Oxidative burst and anticancer activities of rat neutrophils

It is assumed that oxidative damage caused by reactive oxygen species (ROS) from activated neutrophil granulocytes may contribute to pathology of tumors. ROS are crucial in neutrophil-mediated tumor cell lysis. The present study is focused on the oxidative burst and antitumorous activities of neutrophils when challenged with Walker carcinoma W256. Survival and tumor growth dynamics were monitored in vivo, while tumor cell proliferation when mixed with neutrophils was studied in vitro together with the generation/release of neutrophil respiratory burst products, primarily 1O2. Neutrophils were collected upon Sephadex injection. The survival of Sephadex injected animals was slightly improved, while their tumors grew less than in controls. The presence of tumor cells in vitro activated neutrophils to produce singlet oxygen similar to phorbol ester. Neutrophils from Sephadex-bearing animals diminished tumor cell proliferation in vitro (measured by 3H-TdR incorporation), while neutrophils from Sephadex and the tumor-bearing animals did not show such activity in vitro. Our results confirm that in the case of rapidly growing tumors such as murine W256 carcinoma neutrophils have antitumorous effects in the early phase of tumor development.     

Trichomonas vaginalis: reactive oxygen species mediates caspase-3 dependent apoptosis of human neutrophils

There are many neutrophils in the vaginal discharge from women infected with Trichomonas vaginalis. The aim of our study was to determine whether human neutrophil apoptosis may be regulated by reactive oxygen species (ROS) in response to trichomonads infection. Incubation of human neutrophils with live trichomonads caused marked receptor shedding of CD16, decrease of mitochondrial membrane potential (MMP) and caspase-3 activation in human neutrophils. These proapoptotic effects of T. vaginalis on neutrophils were inhibited by pretreatment of neutrophils with an inhibitor of NADPH oxidase, diphenyleneiodonium chloride (DPI), suggesting an important role of intracellular ROS accumulation in T. vaginalis-triggered apoptosis. Indeed, large amounts of ROS levels were detected in neutrophils incubated with live trichomonads, and were also effectively inhibited by DPI. However, pan-caspase inhibitor z-VAD-fmk or caspase-3 inhibitor z-DEVD-fmk did not affect T. vaginalis-induced ROS generation in neutrophils. These results suggest that ROS-dependent caspase-3 activation plays an important role in apoptosis of human neutrophils induced by T. vaginalis.     

Tec kinases regulate actin assembly and cytokine expression in LPS-stimulated human neutrophils via JNK activation

The acute inflammatory response involves neutrophils wherein recognition of bacterial products, such as lipopolysaccharide (LPS), activates intracellular signaling pathways. We have shown that the mitogen-activated protein kinase (MAPK) c-Jun NH₂ terminal kinase (JNK) is activated by LPS in neutrophils and plays a critical role in monocyte chemoattractant protein (MCP)-1 expression and actin assembly. As the Tec family kinases are expressed in neutrophils and regulate activation of the MAPKs in other cell systems, we hypothesized that the Tec kinases are an upstream component of the signaling pathway leading to LPS-induced MAPKs activation in neutrophils. Herein, we show that the Tec kinases are activated in LPS-stimulated human neutrophils and that inhibition of the Tec kinases, with leflunomide metabolite analog (LFM-A13), decreased LPS-induced JNK, but not p38, activity. Furthermore, LPS-induced actin polymerization as well as MCP-1, tumor necrosis factor-α, interleukin-6, and interleukin-1β expression are dependent on Tec kinase activity.     

Antagonistic cross-talk between Rac and Cdc42 GTPases regulates generation of reactive oxygen species

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b(558) and p67(phox) oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b(558) that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b(558) in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.     

In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes

Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan-based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O(2)(-) and HO(-) and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O(2)(-) or HO(-). Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications.     

Inhibitory effect of curcuminoids and tetrahydrocurcuminoids on equine activated neutrophils and myeloperoxidase activity

In the horse, the inflammation response to various pathologies (intestinal strangulations, laminitis, etc.) involves an excessive stimulation of the polymorphonuclear neutrophils releasing reactive oxygen species (ROS) and myeloperoxidase (MPO). The aim of the present work was to study the effect of natural polyphenols, curcuminoids and tetrahydrocurcuminoids (THC) on isolated stimulated equine neutrophils and on the activity of purified MPO. The ROS production and the release of MPO by activated neutrophils were measured by chemiluminescence and ELISA techniques, respectively. The activity of purified MPO was measured by studying its nitration, chlorination or oxidation capacity and by using an original method called SIEFED allowing the study of drug interaction with the enzyme without interferences of the medium. Curcuminoids and THC had dose-dependent inhibitory effects on ROS production and MPO release by activated neutrophils and on purified MPO activity. We suggest that the higher efficacy of curcuminoids versus THC could be explained, at least partially, by its chemical structure: the conjugated double bounds and the plane structure of curcuminoids made easier the neutralization of the radical species generated by activated neutrophils and the interaction of the drug with the active site of MPO. These inhibitory effects of curcuminoids on the oxidant activity of equine neutrophils and on MPO activity open therapeutic perspectives in equine pathologies with excessive inflammatory reactions.     

NADPH-oxidase activation in murine neutrophils via formyl peptide receptors

Neutrophils play a key role at inflammatory sites where, in addition to destroying infecting microorganisms, they may also have deleterious effects on host tissues. Both activities involve activation of the NADPH-oxidase that produces bactericidal and tissue-destructive reactive oxygen species (ROS). We activated the murine NADPH-oxidase using different types of neutrophil activators and characterized the oxidative responses with respect to magnitude, localization, and kinetics. We show that agonist-induced activation of murine neutrophils results exclusively in extracellular release of ROS and no intracellular production could be detected. We also show that the formylated peptide, formyl-Met-Leu-Phe (fMLF), is a much less potent activator of the murine NADPH-oxidase than of the human analogue. Nevertheless, fMLF responses can be primed by pretreating the murine neutrophils with either cytochalasin B or bacterial lipopolysaccharide. Finally, we show that a synthetic hexapeptide, WKYMVM, is a more potent stimulus than fMLF for murine neutrophils and that these two agonists probably act via nonidentical high-affinity receptors.     

S-Glutathionylation of p47phox sustains superoxide generation in activated neutrophils

Post-translational modifications (PTMs) induced conformational changes of proteins can cause their activation or inactivation. Neutrophils clear pathogen through phagocytosis and oxidative burst generation, while participate in inflammation through sustained and uncontrolled generation of ROS. In activated PMNs, cytosolic NOX-2 subunit p47phox following phosphorylation interacts with p67phox, p40phox and along with Rac2 translocate to the membrane. Phosphorylation of p47phox subunit occurs in both short spurts as well as sustained ROS generation, suggesting towards the unidentified molecular mechanism(s) driving these two diverse outcomes by various stimuli. The present study demonstrates that in PMA or NO treated neutrophils a subunit of NOX2, p47phox gets glutathionylated to sustain ROS generation along with a decrease in catalase, Grx-1 activity and change in GSH/GSSG ratio. Surprisingly, fMLP treated cells neither showed sustained ROS production nor glutathionylation of p47phox. S-Glutathionylation was always secondary to phosphorylation of p47phox and inhibition of glutathionylation did not alter phosphorylation but specifically impaired sustained ROS production. Interestingly, forced S-glutathionylation of p47phox converted the fMLP induced ROS generation into sustained release of ROS. We then identified the glutathionylation susceptible cysteine residues of p47phox by LC-MS/MS with IAM switch mapping. Site-directed mutagenesis of cysteine residues further mitigated p47phox S-glutathionylation. Thus, we demonstrate that p47phox S-glutathionylation plays an essential key role in the sustained ROS generation by human neutrophils.     

Conversion of Tyrosine Hydroxylase to Stable and Inactive Form by the End Products

We have reported previously that tyrosine hydroxylase in the crude extract from rat striatum exists in the inactive form showing almost no activity at the physiological pH and that the inactive form is produced by the action of the end products of the enzyme, such as dopamine. The incubation of the enzyme with the end products resulted in not only the inactivation but also a remarkable stabilization of the enzyme. Catechols possessing amino groups but no negatively charged groups on the side chains (catecholamine-type catechols) were effective at a concentration as low as 10(-7) M in both the inactivation and stabilization of the enzyme. In contrast, catechols not possessing positively or negatively charged side chains (3,4-dihydroxyphenylethyleneglycol-type catechols) were ineffective at a concentration of 10(-7) M but effective at a concentration of 10(-6) M for both the inactivation and stabilization. Catechols possessing negatively charged groups (3,4-dihydroxyphenylacetic acid-type catechols) were ineffective even at a concentration of 10(-6) M. Thus, the end products of tyrosine hydroxylase appear to serve to keep the enzyme inactive and stable. The reaction mechanism of the conversion of the enzyme from the active/labile form to the inactive/stable form by dopamine was also investigated.     

PI3Kgamma controls oxidative bursts in neutrophils via interactions with PKCalpha and p47phox

Neutrophils release reactive oxygen species (ROS) as part of the innate inflammatory immune response. Phosphoinositide 3-kinase gamma (PI3Kgamma), which is induced by the bacterial peptide N-formylmethionyl-leucyl-phenylalanine (fMLP), has been identified as an essential intracellular mediator of ROS production. However, the complex signalling reactions that link PI3Kgamma with ROS synthesis by NADPH oxidase have not yet been described in detail. We found that activation of neutrophils by fMLP triggers the association of PI3Kgamma with protein kinase Calpha (PKCalpha). Specific inhibition of PI3Kgamma suppresses fMLP-mediated activation of PKCalpha activity and ROS production, suggesting that the protein kinase activity of PI3Kgamma is involved. Our data suggest that the direct interaction of PI3Kgamma with PKCalpha forms a discrete regulatory module of fMLP-dependent ROS production in neutrophils.     

Both the Fab and Fc domains of IgG are essential for ROS emission from TNF-α-primed neutrophils by IVIG

Intravenous immunoglobulin (IVIG) is currently a very important therapeutic used for not only infectious diseases, but also for autoimmune diseases such as idiopathic thrombocytopenic purpura (ITP). Untoward reactions of IVIG have been thought to result from complement activation by aggregated IgG in IVIG. In addition, the aggregates have been known to activate neutrophils, which may result in the untoward reactions. However, the effect and mechanism of IVIG on neutrophils remain unclear. In this study, we investigated the activation of neutrophils by IVIG in terms of their reactive oxygen species (ROS) emission to elucidate the mechanisms. IVIG-induced ROS emission from purified neutrophils was remarkably augmented by TNF-α priming of the cells. The ROS emission from TNF-α-primed neutrophils occurred by activation with whole gammaglobulin (GG) molecules, but not F(ab')(2), Fc, or a mixture of F(ab')(2) and Fc. ROS emission by GG was inhibited by the F(ab')(2) fragment and an inhibitory antibody against FcγRIII. These results suggest that binding of IVIG to not only surface antigen(s), but also FcγRIII on neutrophils, is involved in IVIG-induced ROS emission from TNF-α-primed neutrophils, and contribute to the untoward reactions of IVIG.     

Effects of activated neutrophils on isolated rings of rat thoracic aorta

Local inflammation and respiratory burst of polymorphonuclear leukocytes generate reactive oxygen species (ROS). The aim of our study was to analyze the effects of peritoneal neutrophils on changes of the muscle tension of isolated aorta and compare their effects with those of different ROS. While native neutrophils did not influence muscle tension, the N-formyl-methionyl-leucyl-phenylalanine activated ones evoked a biphasic response on the KCl-precontracted aorta. The effects of activated neutrophils were in both respects similar to those evoked by xanthine/xanthine oxidase (X/XO) and differed from the effects evoked by H(2)O(2) and Fe(2)SO(4)/H(2)O(2). Using H(2)O(2) we demonstrated that the effects of ROS were dependent on the KCl induced initial tension. To exclude the effect of extensive depolarization the action of different ROS was studied also on tissues precontracted by phenylephrine. Under such condition activated neutrophils caused a marked contraction similar to that evoked by X/XO. Their effects differed however, from those of H(2)O(2) and Fe(2)SO(4)/ascorbic acid. These findings and elimination of activated neutrophil-induced contractions as well as the chemiluminiscence by superoxide dismutase suggest that the primarily activated neutrophil-released ROS was superoxide, which can be transformed to peroxynitrite, and other ROS including H(2)O(2). Reduction of all followed-up contractions caused by nordihydroguaiaretic acid, indicate that 5-lipoxygenase metabolites unselectively reduce contractions. In contrast, selective inhibition of activated neutrophil-evoked contraction by indomethacin suggests that cyclooxygenase metabolites are involved mainly in their action on vascular smooth muscle.     

Critical role of proline-rich tyrosine kinase 2 in reversion of the adhesion-mediated suppression of reactive oxygen species generation by human neutrophils

Neutrophils act as the first line of innate immune defense against invading microorganisms during infection and inflammation. The tightly regulated production of reactive oxygen species (ROS) through activation of NADPH oxidase is a major weapon used by neutrophils and other phagocytic leukocytes to combat such pathogens. Cellular adhesion signals play important physiological roles in regulating the activation of NADPH oxidase and subsequent ROS formation. We previously showed that the initial suppression of the oxidase response of chemoattractant-stimulated adherent neutrophils is mediated via inhibition of Vav1-induced activation of the NADPH oxidase regulatory GTPase Rac2 by adhesion signals. In this study we show that prior exposure of neutrophils to a number of cytokines and inflammatory mediators, including TNF-alpha, GM-CSF, and platelet-activating factor, overcomes the adhesion-mediated suppression of ROS formation. Proline-rich tyrosine kinase 2 (pyk2) activity is enhanced under these conditions, correlating with the restoration of Vav1 and Rac2 activities. Both dominant negative pyk2 and a pyk2-selective inhibitor prevented restoration of ROS production induced by TNF-alpha, GM-CSF, and platelet-activating factor, and this loss of pyk2 activity resulted in decreased Vav1 tyrosine phosphorylation and subsequent Rac2 activation. Our studies identify pyk2 as a critical regulatory component and a molecular switch to overcome the suppression of leukocyte oxidant generation by cell adhesion. This activity constitutes a mechanism by which cytokines might lead to rapid elimination of invading pathogens by adherent neutrophils under normal conditions or enhance tissue damage in pathological states.     

Strong glucose dependence of electron current in human monocytes

Reactive oxygen species (ROS) production by human monocytes differs profoundly from that by neutrophils and eosinophils in its dependence on external media glucose. Activated granulocytes produce vast amounts of ROS, even in the absence of glucose. Human peripheral blood monocytes (PBM), in contrast, are suspected not to be able to produce any ROS if glucose is absent from the media. Here we compare ROS production by monocytes and neutrophils, measured electrophysiologically on a single-cell level. Perforated-patch-clamp measurements revealed that electron current appeared after stimulation of PBM with phorbol myristate acetate. Electron current reflects the translocation of electrons through the NADPH oxidase, the main source of ROS production. The electron current was nearly abolished by omitting glucose from the media. Furthermore, in preactivated glucose-deprived cells, electron current appeared immediately with the addition of glucose to the bath. To characterize glucose dependence of PBM further, NADPH oxidase activity was assessed as hydrogen peroxide (H(2)O(2)) production and was recorded fluorometrically. H(2)O(2) production exhibited similar glucose dependence as did electron current. We show fundamental differences in the glucose dependence of ROS in human monocytes compared with human neutrophils.     

Oscillation of Reactive Oxygen Species Released by Activated Neutrophils

Phagocytic leukocytes, such as neutrophils and macrophages release reactive oxygen species (ROS) to the surrounding medium upon appropriate stimulation as part of their cytocidal activity. The release of ROS by inflammatory neutrophils, obtained by peritoneal injection of 12% caseinate-PBS was measured by the reduction of ferricytochrome c and luminol chemiluminescence (LCL). Neutrophils were harvested every 4 hours with cold PBS and stimulated with phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). On a regimen providing light between 6:00 to 18:00, PMA-stimulated neutrophils (1.0 x 10 7 neutrophil/ml) were found to release twice as much superoxide anion at night as they did during the day (clock time; 2:00 = 1.43 nmol/min vs. clock time 14:00 = 0.65 nmol/min). Neither FMLP- nor OZ-stimulated neutrophils displayed similar fluctuations. Thus, the qualitative and quantitative aspects of ROS generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the effect of neutrophils on microorganisms and the surrounding tissues may differ with the nature of the stimulus and the time the stimulus is given.     

Oxidative stress and expression of p22phox are involved in the up-regulation of tissue factor in vascular smooth muscle cells in response to activated platelets.

Vascular injury after balloon angioplasty results in the rapid activation of platelets leading to the release of growth factors and vasoactive substances. In addition, up-regulation of tissue factor (TF) and an increased production of reactive oxygen species (ROS) have been detected at sites of vascular injury. We investigated whether platelet-derived products (PDP) released from activated human platelets increase ROS production, resulting in the induction of TF expression in vascular smooth muscle cells (SMC). PDP induced a time- and concentration-dependent increase in ROS generation in cultured SMC that was mediated mainly by PDGF-AB and TGF-beta1 and impaired by the flavin inhibitor diphenylene iodonium. Increased ROS formation was associated with enhanced mRNA levels of the small NAD(P)H oxidase subunit p22phox or its smooth muscle isoform. Transient transfection with a p22phox antisense vector decreased PDP-induced ROS generation. PDP up-regulated TF mRNA expression, which was redox sensitive and reduced by transfection of the p22phox antisense vector. In addition, PDP-stimulated reporter gene activity of two TF promoter constructs was decreased by coexpression of the p22phox antisense vector. These results indicate that activated platelets up-regulate TF expression and that this response involves ROS generation and a p22phox-containing NAD(P)H oxidase in SMC.     

环孢霉素A通过ROS-Cyclophilin A-ERK1/2信号途径抑制人脐静脉内皮细胞与中性粒细胞粘附

为研究环孢霉素A(cyclosporin A,CsA)对缺氧／复氧诱导人脐静脉内皮细胞(ECV-304)与中性粒细胞粘附的影响,本工作以缺氧／复氧诱导粘附为模型,采用D-N-乙酰氨基己糖苷酶比色法检测粘附率,流式细胞术检测ECV-304细胞表面粘附分子E-选择素(E-selectin)、细胞间粘附分子-1(ICAM-1)的表达,Fenton反应测定活性氧(reactive oxygen species,ROS)的含量,Westera-blot法检测ECV-304细胞亲环素A(cyclophilin A,CyPA)、磷酸化及总细胞外信号调节激酶(ERK1／2)蛋白的表达。结果发现,ECV-304细胞经缺氧／复氧处理后,ROS释放增多,E-selectin、ICAM-1的表达上调,其表面中性粒细胞的粘附增加,CsA能显著抑制缺氧／复氧的上述作用。缺氧／复氧后,CyPA蛋白表达明显上调,ERK1/2显著活化,细胞总ERK1/2蛋白表达无明显改变。CyPA抑制剂CsA以及CyPA反义寡核苷酸均明显减轻缺氧／复氧诱导的ERK1/2激活,显著减少ECV-304细胞与中性粒细胞柑附。ERK112信号通路特异性阻断剂PD98059亦显著抑制ECV-304细胞与中性粒细胞的粘附。上述结果提示,CsA抑制缺氧气／复氧诱导的ECV-304细胞与中性粒细胞粘附,并可能通过抑制ROS-Cyclophilin A-ERK112的信号转导途径实现。     

Oscillation of Reactive Oxygen Species Released by Activated Neutrophils

Phagocytic leukocytes, such as neutrophils and macrophages release reactive oxygen species (ROS) to the surrounding medium upon appropriate stimulation as part of their cytocidal activity. The release of ROS by inflammatory neutrophils, obtained by peritoneal injection of 12% caseinate-PBS was measured by the reduction of ferricytochrome c and luminol chemiluminescence (LCL). Neutrophils were harvested every 4 hours with cold PBS and stimulated with phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). On a regimen providing light between 6:00 to 18:00, PMA-stimulated neutrophils (1.0 x 10 7 neutrophil/ml) were found to release twice as much superoxide anion at night as they did during the day (clock time; 2:00 = 1.43 nmol/min vs. clock time 14:00 = 0.65 nmol/min). Neither FMLP- nor OZ-stimulated neutrophils displayed similar fluctuations. Thus, the qualitative and quantitative aspects of ROS generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the effect of neutrophils on microorganisms and the surrounding tissues may differ with the nature of the stimulus and the time the stimulus is given.     

Changes in the immunotropic activity of neutrophilokins in the course of experimental staphylococcal infection

The influence of the products secreted by activated neutrophils (neutrophilokins) of mice, both intact and infected with staphylococci, on the activity of mouse spleen cells in the graft-versus-host reaction, immune response to sheep red blood cells and the antigen-presenting function of peritoneal macrophages was studied. Neutrophilokins of intact mice stimulated the activity of immunocompetent cells. Neutrophilokins obtained from infected mice on day 3 after infection produced an immunosuppressing effect. On day 7 after infection the immunostimulating activity of neutrophils was restored and showed practically no difference from the normal level.