Superoxide Production In Human Neutrophils Research Articles

The Prolyl Isomerase Pin1 Controls Lipopolysaccharide-Induced Priming of NADPH Oxidase in Human Neutrophils.

Production of superoxide anion and other reactive oxygen species (ROS) by neutrophils has a vital role in host defense against microbes. However, over-production can induce cell injury participating to inflammation. Superoxide anion is produced by the phagocyte NADPH oxidase/NOX2, a multicomponent enzyme system consisting of six proteins: two trans-membrane proteins (gp91phox and p22phox) and four soluble cytosolic proteins (p40phox, p67phox, p47phox, and the small G-proteins, Rac1/2). Phosphorylation of p47phox on several serines regulates NADPH oxidase activation. LPS released by gram negative bacteria can enhance or prime neutrophil superoxide production in combination with other agonists such as the bacterial peptide formyl-Met-Leu-Phe (fMLP). Since the pathways involved in LPS-induced priming are not completely understood, we investigated the role of the prolyl cis/trans isomerase Pin1 in this process. Two different Pin1 inhibitors, PiB, and Juglone are able to block LPS-induced priming of ROS production by human neutrophils in a concentration dependent manner. PiB and Juglone did not inhibit LPS-induced CD11b translocation neither CD62L shedding. LPS induced an increase of Pin1 activity in neutrophils similar to TNFα and fMLP. Since the phosphorylation of p47phox on Ser345 is critical for NADPH oxidase up-regulation, we investigated the effect of LPS on this process. Results show that LPS induced the phosphorylation of p47phox mainly on serine 345 and induced the activation of p38MAPKinase and ERK1/2. These results suggest that the prolyl cis/trans isomerase Pin1 may control LPS-induced priming of superoxide production in human neutrophils. Pharmacological targeting of Pin1 could be a valuable approach in sepsis.

Dissociations in the effects of β2-adrenergic receptor agonists on cAMP formation and superoxide production in human neutrophils: support for the concept of functional selectivity.

In neutrophils, activation of the β2-adrenergic receptor (β2AR), a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various β2AR ligands on adenosine-3′,5′-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2 •−) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2 •− formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. β2AR agonists were generally more potent in inhibiting fMLP-induced O2 •− production than in stimulating cAMP accumulation. (−)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2 •− production. Moreover, (−)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2 •− assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2 •− production after β2AR-stimulation although cAMP-increasing substances inhibited O2 •− production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the β2AR inhibits O2 •− production in a cAMP-independent manner.

The leukocyte chemotactic receptor FPR2, but not the closely related FPR1, is sensitive to cell-penetrating pepducins with amino acid sequences descending from the third intracellular receptor loop

Lipidated peptides (pepducins) can activate certain G-protein coupled receptors (GPCRs) through a unique allosteric modulation mechanism involving cytosolic receptor domains. Pepducins with the amino acid sequence of the third intracellular loop of the neutrophil formyl peptide receptors (FPRs) as a common denominator were N-terminally conjugated with palmitic acid. F2Pal16, containing the 16 amino acids present in the third intracellular loop of FPR2, induced superoxide production in human neutrophils and the activity was sensitive to FPR2 antagonists. Cells over-expressing FPR2 were similarly responsive and responded with a transient increase in cytosolic calcium. No such effects were observed with the corresponding FPR1 pepducin. The peptide alone, lacking palmitic acid, did not activate neutrophils. A ten amino acid long pepducin F2Pal10, that was a more potent neutrophil activator than F2Pal16, was used for amino acid substitution studies. The sequences of FPR1 and FPR2 in the third intracellular loop differ by only two amino acids, and a pepducin with the FPR2-specific K231 replaced by the FPR1-specific Q231 lost all activity. The active F2Pal10 pepducin also triggered a response in cells expressing a mutated FPR2 with the third intracellular loop identical to that of FPR1. The data presented suggest that the same signaling pathways are activated when the signaling cascade is initiated by a classical receptor agonist (outside-in signaling) and when signaling starts on the cytosolic side of the membrane by a pepducin (inside-in signaling). A fundamental difference is also disclosed between the two neutrophil FPRs regarding their sensitivities to third intracellular loop pepducins.

Design and synthesis of tryptophan containing dipeptide derivatives as formyl peptide receptor 1 antagonist

Our previous studies identified an Fmoc-(S,R)-tryptophan-containing dipeptide derivative, 1, which selectively inhibited neutrophil elastase release induced by formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in human neutrophils. In an attempt to improve pharmacological activity, a series of tryptophan-containing dipeptides were synthesized and their pharmacological activities were investigated in human neutrophils. Of these, five compounds 3, 6, 19a, 24a, and 24b exhibited potent and dual inhibitory effects on FMLP-induced superoxide anion (O2˙(-)) generation and neutrophil elastase release in neutrophils with IC50 values of 0.23/0.60, 1.88/2.47, 1.87/3.60, 0.12/0.37, and 1.32/1.03 μM, respectively. Further studies indicated that inhibition of superoxide production in human neutrophils by these dipeptides was associated with the selective inhibition of formyl peptide receptor 1 (FPR1). Furthermore, the results of structure-activity relationship studies concluded that the fragment N-benzoyl-Trp-Phe-OMe (3) was most suitable as a core structure for interaction with FPR1, and may be approved as a lead for the development of new drugs in the treatment of neutrophilic inflammatory diseases. As some of the synthesized compounds exhibited separable conformational isomers, and showed diverse bioactivities, the conformation analysis of these compounds is also discussed herein.

Tetomilast Attenuates Elastase-Induced Pulmonary Emphysema through Inhibition of Oxidative Stress in Rabbits

Tetomilast was originally identified as a potent inhibitor of superoxide production in human neutrophils, and is of interest because it may relieve oxidative stress related to chronic obstructive pulmonary disease (COPD). Our objective was to determine whether tetomilast effectively protects against the development of porcine pancreatic elastase (PPE)-induced emphysema in rabbits. Rabbits were divided into three groups (sham n=19, PPE n=19, PPE/Tetomilast n=18). The rabbits were once daily orally administered vehicle solution or tetomilast 5 d/week for 4 weeks before the PPE instillation. We compared pulmonary function, inflammatory cell infiltration, oxidative stress, and the incidences of apoptosis among the three groups. Tetomilast suppressed PPE-induced increases in the incidence of apoptosis and the production of 8-hydroxy-deoxyguanosine (8-OHdG) in lung tissues. PPE-instilled rabbits treated with tetomilast showed significantly less mean linear intercept and significantly better pulmonary function than rabbits administered PPE alone. Tetomilast may inhibit the development of emphysema by attenuating pulmonary inflammation and apoptosis caused by PPE-induced oxidative stress.

Indigenous New Zealand honeys exhibit multiple anti-inflammatory activities

Recent evidence suggests a potential role for honeys in mediating clinical inflammation and tissue damage. Here, we investigated the anti-inflammatory activity of a selection of previously untested indigenous New Zealand (NZ) honeys. We found that several, but not all, New Zealand rewarewa, manuka and kanuka honey samples exhibited potent, dose-dependent reduction of human neutrophil superoxide production invitro. This inhibitory activity did not correlate with levels of known phenolic-based free radical scavengers. Furthermore, the active honeys did not scavenge superoxide generated in a cell-free xanthine/xanthine oxidase assay. In C57BL/6 J mice, topical application of manuka and rewarewa honey samples with the highest invitro activity suppressed arachidonic acid-induced ear oedema, and rewarewa honey suppressed both oedema and leukocyte (monocyte and neutrophil) infiltration. Together, these findings demonstrate that some indigenous NZ honeys exhibit clinically relevant anti-inflammatory activity. Further investigation is warranted to identify the active component(s) and mechanisms responsible for these activities and to determine potential applications for anti-inflammatory honeys in the topical treatment of clinical inflammation.

Altered Human Neutrophil Function in Response to Acute Psychological Stress

To examine the effects of an acute laboratory psychological stress task on neutrophil function, specifically phagocytosis of Escherichia coli and stimulated superoxide production in human neutrophils. There is mounting evidence that acute stress is associated with short-term increases in a number of immune indices. Participants were 40 (n = 20 females) university students (mean age, 25.9 ± 4.56 years). Blood samples to determine neutrophil function by flow cytometry were taken at the end of resting baseline, during an acute stress task, and during recovery. The stress task was a 10-minute time-pressured mental arithmetic challenge with social evaluation. There was an acute increase in phagocytic ability, p = .047, η(2)p = 0.076, and a reduction of superoxide production, p = .026, η(2)p = 0.101, associated with the stress task relative to baseline. These findings suggest that neutrophil bactericidal function may be sensitive to mental challenge tasks that provoke acute psychological stress. Further research is needed to replicate the observed psychological stress-induced changes in neutrophil function.

Cytidylyl and Uridylyl Cyclase Activity of Bacillus anthracis Edema Factor and Bordetella pertussis CyaA

Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) are second messengers for numerous mammalian cell functions. The natural occurrence and synthesis of a third cyclic nucleotide (cNMP), cyclic cytidine 3',5'-monophosphate (cCMP), is a matter of controversy, and almost nothing is known about cyclic uridine 3',5'-monophosphate (cUMP). Bacillus anthracis and Bordetella pertussis secrete the adenylyl cyclase (AC) toxins edema factor (EF) and CyaA, respectively, weakening immune responses and facilitating bacterial proliferation. A cell-permeable cCMP analogue inhibits human neutrophil superoxide production. Here, we report that EF and CyaA also possess cytidylyl cyclase (CC) and uridylyl cyclase (UC) activity. CC and UC activity was determined by a radiometric assay, using [alpha-(32)P]CTP and [alpha-(32)P]UTP as substrates, respectively, and by a high-performance liquid chromatography method. The identity of cNMPs was confirmed by mass spectrometry. On the basis of available crystal structures, we developed a model illustrating conversion of CTP to cCMP by bacterial toxins. In conclusion, we have shown both EF and CyaA have a rather broad substrate specificity and exhibit cytidylyl and uridylyl cyclase activity. Both cCMP and cUMP may contribute to toxin actions.

Regulation of phagocyte NADPH oxidase by hydrogen peroxide through a Ca 2+/c-Abl signaling pathway

The importance of H 2O 2 as a cellular signaling molecule has been demonstrated in a number of cell types and pathways. Here we explore a positive feedback mechanism of H 2O 2-mediated regulation of the phagocyte respiratory burst NADPH oxidase (NOX2). H 2O 2 induced a dose-dependent stimulation of superoxide production in human neutrophils, as well as in K562 leukemia cells overexpressing NOX2 system components. Stimulation was abrogated by the addition of catalase, the extracellular Ca 2+ chelator BAPTA, the T-type Ca 2+ channel inhibitor mibefradil, the PKCδ inhibitor rottlerin, or the c-Abl nonreceptor tyrosine kinase inhibitor imatinib mesylate or by overexpression of a dominant-negative form of c-Abl. H 2O 2 induced phosphorylation of tyrosine 311 on PKCδ and this activating phosphorylation was blocked by treatment with rottlerin, imatinib mesylate, or BAPTA. Rac GTPase activation in response to H 2O 2 was abrogated by BAPTA, imatinib mesylate, or rottlerin. In conclusion, H 2O 2 stimulates NOX2-mediated superoxide generation in neutrophils and K562/NOX2 cells via a signaling pathway involving Ca 2+ influx and c-Abl tyrosine kinase acting upstream of PKCδ. This positive feedback regulatory pathway has important implications for amplifying the innate immune response and contributing to oxidative stress in inflammatory disorders.

HIV-1 Nef associates with p22-phox, a component of the NADPH oxidase protein complex

Altered neutrophil function may contribute to the development of AIDS during the course of HIV infection. It has been described that Nef, a regulatory protein from HIV, can modulate superoxide production in other cells, therefore altered superoxide production in neutrophils from HIV infected patients, could be secondary to a direct effect of Nef on components of the NADPH oxidase complex. In this work, we describe that Nef, was capable of increasing superoxide production in human neutrophils. Furthermore, a specific association between Nef and p22-phox, a membrane component of the NADPH oxidase complex, was found. We propose that this association may reflect a capability of Nef to modulate by direct association, the enzymatic complex responsible for one of the most efficient innate defense mechanisms in phagocytes, contributing to the pathogenesis of the disease.

Changes in the ratio between FPR and FPRL1 triggered superoxide production in human neutrophils—A tool in analysing receptor specific events

Neutrophils express the G protein-coupled N-formyl peptide receptor (FPR) as well as its closely related homologue, formyl peptide like receptor 1 (FPRL1), and activation of these receptors induce a release of superoxide anions. The magnitude of the responses induced by the two peptide agonists fMLF and WKYMVM, specific for FPR and FPRL1, respectively, was found to be very variable in different neutrophil populations. The ratio between the FPR and FPRL1 triggered respiratory burst was, however, very constant and close to 1. The ratio was changed in neutrophils that were desensitized as well as when the signaling through either of the receptors was inhibited by receptor specific antagonists or by a PIP 2 binding peptide. The FPR/FPRL1 ratio was not changed in primed neutrophils or in differentiated HL-60 cells. We show that the change in the ratio, calculated from the amount of radical release in neutrophils triggered with FPR and FPRL1 specific agonists can be used as a valuable tool to find/identify receptor specific/selective changes mediated by peptides/proteins/drugs, as well as to identify cells from patients or groups of patients that diverge from normal cells in their FPR/FPRL1 triggered functions.

Gliotoxin Is a Virulence Factor of Aspergillus fumigatus : gliP Deletion Attenuates Virulence in Mice Immunosuppressed with Hydrocortisone

Gliotoxin is an immunosuppressive mycotoxin long suspected to be a potential virulence factor of Aspergillus fumigatus. Recent studies using mutants lacking gliotoxin production, however, suggested that the mycotoxin is not important for pathogenesis of A. fumigatus in neutropenic mice resulting from treatment with cyclophosphomide and hydrocortisone. In this study, we report on the pathobiological role of gliotoxin in two different mouse strains, 129/Sv and BALB/c, that were immunosuppressed by hydrocortisone alone to avoid neutropenia. These strains of mice were infected using the isogenic set of a wild type strain (B-5233) and its mutant strain (gliPDelta) and the the glip reconstituted strain (gliP(R)). The gliP gene encodes a nonribosomal peptide synthase that catalyzes the first step in gliotoxin biosynthesis. The gliPDelta strain was significantly less virulent than strain B-5233 or gliP(R) in both mouse models. In vitro assays with culture filtrates (CFs) of B-5233, gliPDelta, and gliP(R) strains showed the following: (i) deletion of gliP abrogated gliotoxin production, as determined by high-performance liquid chromatography analysis; (ii) unlike the CFs from strains B-5233 and gliP(R), gliPDelta CFs failed to induce proapoptotic processes in EL4 thymoma cells, as tested by Bak conformational change, mitochondrial-membrane potential disruption, superoxide production, caspase 3 activation, and phosphatidylserine translocation. Furthermore, superoxide production in human neutrophils was strongly inhibited by CFs from strain B-5233 and the gliP(R) strain, but not the gliPDelta strain. Our study confirms that gliotoxin is an important virulence determinant of A. fumigatus and that the type of immunosuppression regimen used is important to reveal the pathogenic potential of gliotoxin.

Superoxide Production in Human Neutrophils Is Enhanced by Treatment with Transmembrane Peptides Derived from Human Formyl Peptide Receptor

Formyl peptide receptor (FPR) mediates a number of important host defense functions. Although studies have been performed on the ligand binding site of FPR, FPR dynamic behavior such as receptor dimerization on the cell surface remains unknown. Recently, peptides derived from the transmembrane (TM) domains of GPCRs were shown to disrupt dimer formation by receptors and to result in specific regulation of receptor function. To reveal the function of FPR TM domains, hFPRTM peptides derived from FPR were synthesized, and their biological activities were evaluated with human neutrophils. Synthetic peptides did not exhibit agonistic or antagonistic activity toward superoxide anion production. However, Neutrophils treated with hFPRTM4 produced 4-fold superoxide anion compared with untreated cells when stimulated with FPR agonist fMLP. Short peptide fragments derived from the fourth TM region of FPR did not enhance superoxide anion production, which suggests that hFPRTM4 did not behave as a ligand. CD and fluorescence spectra suggested that hFPRTM peptides were inserted into the membrane. The addition of hFPRTM4 increased the intracellular calcium concentration, which meant the peptide activated some membrane protein on the cell surface. The present study suggests that the fourth TM domain of FPR has a function related to a priming effect.

Taurine chloramine inhibits PMA-stimulated superoxide production in human neutrophils perhaps by inhibiting phosphorylation and translocation of p47 phox

Neutrophils produce microbicidal oxidants to destroy the invading pathogens using nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a membrane-associated enzyme complex that generates superoxide anion (O 2 −). Upon stimulation, the cytosolic components of NADPH oxidase, p47 phox and p67 phox and the small GTPase Rac move to phagosomal and plasma membranes where they become associated with the membrane components of NADPH oxidase, gp91 phox and p22 phox and express enzyme activity. We previously showed that taurine chloramine (Tau-Cl) inhibits O 2 − production in mouse peritoneal neutrophils (Kim, 1996). In the present study, we investigated the mechanisms underlying Tau-Cl-derived inhibition on O 2 − production using a human myeloid leukemia cell line, PLB-985 cell, which has been differentiated into neutrophil-like cell. Tau-Cl inhibited the phorbol myristate acetate (PMA)-elicited O 2 − production as previously observed in murine peritoneal neutrophils. Translocation of p47 phox , p67 phox and Rac was increased in response to PMA, and Tau-Cl inhibited the PMA-stimulated translocation of p47 phox and p67 phox to plasma membrane without affecting the translocation of Rac. In addition, Tau-Cl inhibited the PMA-derived phosphorylation of p47 phox , a requirement for the translocation of cytosolic NADPH oxidase component to the plasma membrane. These results suggest that Tau-Cl inhibits PMA-elicited O 2 − production in PLB-985 granulocytes by inhibiting phosphorylation of p47 phox and translocation of p47 phox and p67 phox , eventually blocking the assembly of NADPH oxidase complex.

Superoxide Production in Galleria mellonella Hemocytes: Identification of Proteins Homologous to the NADPH Oxidase Complex of Human Neutrophils

The insect immune response has a number of structural and functional similarities to the innate immune response of mammals. The objective of the work presented here was to establish the mechanism by which insect hemocytes produce superoxide and to ascertain whether the proteins involved in superoxide production are similar to those involved in the NADPH oxidase-induced superoxide production in human neutrophils. Hemocytes of the greater wax moth (Galleria mellonella) were shown to be capable of phagocytosing bacterial and fungal cells. The kinetics of phagocytosis and microbial killing were similar in the insect hemocytes and human neutrophils. Superoxide production and microbial killing by both cell types were inhibited in the presence of the NADPH oxidase inhibitor diphenyleneiodonium chloride. Immunoblotting of G. mellonella hemocytes with antibodies raised against human neutrophil phox proteins revealed the presence of proteins homologous to gp91phox, p67phox, p47phox, and the GTP-binding protein rac 2. A protein equivalent to p40phox was not detected in insect hemocytes. Immunofluorescence analysis localized insect 47-kDa and 67-kDa proteins throughout the cytosol and in the perinuclear region. Hemocyte 67-kDa and 47-kDa proteins were immunoprecipitated and analyzed by matrix-assisted laser desorption ionization--time of flight analysis. The results revealed that the hemocyte 67-kDa and 47-kDa proteins contained peptides matching those of p67phox and p47phox of human neutrophils. The results presented here indicate that insect hemocytes phagocytose and kill bacterial and fungal cells by a mechanism similar to the mechanism used by human neutrophils via the production of superoxide. We identified proteins homologous to a number of proteins essential for superoxide production in human neutrophils and demonstrated that significant regions of the 67-kDa and 47-kDa insect proteins are identical to regions of the p67phox and p47phox proteins of neutrophils.

Dapsone suppresses human neutrophil superoxide production and elastase release in a calcium-dependent manner

Dapsone (4,4'-diaminodiphenyl sulphone) is a powerful therapeutic tool in many skin diseases including neutrophilic dermatoses. The drug has an outstanding therapeutic efficacy against many skin diseases characterized by neutrophil-rich infiltrates; however, mechanisms of its action are poorly understood. We investigated the effects of dapsone on respiratory and secretory functions of human neutrophils triggered by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), the physiological agonist C5a, and phorbol myristate acetate (PMA). Human neutrophils were isolated from venous blood obtained from healthy donors. We detected extracellular production of superoxide (O(2) (-)) by cytochrome C reduction assay, and intracellular production of O(2) (-) by flow cytometry. Neutrophil elastase release was measured by the cleavage of the specific elastase substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide. Measurement of cytosolic free calcium concentration was performed using the calcium-reactive fluorescence probe, Fluo-3. Dapsone suppressed intra- and extracellular production of O(2) (-) and elastase release triggered by fMLP and C5a, but not by PMA. Both fMLP and C5a signalled the above pathways by inducing calcium influx, but PMA functions bypassed calcium influx. Dapsone was capable of antagonizing the induction of calcium influx. These findings suggest that one mechanism of the anti-inflammatory action of dapsone is inhibition of calcium-dependent functions of neutrophils including release of tissue-damaging oxidants and proteases in the affected skin.

Effect of Boophone disticha on human neutrophils

Boophone disticha (Amaryllidaceae) are mainly used in Southern Africa for inflammatory conditions. It is also known for its toxic effects. Because of the putative effects on components of the immune system and inflammatory response the effects of extracts of the bulb of Boophane disticha were investigated on ATP production in isolated human neutrophils. Furthermore, one possible mechanism of Boophone disticha's therapeutic properties might be its inhibition of superoxide release from neutrophils. The effect of the extracts on superoxide production of human neutrophils was also investigated. Aqueous and ethanol extracts of the outer and inner scales of the bulb of Boophone disticha was investigated for their effect on human neutrophils. It was decided to test the dry other scales separately from the fleshy inner scales as the parts are also used separately by traditional healers for different applications. ATP production was significantly decreased by ethanol extracts of the inner scales of the bulb. Superoxide production was significantly inhibited by aqueous extracts of the inner and outer scales of the bulb.

Irsogladine, an anti-ulcer drug, suppresses superoxide production by inhibiting phosphodiesterase type 4 in human neutrophils

Neutrophil superoxide production is implicated in the pathogenesis of gastric mucosal damage induced by various ulcerative agents and Helicobacter pylori infection. We investigated here the effects of an anti-ulcer drug irsogladine [2, 4-diamino-6-(2, 5-dichlorophenyl)-s-triazine maleate] on cAMP formation in isolated human neutrophils. The cAMP level in human neutrophils was elevated by a phosphodiesterase (PDE) type 4 selective inhibitor rolipram, but not by any inhibitors of PDE1, PDE2 and PDE3. Irsogladine also increased cAMP formation in a concentration-dependent manner in neutrophils. A non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) alone significantly increased cAMP level, whereas irsogladine was unable to further increase cAMP level in the presence of IBMX. Irsogladine inhibited concentration-dependently the superoxide (O2−) production induced by various stimuli including formyl-methionyl-leucyl-phenylalanine, opsonized zymosan, guanosine 5′-[gamma-thio] triphosphate, A23187 and phorbol 12-myristate 13-acetate. These effects of irsogladine were mimicked by rolipram, IBMX and dibutyryl cAMP. The inhibitory effects of irsogladine and rolipram on the O2− production were reversed by a protein kinase A inhibitor H-89. These results indicate that irsogladine inhibits the superoxide production in human neutrophils by the increase of cAMP content by PDE 4 inhibition, which in turn contributing to the anti-ulcer effects of irsogladine on gastric mucosal lesions associated with oxidative stress.

Dieldrin induces human neutrophil superoxide production via protein kinases C and tyrosine kinases.

We have recently found that dieldrin is a potent human neutrophil agonist in vitro and induces neutrophilic inflammation in vivo. Among the responses observed in vitro, dieldrin was found to induce superoxide (O2-) production by a yet unknown mechanism. In the present study, dieldrin- and phorbol 12-myristate 13-acetate (PMA)-induced O2- responses were compared. For this purpose, cells were preincubated with a panel of signal transduction inhibitors including genistein, H-7, HA-1077, pertussis toxin, staurosporine, calphostin C, SB203580, PD098059, and wortmannin. Dieldrin-induced O2- response was significantly reduced with treatment with genistein, H-7, HA-1077, staurosporine, and calphostin C, whereas PMA-induced response was significantly reduced by treatment with H-7, HA-1077, and staurosporine. This indicates that dieldrin mediates its effect via protein kinases C (PKCs) and tyrosine kinases. Involvement of tyrosine kinases in dieldrin-induced human neutrophils was further demonstrated by an increase in tyrosine phosphorylated protein level expression. Finally, we found that treatment with the mitochondrial stabilizer bongkrekic acid and with the inhibitor of vesicular transport brefeldin A did not reverse dieldrin-induced O2- response.

Activation of human neutrophils in vitro and dieldrin-induced neutrophilic inflammation in vivo

Many chemicals of environmental concern are known to alter the immune system and are considered toxic molecules because they affect immune cell functions. Inflammation related to environmental chemical exposure, however, is poorly documented, except that from air pollutants. In this study, we found that the organochlorine insecticide dieldrin could not alter the ability of human neutrophils to phagocytose opsonized sheep red blood cells at nonnecrotic concentrations (0.1, 1, 10, and 50 microM). However, dieldrin was found to increase human neutrophil superoxide production, RNA synthesis, and proinflammatory cytokine interleukin-8 production. The normal apoptotic rate of neutrophils evaluated by both cytology and flow cytometry (CD-16 staining) was not altered by dieldrin treatments, and this was correlated with its inability to inhibit spreading of neutrophils onto glass. Using the murine air pouch model, we found that dieldrin induces a neutrophilic inflammation. Taken together, these results demonstrated that dieldrin is a proinflammatory contaminant. To our knowledge, this is the first report establishing that dieldrin is a contaminant exhibiting proinflammatory properties. In addition, it is the first time that the murine air pouch model has been successfully used to confirm that a chemical of environmental concern can induce an inflammatory response in vivo.