in a luminol enhanced chemiluminescence assay compared to cells, isolated and analyzed in parallel, from healthy controls i.e. the patient cells were primed. The primed state was, as calculated by Scatchard analysis, accompanied by a significantly increased number of FMLP receptors exposed on the cell surface while the receptor binding affinity remained unchanged. There was, however, no correlation between the degree of priming and the degree of receptor upregulation. Furthermore, it was found that stimulation also with phorbol myristate acetate (PMA), a substance lacking specific cell surface receptors on the PMNL, gave rise to an increased metabolic response in the primed cells. These results indicate that the priming activity induced by a bacterial infection can only partly be explained by receptor modulation and that other mechanisms must also be considered. This study was approved by the Ethics Committee of the Medical Faculty, University of Linköping.

Chemotactic peptide activation of human neutrophils and HL-60 cells. Pertussis toxin reveals correlation between inositol trisphosphate generation, calcium ion transients, and cellular activation.

The mechanism of neutrophil activation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) has been studied by pretreatment of human neutrophils with pertussis toxin. Upon stimulation with FMLP, the cytosolic-free calcium concentration, [Ca2+]i, is increased both by stimulation of calcium influx and mobilization of cellular calcium. We have measured [Ca2+]i as well as the generation of the phospholipid breakdown product inositol trisphosphate (IP3), which is thought to mediate Ca2+ mobilization. As the phosphoinositide pool in human neutrophils is difficult to prelabel with [3H]myoinositol, experiments were also carried out in the cultured human promyelocytic leukemia cell line HL-60 after differentiation with dimethylsulfoxide. Pertussis toxin pretreatment of both cell types inhibited FMLP stimulated membrane depolarization, exocytosis, and superoxide production in a dose-dependent manner. This toxin effect was selective for the receptor agonist, since stimulation of these parameters by two substances bypassing the transduction mechanism, the calcium ionophore ionomycin and the phorbolester phorbol myristate acetate, were unaffected. Rises in [Ca2+]i, as well as generation of IP3 in response to FMLP, were inhibited in parallel; for the inhibition of functional responses, slightly lower toxin concentrations were required. The attentuation of the [Ca2+]i rise was more marked in the absence of extracellular calcium, i.e., when the rise is due only to calcium mobilization. The results provide evidence that phospholipase C stimulation by FMLP resulting in IP3 generation is involved in the signal transduction mechanism. Coupling of FMLP receptor occupancy to phospholipase C activation is sensitive to pertussis toxin, suggesting the involvement of a GTP binding protein (N protein), which has been shown to be a pertussis toxin substrate. The parallel changes in [Ca2+]i and IP3 further support the hypothesis that IP3 is the calcium-mobilizing mediator in FMLP-activated cells.

Decreased affinity for a chemotactic factor in stored granulocyte concentrates.

Previous studies in this laboratory demonstrated decreased migration of neutrophils after storage for 24 hours at room temperature. The current work was undertaken to identify a possible mechanism for decreased migration after storage. Initial studies ruled out the possibility that chemotaxis was decreased due to impaired ability to sense a chemotactic factor gradient since chemokinesis was decreased in addition to chemotaxis. Dose-response curves to the synthetic chemotactic agent Formyl-Methionyl-Leucyl-Phenylalanine (FMLP) showed decreased response to submaximal chemokinetic stimuli in stored neutrophils. This suggested the possibility of altered FMLP receptor binding in stored neutrophils. Neutrophil FMLP receptors were measured in 11 fresh and stored granulocyte concentrates. Although there was a small increase in total FMLP receptors per neutrophil after storage, the affinity of FMLP receptors in fresh neutrophils was significantly greater than that in neutrophils stored 24 hours at room temperature. Thus, decreased migration toward submaximal chemotactic stimuli in stored neutrophils may be due to altered membrane FMLP binding. However, decreased migration of stored neutrophils to maximal stimuli cannot be explained by altered FMLP binding affinity.