Human Neutrophil Degranulation Research Articles

Studies with neutralizing antibodies suggest CXCL8-mediated neutrophil activation is independent of C-C motif chemokine receptor-like 2 (CCRL2) ligand binding function.

C-C motif chemokine receptor-like 2 (CCRL2) is a non-signaling 7 transmembrane receptor that binds chemotactic ligands to shape leukocyte recruitment to sites of inflammation. However, there is a lack of consensus on the ligands that directly bind CCRL2 or their functional impact. Studies with CCRL2 knockout mice have demonstrated that neutrophils have impaired degranulation and migration in response to CXCL8, where the underlying molecular mechanism is proposed to be due to the formation of CCRL2 heterodimers with the chemokine receptor CXCR2. Herein, we characterized the ligands that bind directly to CCRL2 and interrogated the impact of CCRL2 neutralization on CXCL8 signaling in neutrophils using pharmacological antibody tools. Using flow cytometry and Surface Plasmon Resonance microscopy (SPRm) cell binding experiments, we confirmed that chemerin, but not previously reported C-C chemokines, binds CCRL2. Furthermore, we identified human and mouse CCRL2 antibodies that neutralized chemerin binding to CCRL2. Unexpectedly, we found that neutralization of CCRL2 with these antibodies did not attenuate CXCL8-induced human neutrophil degranulation nor CXCL8-induced murine neutrophil recruitment to the peritoneum. Based on the observed differences in modulating CCRL2 function with neutralizing antibodies compared to the reported CCRL2 deficient murine models, we hypothesize that the ligand binding function of CCRL2 is dispensable for CXCL8 signaling in neutrophils. Finally, extensive profiling of CCRL2 expression on peripheral blood leukocytes revealed monocytes, dendritic cells (DC), and subpopulations of natural killer T (NKT) cells as additional targets, highlighting potential roles for CCRL2 in human cell types beyond neutrophils that warrants future investigation.

DUOX2 regulates secreted factors in virus-infected respiratory epithelial cells that contribute to neutrophil attraction and activation.

The first line of defense against respiratory viruses relies on the antiviral and proinflammatory cytokine response initiated in infected respiratory epithelial cells. The cytokine response not only restricts virus replication and spreading, but also orchestrates the subsequent immune response. The epithelial Dual Oxidase 2 (DUOX2) has recently emerged as a regulator of the interferon antiviral response. Here, we investigated the role of DUOX2 in the inflammatory cytokine response using a model of A549 cells deficient in DUOX2 generated using Crispr-Cas9 and infected by Sendai virus. We found that the absence of DUOX2 selectively reduced the induction of a restricted panel of 14 cytokines and chemokines secreted in response to Sendai virus by 20 to 89%. The secreted factors produced by epithelial cells upon virus infection promoted the migration, adhesion, and degranulation of primary human neutrophils, in part through the DUOX2-dependent secretion of TNF and chemokines. In contrast, DUOX2 expression did not impact neutrophil viability or NETosis, thereby highlighting a selective impact of DUOX2 in neutrophil functions. Overall, this study unveils previously unrecognized roles of epithelial DUOX2 in the epithelial-immune cells crosstalk during respiratory virus infection.

Untargeted GC/MS-based approach for identification of anti-inflammatory alkaloids from Hippeastrum elegans (Amaryllidaceae) using a human neutrophil model

Hippeastrum elegans is an Amaryllidaceae species producing alkaloids with pharmaceutical potential including lycorine and galanthamine. Herein, we developed a non-targeted metabolomic study associated to chemometrics and biological evaluations to identify the H. elegans constituents that were able to reduce the human neutrophils proinflammatory mechanisms. The alkaloid fractions were extracted from bulbs cultivated for 15 months (m) and harvested in six harvest periods (5, 7, 9, 11, 13, and 15 m). The GC–MS analysis allowed the detection of 41 alkaloids being 31 identified. All alkaloid components varied over the cultivation and most of them were lycorine-type skeletons. Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) distinguished three groups according to the chemical profile (group I: 5, 7, and 9 m; group II: 11 m and group III: 13 and 15 m). Therefore, the biological assays were only performed with one of the representative samples of each group: 7 m, 11 m and 15 m. None of them was toxic to human neutrophils by LDH activity and MTT test. The 7 m and 15 m-alkaloid fractions showed anti-inflammatory effects by reducing human neutrophil degranulation. However, the former one was more effective in inhibiting the cell activation based on the reduction of myeloperoxidase (MPO) release and reactive oxygen species (ROS) production. Afterwards, Partial Least Squares analysis (PLS) indicated lycorine and 11,12-dehydro-2-methoxy-assoanine as the compounds responsible for the anti-inflammatory activity of the bioactive fraction. Thus, the 7 m-alkaloid fraction of H. elegans seems to be a promising anti-inflammatory drug that deserves additional research.

Zinc Supplementation Modulates NETs Release and Neutrophils’ Degranulation

Zinc plays an important physiological role in the entire body, especially in the immune system. It is one of the most abundant microelements in our organism and an essential component of enzymes and antibacterial proteins. Zinc levels were reported to be correlated with the intensity of innate immunity responses, especially those triggered by neutrophils. However, as the results are fragmentary, the phenomenon is still not fully understood and requires further research. In this study, we aimed to perform a comprehensive assessment and study the impact of zinc on several basic neutrophils’ functions in various experimental setups. Human and murine neutrophils were preincubated in vitro with zinc, and then phagocytosis, oxidative burst, degranulation and release of neutrophil extracellular traps (NETs) were analyzed. Moreover, a murine model of zinc deficiency and zinc supplementation was introduced in the study and the functions of isolated cells were thoroughly studied. We showed that zinc inhibits NETs release as well as degranulation in both human and murine neutrophils. Our study revealed that zinc decreases NETs release by inhibiting citrullination of histone H3. On the other hand, studies performed in zinc-deficient mice demonstrated that low zinc levels result in increased release of NETs and enhanced neutrophils degranulation. Overall, it was shown that zinc affects neutrophils’ functions in vivo and in vitro. Proper zinc level is necessary to maintain efficient functioning of the innate immune response.

Convenient approach for the synthesis of ONO-LB-457, a potent leukotriene B4 receptor antagonist

Abstract This study reports a new approach for the synthesis of 5-[2-(2-carboxyethyl)-3-[6-(4-methoxyphenyl)-(5E)-hexen-1-yloxy]phenoxy]pentanoic acid V (ONO-LB-457), previously described by Konno and col. and which is considered a highly potent and orally active LTB4 receptor antagonist. This compound acts as an inhibitor of aggregation and chemotaxis, in addition to LTB4-induced human neutrophil degranulation. In this work, the preparation of ONO-LB-457 was proposed through a convergent synthesis focused on the preparation of two fragments. First, the preparation of 5-hydroxychroman-2-one (4) from 2,6-dimethoxybenzaldehyde and malonic acid, involving a Knoevenagel reaction, followed by a reduction of the olefin and intramolecular cyclization catalyzed by Lewis acid (tribromide) was achieved with an overall yield of 57%. Second, preparation of (E)-6-(4-methoxyphenyl)hex-5-en-1-yl-methanesulfonate (18) from 5-bromovaleric acid (15) involving a Wittig reaction. The desired compound V (ONO-LB-457) was obtained by nucleophilic substitution of (E)-6-(4-methoxyphenyl)hex-5-en-1-yl-methanesulfonate (18) with the ring-opened phenolic diester 14 followed by hydrolysis, in seven steps with an overall yield of about 11%.

Vitamin D (VD3) antioxidative and anti-inflammatory activities: Peripheral and central effects

Vitamin D (VD3, cholecalciferol), besides its role on bone calcium homeostasis, has also been shown to present anti-inflammatory actions. The objectives of the present work were to further extend these findings, focusing onVD3action mechanisms at the molecular level and onits central and peripheral effects. For that, VD3 antinociceptive and mainly anti-inflammatory activities were evaluated by acute models of nociception (formalin test) and inflammation (carrageenan-induced paw edema), in mice pretreated orally for 7 days with VD3 (0.5 and 1.0 mg/kg). Afterwards, the edematous paws were evaluated by immunohistochemical assays for TNF-alpha. In addition, brains from mice pretreated with VD3, at the same conditions, were harvested for iNOS andCOX-2 immunohistochemical (IHC) assays. The anti-inflammatory effect of VD3 on human neutrophil degranulation was evaluated by the release of myeloperoxidase (MPO) activity, as well as by the reactive oxygen species production. VD3 significantly reduced the licking time in the formalin test, at the second phase (inflammatory pain). VD3 also reduced the edema volume and the number of polymorphonuclear (PMN) cells, as well as the TNF-alpha expression in the edematous paws, compared with the control group. Furthermore, VD3 significantly decreased iNOS and COX-2 expressions in brain areas, such as hippocampus and prefrontal cortex, and inhibited the degranulation of activated neutrophils by the reduction of ROS production and MPO release. Based in these results, VD3 presents anti-inflammatory and antioxidative effects, manifested at peripheral and central sites as showed in the present work for the first time.

Triplaris gardneriana seeds extract exhibits in vitro anti-inflammatory properties in human neutrophils after oxidative treatment.

Triplaris gardneriana Wedd. (Polygonaceae family) is a plant species from Brazilian semiarid region which is used in local traditional medicine for the treatment of inflammatory conditions such as hemorrhoids. In this study, the in vitro anti-inflammatory activity of different concentrations of ethanolic extract from T. gardneriana seeds (EETg) was performed in order to contribute to the knowledge about etnomedicinal use of this plant species. The anti-inflammatory properties were evaluated through different approaches, such as in vitro protein anti-denaturation test, scavenging of reactive oxygen species (ROS) and myeloperoxidase (MPO) inhibition in human neutrophils activated by phorbol-12-myristate-13-acetate (PMA). Besides that, molecular docking was performed to provide new insights about the interaction between the major phenolic components in the plant extract and MPO. EETg was characterized showing a total phenol content of 153.5±6.3μg gallic acid equivalent/mg extract, ability to remove hydrogen peroxide (H2O2) in a concentration-dependent manner and had a spectroscopic profile which suggests the presence of hydroxyl groups. EETg was able to prevent protein denaturation ranging from 40.17 to 75.09%. The extract, at 10 and 20μg/mL, was able to modulate neutrophils pro-inflammatory functions, such as degranulation and burst respiratory. In both assays, the EETg had anti-inflammatory effect comparable to nonsteroidal anti-inflammatory drugs. Among the main phenolic compounds of EETg, quercitrin, quercetin and catechin showed the highest binding affinity in silico to MPO. This study demonstrated, for the first time, that the anti-inflammatory effect of T. gardneriana seeds occurs due to its modulatory effect on human neutrophil degranulation and free-radical scavenging activity.

Targeting allosteric site of AKT by 5,7-dimethoxy-1,4-phenanthrenequinone suppresses neutrophilic inflammation.

BackgroundAcute lung injury (ALI) is a severe life-threatening inflammatory disease. Neutrophil activation is a major pathogenic factor in ALI. Protein kinase B (PKB)/AKT regulates diverse cellular responses, but the significance in neutrophilic inflammation and ALI remains unknown.MethodsHuman neutrophils and neutrophil-like differentiated HL-60 (dHL-60) cells were used to examine the anti-inflammatory effects of 5,7-dimethoxy-1,4-phenanthrenequinone (CLLV-1). The therapeutic potential of CLLV-1 was determined in a mouse model of lipopolysaccharide (LPS)-induced ALI.FindingsCLLV-1 inhibited respiratory burst, degranulation, adhesion, and chemotaxis in human neutrophils and dHL-60 cells. CLLV-1 inhibited the phosphorylation of AKT (Thr308 and Ser473), but not of ERK, JNK, or p38. Furthermore, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT309–313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Significantly, CLLV-1 ameliorated LPS-induced ALI, neutrophil infiltration, and AKT activation in mice.InterpretationOur results identify CLLV-1 as a covalent allosteric AKT inhibitor by targeting AKT Cys310. CLLV-1 shows potent anti-inflammatory activity in human neutrophils and LPS-induced mouse ALI. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation.

Chemical Composition and Anti-Inflammatory Activity of the Decoction from Leaves of a Cultivated Specimen of Myracrodruon urundeuva

Myracrodruon urundeuva (Anacardiaceae) is the scientific designation of “aroeira-do-sertao”, a tree well dispersed at the northeastern Brazil. The medicinal use of this plant is widespread throughout the rural population and the inner trunk bark decoction is used to treat cutaneous affections, urinary and respiratory diseases, and gynecological problems. “Aroeira-do-sertao” has been included in the list of native species under extinction threat. In order to find a sustainable source of the plant, seedlings have been cultivated. The purpose of this work was to investigate the chemical composition of the decoction from leaves of cultivated plants (aqueous extract from Myracrodruon urundeuva (AEMU)) and its anti-inflammatory activity on human neutrophils. The addition of AEMU on cells suspension inhibited significantly its activation/degranulation induced by phorbol myristate acetate. AEMU since at 50 µg mL-1 showed better pattern of inhibitions when related to indomethacin, non-selective cyclooxygenase inhibitor used as reference anti-inflammatory. The decoction fractionation was performed by high-speed counter-current chromatography (HSCCC) and semi-preparative high-performance liquid chromatography (HPLC) using a phenyl column as the stationary phase, allowing the isolation of 24 compounds. These results showed how the AEMU is rich in bioactive metabolites, which probably contribute for the anti-inflammatory effects of this plant extract, modulating the degranulation of human neutrophils, one of the pro-inflammatory mechanism of these cells.

Targeting Allosteric Site of AKT by 5,7-Dimethoxy-1,4-Phenanthrenequinone Suppresses Neutrophilic Inflammation

Background: Acute lung injury (ALI) is a severe life-threatening inflammatory disease. Neutrophil activation is a major pathogenic factor in ALI. Protein kinase B (PKB)/AKT regulates diverse cellular responses, but the significance in neutrophilic inflammation and ALI remains unknown. Methods: Human neutrophils and neutrophil-like differentiated HL-60 (dHL-60) cells were used to examine the anti-inflammatory effects of 5,7-dimethoxy-1,4-phenanthrenequinone (CLLV-1). The therapeutic potential of CLLV-1 was determined in a mouse model of lipopolysaccharide (LPS)-induced ALI. Findings: CLLV-1 inhibited respiratory burst, degranulation, adhesion, and chemotaxis in human neutrophils and dHL-60 cells. CLLV-1 inhibited the phosphorylation of AKT (Thr308 and Ser473), but not of ERK, JNK, or p38. Furthermore, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT309-313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Significantly, CLLV-1 ameliorated LPS-induced ALI, neutrophil infiltration, and AKT activation in mice. Interpretation: Our results identify CLLV-1 as a covalent allosteric AKT inhibitor by targeting AKT Cys310. CLLV-1 shows potent anti-inflammatory activity in human neutrophils and LPS-induced mouse ALI. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation. Funding Statement: This research was financial supported by the grants from the Ministry of Science Technology (MOST 106-2320-B-255-003-MY3 and MOST 104-2320-B-255-004-MY3), Ministry of Education (EMRPD1G0231 and EMRPD1H0381), and Chang Gung Memorial Hospital (CMRPF1F0011~3, CMRPF1F0061~3, CMRPF1G0241~3, and BMRP450), Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of Interests: The authors declare that no competing interests exist. Ethics Approval Statement: The procedure of neutrophil isolation was approved by the Institutional Review Board at Chang Gung Memorial Hospital. ALI was induced by intra-tracheal spray of 2 mg/kg LPS (Escherichia coli 0111:B4) in seven to eight weeks old C57BL/6 male mice, according to the guidelines and approved by Institutional Animal Care and Use Committee of Chang Gung University, Taiwan.

Baccharis dracunculifoliaDC (Asteraceae) selectively modulates the effector functions of human neutrophils

To examine whether the hydroalcoholic extract from Baccharis dracunculifolia leaves (BdE) modulates the human neutrophil oxidative metabolism, degranulation, phagocytosis and microbial killing capacity. In-vitro assays based on chemiluminescence, spectrophotometry, flow cytometry and polarimetry were used, as well as docking calculations. At concentrations that effectively suppressed the neutrophil oxidative metabolism elicited by soluble and particulate stimuli (<10 μg/ml), without clear signs of cytotoxicity, BdE (1) inhibited NADPH oxidase and myeloperoxidase activity; (2) scavenged H2 O2 and HOCl; (3) weakly inhibited phagocytosis; and (4) did not affect neutrophil degranulation and microbial killing capacity, the expression levels of TLR2, TLR4, FcγRIIa, FcγRIIIb and CR3 and the activity of elastase and lysozyme. Caffeic acid, one of the major B. dracunculifolia secondary metabolites, did not inhibit phagocytosis but interfered in the myeloperoxidase-H2 O2 -HOCl system by scavenging H2 O2 and HOCl, and interacting with the catalytic residues His-95, Arg-239 and Gln-91. BdE selectively modulates the effector functions of human neutrophils, inhibits the activity of key enzymes and scavenges physiological oxidant species. Caffeic acid contributes to lower the levels of oxidant species. Our findings help to unravel the mechanisms by which these natural products exert immunomodulatory action towards neutrophils.

Early reduction of matrix metalloproteinase-8 serum levels is associated with leptin drop and predicts diabetes remission after bariatric surgery

BackgroundWe aimed at investigating whether the acute abrogation of leptin after bariatric surgery is able to reduce neutrophil activation and potentially affect type 2 diabetes mellitus (T2DM) remission. MethodsMetabolic and inflammatory parameters (i.e. leptin, IL-6 and neutrophil products) were compared at baseline (before bariatric surgery), one month, one and three years after surgery in morbid obese (MOB) T2DM patients (n=12) and non-MOB controls (n=32). In vitro, the effects of leptin on Il-6-induced human neutrophil degranulation and integrin upregulation were assessed. ResultsAt baseline, MOB T2DM patients had a similar demographic, lipid and glycemic profiles than non-MOB T2DM controls, but higher levels of inflammatory mediators, such as CRP, fibrinogen, neutrophil-to-lymphocyte ratio (NLR), matrix metalloproteinase (MMP)-8 and leptin. One month after surgery, CRP, fibrinogen and MMP-8 were reduced only in MOB T2DM patients, while serum leptin was reduced in both groups. In the overall cohort, leptin and MMP-8 drops from baseline to one month post-surgery were positively correlated (Δleptin vs. ΔMMP8: r=0.391, p=0.025). Moreover, ΔMMP8 inversely correlated with fasting glucose levels at one-year follow-up and with glycated hemoglobin at both one- and three-year. At the cut-off point identified by ROC curve analysis (>0ng/mL), ΔMMP8 predicted complete T2DM remission at 3-year follow-up. In vitro, leptin increased IL-6-induced MMP-8 release and abrogated CD18 up-regulation. ConclusionBariatric surgery is associated to an acute abrogation of leptin that could affect MMP-8 levels, particularly in MOB T2DM patients. This beneficial event is associated with T2DM remission at 3-year follow-up.

Mitochondrial reactive oxygen species are involved in chemoattractant-induced oxidative burst and degranulation of human neutrophils in vitro

Activation of neutrophils is accompanied by the oxidative burst, exocytosis of various granule types (degranulation) and a delay in spontaneous apoptosis. The major source of reactive oxygen species (ROS) in human neutrophils is NADPH oxidase (NOX2), however, other sources of ROS also exist. Although the function of ROS is mainly defensive, they can also play a regulatory role in cell signaling. However, the contribution of various sources of ROS in these processes is not clear. We investigated a possible role of mitochondria-derived ROS (mtROS) in the regulation of neutrophil activation induced by chemoattractant fMLP in vitro. Using the mitochondria-targeted antioxidant SkQ1, we demonstrated that mtROS are implicated in the oxidative burst caused by NOX2 activation as well as in the exocytosis of primary (azurophil) and secondary (specific) granules. Scavenging of mtROS with SkQ1 slightly accelerated spontaneous apoptosis and significantly stimulated apoptosis of fMLP-activated neutrophils. These data indicate that mtROS play a critical role in signal transduction that mediates the major neutrophil functional responses in the process of activation.

Immunomodulatory effects of Santolina chamaecyparissus leaf extracts on human neutrophil functions

Context: Santolina chamaecyparissus L. (Asteraceae) is an aromatic plant wide spread in the Mediterranean region. It is used in folk medicine for its anti-inflammatory properties.Objective: The effects of S. chamaecyparissus aqueous extract (SCAE) and polyphenolic extract (SCPE) on human polymorphonuclear neutrophil (PMN) degranulation, chemotaxis, phagocytosis, and microbicidal capacity were examined in vitro.Materials and methods: Aqueous and polyphenolic extracts were prepared from S. chamaecyparissus leaves. The elastase release was used as a marker for measuring PMN degranulation, while chemotaxis was performed using a 48-microwell chemotaxis chamber. The phagocytosis and the microbicidal capacity were evaluated using fresh cultures of Candida albicans.Results: The treatment of neutrophils with different concentrations (10–200 µg/ml) of SCAE and SCPE caused a significant (p < 0.001) and dose-dependent inhibitory effect on elastase release in fMLP/Cytochalasin B (CB)-stimulated neutrophils. Indeed, 100 µg/ml of SCAE exerted an inhibitory effect of 51.97 ± 6.2%, whereas SCPE at the same concentration abolished completely PMN degranulation. Moreover, both extracts inhibited markedly (p < 0.01) fMLP-induced chemotactic migration. At 200 µg/ml, SCAE and SCPE exerted an inhibitory effect of 54.61 ± 7.3% and 57.71 ± 7.44%, respectively. In addition, a decline in both phagocytosis and microbicidal capacity against Candida albicans was observed when PMNs were exposed to 100 and 200 µg/ml of SCAE or SCPE.Conclusion: The exerted effects on neutrophil functions support the anti-inflammatory activity and show new mechanisms of action and effectiveness of S. chamaecyparissus leaf extracts. This plant may be considered as an interesting source of anti-inflammatory and immunomodulatory agents.

Curcumin increases gelatinase activity in human neutrophils by a p38 mitogen-activated protein kinase (MAPK)-independent mechanism

Curcumin has been found to possess anti-inflammatory activities and neutrophils, key players in inflammation, were previously found to be important targets to curcumin in a few studies. For example, curcumin was found to induce apoptosis in neutrophils by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. However, the role of curcumin on the biology of neutrophils is still poorly defined. To study the role of curcumin on neutrophil degranulation and to determine the role of p38 MAPK, human neutrophils were freshly isolated from healthy individuals and incubated in vitro with curcumin. Degranulation was studied at three levels: surface expression of granule markers by flow cytometry; release of matrix metallopeptidase-9 (MMP-9 or gelatinase B) enzyme into supernatants by Western blot; and gelatinase B activity by zymography. Activation of p38 MAPK was studied by monitoring its tyrosine phosphorylation levels by western blot and its role by the utilization of a pharmacological inhibitor. The results indicate that curcumin increased the cell surface expression of CD35 (secretory vesicle), CD63 (azurophilic granules), and CD66b (gelatinase granules) in neutrophils. Also, curcumin increased the release and enzymatic activity of gelatinase B in the extracellular milieu and activated p38 MAP kinase in these cells. However, in contrast to fMLP, curcumin-induced enzymatic activity and secretion of gelatinase B were not reversed by use of a p38 inhibitor. Finally, it was found that curcumin was able to enhance phagocytosis. Taken together, the results here demonstrate that curcumin induced degranulation in human neutrophils and that the increased gelatinase activity is not dependent on p38 MAPK activation. Therefore, degranulation is another human neutrophil function that could be modulated by curcumin, as well as phagocytosis.

The histamine H4 receptor is a potent inhibitor of adhesion-dependent degranulation in human neutrophils.

The histamine H4 receptor regulates the inflammatory response. However, it is not known whether this receptor has a functional role in human neutrophils. We found that fMLP (1 μM), but not histamine (0.1-1 μM), induced Mac-1-dependent adhesion, polarization, and degranulation (release of lactoferrin). A pretreatment of neutrophils with histamine (0.001-1 μM) or JNJ 28610244 (0.1-10 μM), a specific H4 receptor agonist, led to inhibition of degranulation. Total inhibition of degranulation was obtained with 0.1 μM histamine and 10 μM JNJ 28610244. Furthermore, such inhibition by histamine of degranulation was reversed by JNJ 7777120 and JNJ 28307474, two selective H4 receptor antagonists. However, neither histamine nor the H4 receptor agonist JNJ 28610244 prevented fMLP-induced, Mac-1-dependent adhesion, indicating that the H4 receptor may block signals emanating from Mac-1-controlling degranulation. Likewise, engagement of the H4 receptor by the selective agonist JNJ 28610244 blocked Mac-1-dependent activation of p38 MAPK, the kinase that controls neutrophil degranulation. We also show expression of the H4 receptor at the mRNA level in ultrapure human neutrophils and myeloid leukemia PLB-985 cells. We concluded that engagement of this receptor by selective H4 receptor agonists may represent a good, therapeutic approach to accelerate resolution of inflammation.

7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils

In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methylcoumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate superoxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimulated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxylated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemiluminescence assay. These species also oxidized ascorbic acid and the spin traps α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reactive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperoxidase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neutrophils to produce relevant anti-inflammatory effects.

Correction: Overhauser-Enhanced MRI of Elastase Activity from In Vitro Human Neutrophil Degranulation

A funding organization and grant for the fifth author were incorrectly omitted from the Funding Statement. The following sentence should be read as the second sentence of the Funding Statement: Neha Koonjoo was supported by TRAILDNP from the public grant of the French 'Agence Nationale de la Recherche' within the context of the Investments for the Future Program, referenced ANR-10-LABX-57 and named TRAIL.

Afrormosin, an Isoflavonoid from Amburana cearensis A. C. Smith, Modulates the Inflammatory Response of Stimulated Human Neutrophils

Isoflavones are phytoestrogens known by their anti-inflammatory, antioxidant and immunomodulatory properties. Presently, there is no information on whether afrormosin, an isoflavone from Amburana cearensis A.C. Smith (Fabaceae), has some effect on the inflammatory response from stimulated human neutrophils. Thus, the aim of this study was to evaluate the anti-inflammatory and antioxidant potentials of afrormosin on human neutrophils. Neutrophils (2.5×10(6) cells/mL) were incubated with afrormosin (3.35-335.2μM) prepared from a product isolated from Amburana cearensis A.C. Smith with a 78.5% degree of purity and stimulated by the addition of cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate-13-acetate (PMA). Afrormosin inhibited the neutrophil degranulation induced by fMLP (10.47-335.2μM) or PMA (0.33-167.6μM), myeloperoxidase activity (3.3-335.2μM), TNF-α secretion (16.7-335.2μM) and the reactive oxygen species (ROS) generation (16.7-335.2μM). On the other hand, afrormosin did not show any effect either on elastase or as a free radical scavenger. These data suggest that afrormosin modulates intermediary steps of the neutrophil ROS generation process. In addition, the modulatory effect of afrormosin on human neutrophil degranulation seems to be directed towards PMA-induced activation, indicating a potent inhibition of the protein kinase C activity. This study provided evidence, for the first time, to support the anti-inflammatory and antioxidant activities of afrormosin, creating novel insights into the pharmacological actions of this natural isoflavone.

TiO2, CeO2 and ZnO nanoparticles and modulation of the degranulation process in human neutrophils

Inflammation is frequently associated with nanoparticle (NP) exposures. Given that excessive polymorphonuclear neutrophil cell degranulation is a common feature of inflammatory disorders, and since these cells are key players in inflammation, we decided to test the hypothesis that NPs could act as modulators of degranulation in human neutrophils. TiO2, CeO2 and ZnO NPs slightly down-regulated cell surface expression of the granule marker CD35, but increased CD66b and CD63 expression, as assessed by flow cytometry. In addition, expression of myeloperoxidase, MMP-9 and albumin stored in azurophil, specific/gelatinase and secretrory granules, respectively, was significantly increased in the supernatants of NPs-induced neutrophils when compared to untreated cells. Moreover, NPs were more potent than the classical bacterial tripeptide N-formyl-methionine-leucine-phenylalanine (fMLP) agonist. Finally, TiO2 and CeO2 markedly increased the enzymatic activity of MMP-9 released into the supernatant, as assessed by gelatin zymography, while ZnO exerted only a modest effect. We conclude that NPs can differentially affect all steps involved during neutrophil degranulation, namely, cell surface expression of granule markers, liberation of proteins in the supernatants and enzymatic activity. These results are expected to be helpful to understand the toxicity of TiO2, CeO2 and ZnO.