Neutrophil Superoxide Generation Research Articles

Inflammation Modulatory Phorbol Esters from the Seeds of Aquilaria malaccensis.

The tree Aquilaria malaccensis is a valuable source of agarwood, which is used in herbal medicinal preparations. Phytochemical research on A.malaccensis seeds has led to the isolation of four new phorbol esters (1-4), two known phorbol esters (5, isolated from Nature for the first time, and 6), and two known glycerides (7 and 8). The structures of these isolates were elucidated by means of spectroscopic data interpretation. The inflammation-modulatory activities of the isolates on elastase release and superoxide anion generation in human neutrophils were evaluated. Interestingly, phorbol esters 1, 5, and 6 showed potent inhibitory activity on elastase release in human neutrophils, with IC50 values of 2.7, 0.8, and 2.1 μM, respectively. All isolated phorbol esters exerted enhancing activity on superoxide anion generation. The results indicated that phorbol esters may play a bilateral modulatory role in the processes of inflammation. In addition, the compounds were evaluated for their cytotoxic properties against HepG2 (hepatoma), MDA-MB-231 (breast), and A549 (lung) cancer cells, but all compounds were inactive for all cell lines used (IC50 > 10 μM).

Strain-Specific Impact of Fusobacterium nucleatum on Neutrophil Function.

Neutrophil function is critical for initiation and progression of infecto-inflammatory diseases. Key quorum-sensing plaque bacteria, such as Fusobacterium nucleatum, act as bridging species between early and late colonizer pathogens, such as Porphyromonas gingivalis, as the biofilm ages and periodontal inflammation increases. This study is designed to determine impact of different F. nucleatum strains on neutrophil function. Cells of human promyelocytic leukemia cell line-60 were differentiated into neutrophil-like cells and cultured with F. nucleatum strains of subspecies (ssp.) nucleatum ATCC 25586, ssp. polymorphum ATCC 10953, and ssp. vincentii ATCC 49256. Neutrophil phagocytosis of F. nucleatum strains and neutrophil apoptosis were analyzed by flow cytometry. Superoxide generation was measured by cytochrome C reduction in the presence and absence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 μM) stimulation. Proinflammatory cytokine release was determined after 2, 6, and 24 hours of culture in the presence/absence of different F. nucleatum strains. Expression of Toll-like receptor (TLR)2, TLR4, and nuclear factor (NF)-kappa B mRNA levels were analyzed using real-time quantitative polymerase chain reaction. Each experiment was repeated at least three times in triplicate. Data were analyzed using analysis of variance followed by post hoc Bonferroni correction. All strains of F. nucleatum significantly increased phagocytic capacity of neutrophils. Neutrophil phagocytosis of F. nucleatum ssp. polymorphum was significantly greater than that of F. nucleatum ssp. vincentii and ssp. nucleatum (P <0.001). F. nucleatum ssp. nucleatum and ssp. polymorphum significantly blocked fMLP-induced superoxide generation (P <0.001). Although F. nucleatum vincentii also reduced superoxide generation (25%), the impact was not as strong as that of ssp. nucleatum (83%) and ssp. polymorphum (100%). All F. nucleatum strains stimulated significant increase in neutrophil apoptosis compared with control (P <0.001) and significantly increased expression of NF-κB mRNA in neutrophils (P <0.05). Levels of interleukin-8 and tumor necrosis factor-α produced by neutrophils were significantly increased in all F. nucleatum groups compared with control (P <0.001). These findings suggest that different strains of F. nucleatum impact neutrophil function in different ways. Two of three subspecies blocked neutrophil superoxide generation in response to a secondary stimulus, preventing oxidative killing by neutrophils. The direct role of bridging species in pathogenesis of periodontitis may be greater than previously suspected in which they create a favorable environment for pathogenic transition of the dental ecosystem.

Dipeptide HCH6-1 inhibits neutrophil activation and protects against acute lung injury by blocking FPR1

Formyl peptide receptor 1 (FPR1) is an emerging therapeutic target for the discovery of drugs to treat neutrophilic inflammatory diseases. However, development of FPR1 antagonists for clinical use is still inadequate. The purpose of this study was to identify a synthetic dipeptide N-(N-benzoyl-L-tryptophanyl)-D-phenylanlanine methyl ester (HCH6-1) as a FPR1 inhibitor and to investigate its protective effects against acute lung injury (ALI). HCH6-1 inhibited superoxide anion generation, elastase release, and chemotaxis in human neutrophils specifically activated by formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF), an FPR1 agonist. HCH6-1 produced right shifts in the concentration-response curves of fMLF, suggesting that HCH6-1 was a competitive antagonist of FPR1. Indeed, HCH6-1 bound to FPR1 in human neutrophils and neutrophil-like THP-1 as well as hFPR1-transfected HEK293 cells. Also, the FPR1 downstream signaling pathways were competitively inhibited by HCH6-1. Furthermore, HCH6-1 prevented pulmonary neutrophil infiltration and edema along with alveolar damage in LPS-induced ALI in mice. Our findings suggest that HCH6-1, a FPR1 antagonist, may have potential as a new therapeutic agent for treating FPR1-involved inflammatory lung diseases.

The Role of Acute Intermittent Hypoxia in Neutrophil-Generated Superoxide, Sympathovagal Balance, and Vascular Function in Healthy Subjects.

Introduction: Recurrent hypoxia (HPX), a hallmark of the obstructive sleep apnea (OSA), impairs autonomic balance, and increases arterial blood pressure (BP). Oxidative stress is one of the mechanisms involved in these alterations. The cumulative effect of acute intermittent HPX and the chronicity may determine whether the response crosses the threshold from having protective value to pathology. However, the impact of acute intermittent HPX–reoxygenation on markers of oxidative stress in healthy individuals remains to be fully understood.Objective: To analyze the effects of the acute intermittent HPX on the generation of neutrophil-derived superoxide, sympathovagal balance, and vascular function in healthy subjects.Methods: We applied six cycles of intermittent HPX (10% O2 and 90% N2) for 5 min followed by 2 min of room-air in 15 healthy volunteers (34 ± 2 years; 22.3 ± 0.46 kg/m2), without OSA (polysomnography), during wakefulness. During the experimental protocol, we recorded O2 saturation, end-tidal CO2, heart rate (HR), systolic, and diastolic BP, cardiac output (CO) and peripheral resistance (PR). Cardiac sympathovagal balance was determined by HR variability analysis (low frequency and high frequency bands, LF/HF). Superoxide generation in polymorphonuclear neutrophil cells were established using relative luminescence units (PMNs RLU) at baseline (pre-HPX) and immediately after hypoxia induction (post-HPX6).Results: The studied subjects had normal levels of BP, plasma glucose, lipid profile, and inflammatory marker (C-reactive protein). Acute intermittent HPX increased HR, systolic BP, CO, and decreased PR. Additionally, acute intermittent HPX increased PMNs RLU, measured post-HPX6 (470 ± 50 vs. 741 ± 135, P < 0.05). We found a similar increase in LF/HF post-HPX6 (0.91 ± 0.11 vs. 2.85 ± 0.40, P < 0.05). PR was diminished from pre-HPX to post-HPX6 (1.0 ± 0.03 vs. 0.85 ± 0.06, P < 0.05). Further analysis showed significant association between O2 saturation and PMNs RLU (R = −0.62, P = 0.02), and with LF/HF (R = −0.79, P = 0.02) post-HPX6. In addition, an association was found between PMNs RLU and PR post-HPX6 (R = 0.58, P = 0.04).Conclusion: Acute exposure to intermittent HPX not only increased superoxide generation in neutrophils, but also impaired cardiac sympathovagal balance in healthy subjects. These data reinforce the role of intermittent HPX in superoxide generation on neutrophils, which may lead to an impairment in peripheral vascular resistance.

Gut Microbiota Conversion of Dietary Ellagic Acid into Bioactive Phytoceutical Urolithin A Inhibits Heme Peroxidases.

Numerous studies signify that diets rich in phytochemicals offer many beneficial functions specifically during pathologic conditions, yet their effects are often not uniform due to inter-individual variation. The host indigenous gut microbiota and their modifications of dietary phytochemicals have emerged as factors that greatly influence the efficacy of phytoceutical-based intervention. Here, we investigated the biological activities of one such active microbial metabolite, Urolithin A (UA or 3,8-dihydroxybenzo[c]chromen-6-one), which is derived from the ellagic acid (EA). Our study demonstrates that UA potently inhibits heme peroxidases i.e. myeloperoxidase (MPO) and lactoperoxidase (LPO) when compared to the parent compound EA. In addition, chrome azurol S (CAS) assay suggests that EA, but not UA, is capable of binding to Fe3+, due to its catechol-like structure, although its modest heme peroxidase inhibitory activity is abrogated upon Fe3+-binding. Interestingly, UA-mediated MPO and LPO inhibition can be prevented by innate immune protein human NGAL or its murine ortholog lipocalin 2 (Lcn2), implying the complex nature of host innate immunity-microbiota interactions. Spectral analysis indicates that UA inhibits heme peroxidase-catalyzed reaction by reverting the peroxidase back to its inactive native state. In support of these in vitro results, UA significantly reduced phorbol myristate acetate (PMA)-induced superoxide generation in neutrophils, however, EA failed to block the superoxide generation. Treatment with UA significantly reduced PMA-induced mouse ear edema and MPO activity compared to EA treated mice. Collectively, our results demonstrate that microbiota-mediated conversion of EA to UA is advantageous to both host and microbiota i.e. UA-mediated inhibition of pro-oxidant enzymes reduce tissue inflammation, mitigate non-specific killing of gut bacteria, and abrogate iron-binding property of EA, thus providing a competitive edge to the microbiota in acquiring limiting nutrient iron and thrive in the gut.

Estimation of the Bioactivity of Seven Aromatic Compounds Isolated from Niihime Fruit by Simultaneously Monitoring Superoxide Generation and Intracellular Calcium Ion Levels in Neutrophils

Estimation of the Bioactivity of Seven Aromatic Compounds Isolated from Niihime Fruit by Simultaneously Monitoring Superoxide Generation and Intracellular Calcium Ion Levels in Neutrophils

Inhibition of neutrophil superoxide generation by shikonin is associated with suppression of cellular Ca(2+) fluxes.

Shikonin, an anti-inflammatory compound of “Shikon”, inhibits the neutrophil superoxide (O2•−) generation by NADPH oxidase 2 (Nox2); however, the mechanisms of how shikonin affects Nox2 activity remained unclear. We aimed to elucidate the relationship between the inhibition of Nox2 activity and influences on intracellular Ca2+ concentration ([Ca2+]i) by shikonin. For this purpose, we used a simultaneous monitoring system for detecting changes in [Ca2+]i (by fluorescence) and O2•− generation (by chemiluminescence) and evaluated the effects of shikonin on neutrophil-like HL-60 cells stimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Since fMLP activates Nox2 by elevation in [Ca2+]i via fluxes such as inositol 1,4,5-trisphosphate-induced Ca2+ release (IICR) and store-operated Ca2+ entry (SOCE), we also evaluated the effects of shikonin on IICR and SOCE. Shikonin dose-dependently inhibited the fMLP-induced elevation in [Ca2+]i and O2•− generation (IC50 values of 1.45 and 1.12 µM, respectively) in a synchronized manner. Analyses of specific Ca2+ fluxes showed that shikonin inhibits IICR and IICR-linked O2•− generation (IC50 values: 0.28 and 0.31 µM for [Ca2+]i and O2•−, respectively), as well as SOCE and SOCE-linked O2•− generation (IC50 values: 0.39 and 0.25 µM for [Ca2+]i and O2•−, respectively). These results suggested that shikonin inhibits the O2•− generation by Nox2 in fMLP-stimulated neutrophils by targeting Ca2+ fluxes such as IICR and SOCE.

Triterpenoids and an alkamide from Ganoderma tsugae

Ganoderma tsugae is a medicinal mushroom. In a continual study on the bioactive constituents of this fungus, a new lanostanoid, 3β-acetoxy-16α-hydroxy-24ξ-methyl-5α-lanosta-8,25-dien-21-oic acid, named tsugaric acid F (1) and a novel palmitamide, N-(3′α,4′β-dihydroxy-2′β-(hydroxymethyl)-1′β-(cyclobutyl)palmitamide (2) were isolated and characterized from the fruit bodies of G. tsugae, and three novel seco-lanostanoids, 3,4-seco-8α,9α-epoxy-5α-lanosta-21-oic acid 3,4 lactone (5), 3,4-seco-5β-lanosta-7,9(11),4(29)-trien-3,21-dioic acid-3-methyl ester (6), 3,4-seco-5β-lanosta-7,9(11),4(29)-trien-3,21-dioic acid (7), and a known compound, 3-oxo-5α-lanosta-8-en-21-oic acid (4) were prepared from 3. The structures of new compounds, 1, 2, 5–7 were determined by spectroscopic methods. Compounds 1 and 4 showed inhibitory effects on xanthine oxidase (XO) with an IC50 values of 313.3±80.0 and 43.9±29.9μM, respectively when 7 exhibited potent inhibitory effect on superoxide anion generation in rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB) with an IC50 values of 1.3±0.2μM. Compounds 4–7 showed weak cytotoxic activities against PC3 cells. These results indicated that 4 and 7 may be used as cancer chemopreventive agents.

Glaucumolides A and B, Biscembranoids with New Structural Type from a Cultured Soft Coral Sarcophyton glaucum.

Glaucumolides A (1) and B (2), novel biscembranes composed of an unprecedented α,β-unsaturated ε-lactone, along with the known metabolites ximaolide A (3) and isosarcophytonolide D (4), were isolated from the cultured soft coral Sarcophyton glaucum. The structures of the new metabolites were determined by extensive spectroscopic analyses. Compounds 1 and 2 were shown to exhibit cytotoxicity against a limited panel of cancer cell lines. In anti-inflammation assay, compounds 1 and 2 displayed strong inhibition of superoxide anion generation and elastase release in human neutrophils stimulated by fMLP/CB. Furthermore, both 1 and 2 were shown to significantly inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase protein, and compounds 1−3 were found to effectively reduce the expression of cyclooxygenase-2 protein, in lipopolysaccharide-stimulated RAW264.7 macrophage cells.

Ruscogenin suppresses mouse neutrophil activation: Involvement of protein kinase A pathway

Ruscogenin, a natural steroidal sapogenin, presents in both food and medicinal plants. It has been found to exert significant anti-inflammatory activities. Considering that activation of neutrophil is a key feature of inflammatory diseases, this study was performed to investigate the inhibitory effect of ruscogenin and its underlying mechanisms responsible for neutrophil activation. Ruscogenin displayed potent antioxidative effects against Formyl-Met-Leu-Phe (FMLP)-induced extra- and intracellular superoxide generation in mouse bone marrow neutrophils, with IC50 values of 1.07±0.32μM and 1.77±0.46μM, respectively. Phorbol myristate acetate (PMA)-elicited extra- and intracellular superoxide generation were also suppressed by ruscogenin, with IC50 values of 1.56±0.46μM and 1.29±0.49μM, respectively. However, ruscogenin showed weak inhibition in NaF-induced response. Inhibition of superoxide generation was mediated neither by a superoxide-scavenging ability nor by a cytotoxic effect. Furthermore, ruscogenin inhibited the membrane translocation of p47phox and p67phox. It reduced FMLP-induced phosphorylation of cytosolic phospholipase A2 (cPLA2) and p21-activated kinase (PAK). The cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were increased by ruscogenin. Moreover, ruscogenin inhibited phosphorylation of protein kinase B (Akt), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK). In addition, the inhibitory effects of ruscogenin on superoxide production and the phosphorylation of Akt, p38MAPK, and ERK1/2 were reversed by PKA inhibitor (H89), suggesting a PKA-dependent mechanism. In summary, our data suggest that ruscogenin inhibits activation of neutrophil through cPLA2, PAK, Akt, MAPKs, cAMP, and PKA signaling pathways. Increased PKA activity is associated with suppression of the phosphorylation of Akt, p38MAPK, and ERK1/2 pathways.

Suppression of neutrophil superoxide generation by BNP is attenuated in acute heart failure: a case for 'BNP resistance'.

The release of the B-type natriuretic peptide (BNP) is increased in heart failure (HF), a condition associated with oxidative stress. BNP is known to exert anti-inflammatory effects including suppression of neutrophil superoxide (O2(-)) release. However, BNP-based restoration of homeostasis in HF is inadequate, and the equivocal clinical benefit of a recombinant BNP, nesiritide, raises the possibility of attenuated response to BNP. We therefore tested the hypothesis that BNP-induced suppression of neutrophil O2(-) generation is impaired in patients with acute HF. We have recently characterized suppression of neutrophil O2(-) generation (PMA- or fMLP-stimulated neutrophil burst) by BNP as a measure of its physiological activity. In the present study, BNP response was compared in neutrophils of healthy subjects (n = 29) and HF patients (n = 45). Effects of BNP on fMLP-induced phosphorylation of the NAD(P)H oxidase subunit p47phox were also evaluated. In acute HF patients, the suppressing effect of BNP (1 µmol/L) on O2(-) generation was attenuated relative to that in healthy subjects (P < 0.05 for both PMA and fMLP). Analogously, BNP inhibited p47phox phosphorylation in healthy subjects but not in HF patients (P < 0.05). However, O2(-)-suppressing effects of the cell-permeable cGMP analogue (8-pCPT-cGMP) were preserved in acute HF. Conventional HF treatment for 5 weeks partially restored neutrophil BNP responsiveness (n = 25, P < 0.05), despite no significant decrease in plasma NT-proBNP levels. BNP inhibits neutrophil O2(-) generation by suppressing NAD(P)H oxidase assembly. This effect is impaired in acute HF patients, with partial recovery during treatment.

Ursolic acid inhibits superoxide production in activated neutrophils and attenuates trauma-hemorrhage shock-induced organ injury in rats.

Neutrophil activation is associated with the development of organ injury after trauma–hemorrhagic shock. In the present study, ursolic acid inhibited the superoxide anion generation and elastase release in human neutrophils. Administration of ursolic acid attenuated trauma–hemorrhagic shock-induced hepatic and lung injuries in rats. In addition, administration of ursolic acid attenuated the hepatic malondialdehyde levels and reduced the plasma aspartate aminotransferase and alanine aminotransferase levels after trauma–hemorrhagic shock. In conclusion, ursolic acid, a bioactive natural compound, inhibits superoxide anion generation and elastase release in human neutrophils and ameliorates trauma–hemorrhagic shock-induced organ injury in rats.

Diosgenin inhibits superoxide generation in FMLP-activated mouse neutrophils via multiple pathways

Diosgenin possesses anti-inflammatory and anticancer properties. Activated neutrophils produce high concentrations of the superoxide anion which is involved in the pathophysiology of inflammation-related diseases and cancer. In the present study, the inhibitory effect and possible mechanisms of diosgenin on superoxide generation were investigated in mouse bone marrow neutrophils. Diosgenin potently and concentration-dependently inhibited the extracellular and intracellular superoxide anion generation in Formyl-Met-Leu-Phe (FMLP)- activated neutrophils, with IC50 values of 0.50 ± 0.08 μM and 0.66 ± 0.13 μM, respectively. Such inhibition was not mediated by scavenging the superoxide anion or by a cytotoxic effect. Diosgenin inhibited the phosphorylation of p47phox and membrane translocation of p47phox and p67phox, and thus blocking the assembly of nicotinamide adenine dinucleotide phosphate oxidase. Moreover, cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were also effectively increased by diosgenin. It attenuated FMLP-induced increase of phosphorylation of cytosolic phospholipase A (cPLA2), p21-activated kinase (PAK), Akt, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK). Our data indicate that diosgenin exhibits inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA2, PAK, Akt and MAPKs signaling pathways. The results may explain the clinical implications of diosgenin in the treatment of inflammation-related disorders.

Antioxidant effect of the active metabolites of tibolone

Certain steroidal compounds have an antioxidant effect in humans. Our aim was to test whether the synthetic steroid tibolone and its metabolites are also able to display such a property. For this, granulocytes from healthy men and women were incubated for two hours with different concentrations (10−7, 10−8, 10−9 M) of either estradiol, tibolone, 3α-hydroxytibolone, 3β-hydroxytibolone, Δ4-tibolone, 3α-sulfated-tibolone, 3α-17β-disulfated-tibolone, 3β-sulfated-tibolone or 3β-17β-disulfated-tibolone. Superoxide anion generation of neutrophils was measured by photometry. Results of different steroids were given as percentages of their controls. A more simple superoxide generating system, the xanthine–xanthine oxidase reaction was also tested. We found that granulocyte superoxide production did not differ from the control using 10−9 M of steroids. Using 10−8 M concentration: estradiol (80.9 ± 2.5%); 3β-sulfated-tibolone (83.3 ± 4.7%); 3β-17β-disulfated-tibolone (81.0 ± 4.2%) caused a significant decrease in superoxide production, compared to the control. In addition at 10−7 M, 3β-hydroxytibolone and 3α-sulfated-tibolone also showed antioxidant effects. In the xanthine–xanthine oxidase system estradiol (67.4 ± 1.0%), 3α-sulfated-tibolone (85.8 ± 5.3%), 3α-17β-disulfated-tibolone (71.9 ± 2.5%), 3β-sulfated-tibolone (73.9 ± 5.0%), and 3β-17β-disulfated-tibolone (65.8 ± 3.4%) caused a significant decrease in superoxide production. Conclusively, although tibolone itself did not show significant antioxidant capacity, most of its active metabolites have antioxidant effects.

The effects of Bruton tyrosine kinase inhibition on chemotaxis and superoxide generation in human neutrophils.

The role of the Bruton tyrosine kinase (Btk) protein in neutrophil function has been evaluated using neutrophils from healthy volunteers after incubation with a Btk inhibitor, leflunomide metabolite analog (LFM-A13), suggesting an important role for Btk in neutrophil function. We sought to determine the role of Btk protein on neutrophil superoxide generation and chemotaxis stimulated by N-formyl-methionine-leucine-phenylalanine (fMLP). Chemotaxis was assayed on agarose gel and superoxide generation by cytochrome C reduction. The affects of LFM-A13 on chemotaxis and superoxide generation in unstimulated and fMLP stimulated neutrophils were studied in Btk deficient neutrophils from XLA patients compared with matched controls analyzed simultaneously. Chemotaxis and stimulated superoxide production were similar in the normal and Btk deficient neutrophils and were similarly inhibited by LFM-A13. In one patient, LFMA13 had no effect on superoxide generation in Btk deficient neutrophils up to a concentration of 25microM, while inhibited superoxide production by control neutrophils. Our results suggest that Btk does not have a specific role in neutrophil fMLP-stimulated superoxide generation and chemotaxis since these activities were similarly inhibited by LFM-A13 in Btk deficient and normal neutrophils. The lack of superoxide generation following Btk inhibition by LFM-A13 in Btk deficient neutrophils from one patient may suggest some heterogeneity in the role of Btk in fMLP induced neutrophil superoxide generation.

P.3.006 Genetic risk factors for cytokine-induced depression

Ageing is accompanied by reduced functioning of the immune system, termed immunesenescence which is associated with increased risk of infection and mortality. However the immune system does not operate in isolation and can be modified by many environmental factors, including stress. In this study we determined whether physical stress (hip fracture) and psychological distress (depressive symptoms) had additive effects upon the aged immune system, specifically on monocyte numbers and function. We assessed immune function in 101 hip fracture patients (81 female) 6 weeks and 6 months after injury and 43 healthy age matched controls (28 females). Thirty-eight of the hip fracture group were found to be depressed at the 6 week sampling. No differences in peripheral monocyte count, distribution of monocyte subsets or TNFα secretion were observed between hip fracture patients and healthy controls. However we observed significantly reduced superoxide production in response to Escherichia coli in the monocytes of hip fracture patients who developed depressive symptoms compared with non-depressed hip fracture patients (p = 0.002) or healthy controls (p = 0.008) 6 weeks after the fracture which remained decreased 6 months following injury. In previous studies we have shown an effect of depression on neutrophil superoxide generation in hip fracture patients, suggesting a particular susceptibility of this aspect of immune cell function to psychological stress.

Depressive symptoms in hip fracture patients are associated with reduced monocyte superoxide production

Ageing is accompanied by reduced functioning of the immune system, termed immunesenescence which is associated with increased risk of infection and mortality. However the immune system does not operate in isolation and can be modified by many environmental factors, including stress. In this study we determined whether physical stress (hip fracture) and psychological distress (depressive symptoms) had additive effects upon the aged immune system, specifically on monocyte numbers and function. We assessed immune function in 101 hip fracture patients (81 female) 6weeks and 6months after injury and 43 healthy age matched controls (28 females). Thirty-eight of the hip fracture group were found to be depressed at the 6week sampling. No differences in peripheral monocyte count, distribution of monocyte subsets or TNFα secretion were observed between hip fracture patients and healthy controls. However we observed significantly reduced superoxide production in response to Escherichia coli in the monocytes of hip fracture patients who developed depressive symptoms compared with non-depressed hip fracture patients (p=0.002) or healthy controls (p=0.008) 6weeks after the fracture which remained decreased 6months following injury. In previous studies we have shown an effect of depression on neutrophil superoxide generation in hip fracture patients, suggesting a particular susceptibility of this aspect of immune cell function to psychological stress.

Effect of granulocyte‐macrophage colony‐stimulating factor on neutrophil function in idiopathic bronchiectasis

Neutrophils are consistently found in inflamed and infected airways in idiopathic bronchiectasis, but relatively little is known about the function of blood neutrophils in this condition. We hypothesized that peripheral blood neutrophil (PBN) phagocytosis and superoxide generation are impaired in bronchiectasis, and that granulocyte-macrophage colony-stimulating factor (GM-CSF) is capable of improving neutrophil function. Neutrophils were isolated from the peripheral blood of patients with idiopathic bronchiectasis who were free of exacerbation, and from healthy controls of similar age (n = 21 in both groups). Ingestion of serum-opsonized zymosan by neutrophils was used to quantify phagocytic capacity. Superoxide generation in neutrophils was measured in response to addition of platelet activating factor and formyl-methionyl-leucyl-phenylalanine. Experiments were performed in the presence or absence of GM-CSF. No differences were observed in either phagocytic capacity (P = 0.99) or superoxide generation (P = 0.81) when comparing patients and controls. However, a significant increase in phagocytic capacity above baseline levels in both patients (P < 0.005) and controls (P < 0.005) was induced by GM-CSF. Similarly, the superoxide generation in patients (P < 0.005) and controls (P = 0.001) was significantly increased by GM-CSF. PBN function was preserved in idiopathic bronchiectasis. Enhancement of neutrophil phagocytosis and superoxide generation by GM-CSF requires further study.

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.

Bioactive Secondary Metabolites of a Marine Bacillus sp. Inhibit Superoxide Generation and Elastase Release in Human Neutrophils by Blocking Formyl Peptide Receptor 1

It is well known that overwhelming neutrophil activation is closely related to acute and chronic inflammatory injuries. Formyl peptide receptor 1 (FPR1) plays an important role in activation of neutrophils and may represent a potent therapeutic target in inflammatory diseases. In the present study, we demonstrated that IA-LBI07-1 (IA), an extract of bioactive secondary metabolites from a marine Bacillus sp., has anti-inflammatory effects in human neutrophils. IA significantly inhibited superoxide generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated neutrophils, but failed to suppress the cell responses activated by non-FPR1 agonists. IA did not alter superoxide production and elastase activity in cell-free systems. IA also attenuated the downstream signaling from FPR1, such as the Ca2+, MAP kinases and AKT pathways. In addition, IA inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analogue of FMLP, to FPR1 in human neutrophils and FPR1-transfected HEK293 cells. Taken together, these results show that the anti-inflammatory effects of IA in human neutrophils are through the inhibition of FPR1. Also, our data suggest that IA may have therapeutic potential to decrease tissue damage induced by human neutrophils.