Neutrophil Superoxide Release Research Articles

Onion bulb extract can both reverse and prevent colitis in mice via inhibition of pro-inflammatory signaling molecules and neutrophil activity.

Onion is one of the most commonly used plants in the traditional medicine for the treatment of various diseases. We recently demonstrated the anti-inflammatory properties of onion bulb extract (OBE) in reducing colitis severity in mice when administered at the same time of colitis induction. However, whether onion can reverse established colitis or even prevent its development has not been investigated. To test 1. whether OBE can reduce colitis severity when given either before (preventative approach) or after (treatment approach) colitis induction and if so, 2. what are the mechanisms by which onion can achieve these effects. Colitis was induced by dextran sulfate sodium (DSS) administration using treatment and preventative approaches. The severity of the inflammation was determined by the gross and histological assessments. The colonic level/activity of pro-inflammatory molecules and immune cell markers was assessed by immunofluorescence and western blotting analysis. In vitro neutrophil superoxide release and survival was assessed by chemilumenecense and Annexin-V/7AAD assays respectively. OBE treatment significantly reduced colitis severity in both approaches, the colonic expression/activity profile of pro-inflammatory molecules, inhibited WKYMVm-induced superoxide release, and increased spontaneous apoptosis of neutrophils in vitro. OBE can be used as an effective option in the prevention and/or the treatment of established colitis.

Manuka Honey Modulates the Inflammatory Behavior of a dHL-60 Neutrophil Model under the Cytotoxic Limit

Recent work has shown that Manuka honey, an increasingly popular wound additive with potent antibacterial properties, also has anti-inflammatory properties. However, little research has been done examining its effect on neutrophils. This study investigates the hypothesis that Manuka honey reduces neutrophil superoxide release and chemotaxis and reduces the activation of the inflammatory nuclear factor-κB (NF-κB) signaling pathway under honey's cytotoxic limit. A differentiated HL-60 cell line was used as a neutrophil model and cultured in various concentrations of Manuka honey for 3 and 24 hours to measure cytotoxicity via mitochondrial activity and visual trypan-exclusion count. Cytochrome C and Boyden chamber assays were used to measure the effect of Manuka honey on superoxide release and chemotaxis toward fMLP, respectively. Additionally, a Western blot for NF-κB inhibitor α (IκBα) was performed to measure Manuka honey's effect on the NF-κB pathway via IκBα phosphorylation. The results indicate a cytotoxic limit of 3-5% v/v. The presence of 1% honey decreased superoxide release at 24 hours. The 0.5, 1, and 3% honey concentrations reduced chemotaxis and IκBα phosphorylation in a dose-dependent fashion. These results suggest that Manuka honey significantly reduces neutrophil recruitment and inflammatory behavior in the wound site in a dose-dependent fashion under the cytotoxic limit.

Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities.

Aeromonas sobria serine protease (ASP) is secreted from Aeromonas sobria, a pathogen causing gastroenteritis and sepsis. ASP resembles Saccharomyces cerevisiae Kex2, a member of the subtilisin family, and preferentially cleaves peptide bonds at the C-terminal side of paired basic amino acid residues; also accepting unpaired arginine at the P1 site. Unlike Kex2, however, ASP lacks an intramolecular chaperone N-terminal propeptide, instead utilizes the external chaperone ORF2 for proper folding, therefore, ASP and its homologues constitute a new subfamily in the subtilisin family. Through activation of the kallikrein/kinin system, ASP induces vascular leakage, and presumably causes edema and septic shock. ASP accelerates plasma clotting by α-thrombin generation from prothrombin, whereas it impairs plasma clottability by fibrinogen degradation, together bringing about blood coagulation disorder that occurs in disseminated intravascular coagulation, a major complication of sepsis. From complement C5 ASP liberates C5a that induces neutrophil recruitment and superoxide release, and mast cell degranulation, which are associated with pus formation, tissue injury and diarrhea, respectively. Nicked two-chain ASP also secreted from A. sobria is more resistant to inactivation by α2-macroglobulin than single-chain ASP, thereby raising virulence activities. Thus, ASP is a potent virulence factor and may participate in the pathogenesis of A. sobria infection.

Anti-inflammatory action of angiotensin 1-7 in experimental colitis may be mediated through modulation of serum cytokines/chemokines and immune cell functions

We recently demonstrated Ang 1–7 reduced inflammation in the dextran sulfate sodium (DSS) colitis model. In this study we examined the effect of Ang 1–7 on modulation of plasma levels of selected cytokines and chemokines and immune cell effector functions (apoptosis, chemotaxis and superoxide release) in vitro. The degree of neutrophil recruitment to the colon was assessed by immunofluorescence and myeloperoxidase activity. Daily Ang 1–7 treatment at 0.01 mg/kg dose which previously ameliorated colitis severity, showed a significant reduction in circulating levels of several cytokines and chemokines, and neutrophil recruitment to the colonic tissue. It also significantly enhanced immune cell apoptosis, and reduced neutrophil chemotaxis and superoxide release in vitro. In contrast, daily administration of the Ang 1–7R antagonist A779 which previously worsened colitis severity showed significant up-regulation of specific mediators. Our results demonstrate a novel anti-inflammatory action of Ang 1–7 through modulation of plasma levels of cytokines/chemokines and immune cell activity.

The effects of Caffeic Acid Phenethyl Ester (CAPE) on oxidative stress and hypoxia‐induced cell damage

Oxidative stress has been implicated in pathogenesis of hypoxia and ischemia/reperfusion (I/R) injury. In previous studies, we have shown that the antioxidant CAPE exerted cardioprotection in an isolated rat heart I (30 min)/R (60 min) injury model. In this study, we further evaluated the effects of CAPE on oxidative stress and hypoxia‐induced cell damage. We evaluated the inhibition of absorbance in the phorbol 12‐myristate 13‐acetate (30 nM) induced superoxide production spectrophotometrically in isolated rat neutrophils via reduction of exogenous cytochrome C. We found that CAPE (0.5 μM–40 μM; n=4–13) reduced phorbol 12‐myristate 13‐acetate induced neutrophil superoxide release dose‐dependently from 29±3% to 95±2%. In a rat hind limb I (30 min)/R (60 min) model, blood hydrogen peroxide levels serves as an indicator of blood oxidative stress and was measured in real‐time via a hydrogen peroxide microsensor (100 μm) inserted into both femoral veins (one served as sham, the other as I/R). We found that in the control group, I/R significantly increased blood hydrogen peroxide levels to 2.1±0.8 μM relative to the sham limb at 60 minutes reperfusion when saline was given at the beginning of reperfusion (n=5). By contrast, CAPE when given at reperfusion (40 μM, n=5) significantly reduced blood hydrogen peroxide levels from 30 min reperfusion and throughout the rest of experiment (p<0.05). The relative change was −0.4±0.3 μM between the I/R and sham limbs at 60 minutes reperfusion. In H9C2 rat myoblast cells, cobalt chloride simulated hypoxia and induced cell damage. Tetrazolium, which can differentiate metabolically active and inactive cells/tissues, was used to measure cell damage by measuring absorbance at 450 nm. We found that incubation of cobalt chloride (800 μM, n=8) for 24 hr induced 55±5% cell death. By contrast, pretreatment of CAPE (0.5 μM–10 μM; n=1–3) for 24 hr dose‐dependently improved cell survival up to 30±9% relative to cobalt chloride treatment. In summary, this study suggests that CAPE attenuates oxidative stress from neutrophils and I/R, which may contribute to improvement in cell survival under hypoxia conditions.Support or Funding InformationThis study was supported by Division of Research and Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

Anti-Inflammatory Properties of the Enaminone E121 in the Dextran Sulfate Sodium (DSS) Colitis Model.

BackgroundEnaminones are synthetic compounds with an established role in the prevention of various forms of seizures. Recent evidence suggests potent anti-tussive, bronchodilation and anti-inflammatory properties. Pre-treatment with particularly E121 compound resulted in a decrease in leukocyte recruitment in the ovalbumin induced-model of asthma, immune cell proliferation and cytokine release in vitro. We hypothesize that E121 might serve as a therapeutic potential in intestinal inflammation through modulating immune cell functions.MethodsColitis was induced by daily dextran sulfate sodium (DSS) administration for 5 days, and its severity was determined by gross and histological assessments. The plasma level of various cytokines was measured using flow cytometry-based assay. The colonic expression/ phosphorylation level of various molecules was determined by immunofluorescence and western blotting. The effects of E121 treatment on in vitro neutrophil chemotaxis (under-agarose assay), superoxide release (luminol oxidation assay) and apoptosis (annexin V/7AAD) were also determined.ResultsDSS-induced colitis in mice was significantly reduced by daily E121 treatment (30–100 mg/kg) at gross and histological levels. This effect was due to modulated plasma levels of interleukin (IL-2) and colonic expression levels of various signaling molecules and proteins involved in apoptosis. In vitro neutrophil survival, chemotaxis, and superoxide release were also reduced by E121 treatment.ConclusionOur results indicate important anti-inflammatory actions of E121 in the pathogenesis of IBD.

Ugonin U stimulates NLRP3 inflammasome activation and enhances inflammasome-mediated pathogen clearance

The NOD-like receptor pyrin domain 3 (NLRP3) inflammasome contains Nod-like receptors, a subclass of pattern recognition receptors, suggesting that this complex has a prominent role in host defenses. Various structurally diverse stimulators activate the NLRP3 inflammasome through different signaling pathways. We previously reported that ugonin U (UgU), a natural flavonoid isolated from Helminthostachys zeylanica (L) Hook, directly stimulates phospholipase C (PLC) and triggers superoxide release in human neutrophils. In the present study, we showed that UgU induced NLRP3 inflammasome assembly and subsequent caspase-1 and interleukin (IL)-1β processing in lipopolysaccharide-primed human monocytes. Moreover, UgU elicited mitochondrial superoxide generation in a dose-dependent manner, and a specific scavenger of mitochondrial reactive oxygen species (ROS) diminished UgU-induced IL-1β and caspase-1 activation. UgU induced Ca2+ mobilization, which was inhibited by treatment with inhibitors of PLC or inositol triphosphate receptor (IP3R). Blocking Ca2+ mobilization, PLC, or IP3R diminished UgU-induced IL-1β release, caspase-1 activation, and mitochondrial ROS generation. These data demonstrated that UgU activated the NLPR3 inflammasome activation through Ca2+ mobilization and the production of mitochondrial ROS. We also demonstrated that UgU-dependent NLRP3 inflammasome activation enhanced the bactericidal function of human monocytes. The ability of UgU to stimulate human neutrophils and monocytes, both of which are professional phagocytes, and its capacity to activate the NLRP3 inflammasome, which is a promising molecular target for developing anti-infective medicine, indicate that UgU treatment should be considered as a possible novel therapy for treating infectious diseases.

Neutrophil superoxide release and plasma C-reactive protein levels pre- and post-periodontal therapy.

To determine peripheral blood neutrophil superoxide release and C-reactive protein (CRP) concentration in chronic periodontitis patients, before and after non-surgical periodontal treatment. Neutrophils were isolated from patient and control volunteers (n=20) and superoxide measured by lucigenin-enhanced chemiluminescence with and without stimulation with unopsonized Porphyromonas gingivalis, unopsonized Fusobacterium nucleatum and phorbol 12-myristate 13-acetate (PMA) before and 2-months following non-surgical therapy. Corresponding high-sensitivity plasma CRP concentrations were also determined. At pre-treatment baseline, patient neutrophils released more superoxide in the absence (p≤0.032) and presence of periodontal bacteria (p≤0.013) and after PMA stimulation (p=0.041) compared to control cells. Post-therapy, patient neutrophil superoxide release was reduced to control cell levels. Median patient plasma CRP concentrations were non-significantly higher than control values and were reduced after therapy (1.80-1.36mg/l). Patient pre-treatment baseline, unstimulated neutrophil superoxide release showed a significant, positive correlation with plasma CRP concentration (p=0.01). Chronic periodontitis is characterized by peripheral neutrophils exhibiting superoxide hyperactivity and hyper-reactivity to periodontal pathogens that is not a constitutive feature of periodontitis patients. The positive, pre-therapy relationship between unstimulated neutrophil superoxide release and plasma CRP is consistent with a protective role for CRP in reducing oxidative stress and systemic inflammation invivo.

P95 - Differential activation of neutrophil extracellular traps by specific periodontal bacteria

Periodontitis is a chronic inflammatory disease initiated by periodontal pathogens. Neutrophils play a pivotal role within the periodontal lesion, where they have a cytotoxic arsenal at their disposal, including Reactive oxygen species (ROS) and release of neutrophil extracellular trap (NETs). ROS production is essential for NET release and neutrophils in periodontitis patients are hyperactive and hyper-reactive with regard to ROS release (Matthews 2007), which may lead to disease progression. Here we characterise NET and ROS release in response to specific periodontal bacteria.Peripheral blood neutrophils were incubated with a panel of bacteria, including F. nucleatum and C. gingivalis. Neutrophil total ROS and superoxide release were measured (2hours) by luminol and lucigenin-enhanced chemiluminescence respectively. NET release was assayed by incubating the cells with bacteria for 4hours and fluorometrically quantifying NET DNA with sytox green (Palmer 2012). In all assays phorbol myristate acetate [PMA] was used as a positive control.Relative to unstimulated neutrophils S. Gordinii, F. nucleatum, V. parvula and A. viscosus stimulated significantly greater total ROS release (n=5, p=<0.05). S. Gordinii and V. parvula (n=5, p=<0.05) also stimulated significantly higher levels of superoxide.Bacterial stimulation of NETs (n=5) showed marked heterogeneity, with F. nucleatum and S. Gordinii stimulating higher levels of NETs than other species.Collectively, these data demonstrate variability between periodontal bacteria in their ability to stimulate neutrophil total ROS production, superoxide and NET responses. This variability may contribute to altered NET production in-vivo by specific periodontal bacteria and may contribute to the pathogenesis of periodontitis. Mechanisms may include bacterial avoidance of NET stimulation and thus persistence of infection, or excess NET release with associated autoimmunity.

O066 Mechanisms of BNP interactions with neutrophil superoxide release: attenuation in acute and chronic heart failure

O066 Mechanisms of BNP interactions with neutrophil superoxide release: attenuation in acute and chronic heart failure

Protein Kinase C Mediates UVB Light Induced Changes In Human Platelets that Lead to Inflammation In a Two-Event Animal Model of Transfusion Mediated Acute Lung Injury

AbstractAbstract 3358Pathophysiology of acute lung injury (ALI) includes an inflammatory component with recruitment and activation of neutrophils to the lungs. One proposed mechanism of transfusion related acute lung injury (TRALI) involves two events; the first is a generalized inflammatory response, as would occur in sepsis, which leads to activation of endothelial cells and sequestration of neutrophils to the lungs. The second is an infusion of a transfusion product that contains HLA or HNA antibodies or biologic modifiers such as lipids from stored cells. The second event activates the neutrophils sequestered in the lungs which lead to neutrophil degranulation, superoxide release and localized tissue damage. Growing evidence suggests that platelets exert proinflammatory actions which include supporting tissue infiltration of neutrophils in septic lung injury. In a separate 2010 ASH abstract we show that ultraviolet B light (UVB, 2.4 J/cm2) exposed human platelets (HPs) mediate lung injury in a two-event animal model of ALI. UVB exposure has been reported to activate platelet protein kinase C (PKC). We compared the effects of UVB exposure to PKC activation by a PKC agonist, PMA (30 nM), in aggregation, activation and potential to cause lung injury in the two-event animal model. HPs were collected by apheresis and stored overnight with experiments performed on day 1 post collection. Platelet aggregation induced by increasing concentrations of ADP (5-20 mM) was potentiated by pretreatment with UVB or PMA. TRAP (20 mM) induced aggregation was inhibited by UVB, but unchanged by PMA pretreatment. Both UVB and PMA increased platelet PAC-1 binding and p-selectin expression. Pretreating HPs with a PKC inhibitor prevented all of PMA induced PAC-1 binding and inhibited UVB induced PAC-1 binding by 40%. Furthermore, the PKC inhibitor partially reduced p-selectin expression on PMA and UVB treated HPs, whereas p-selectin expression on control HPs remained unchanged. The UVB HPs or PMA HPs were evaluated in the two-event animal model of ALI. Immunodeficient (SCID) mice were used to minimize the species difference (Piper et al., Transfusion 47:1540-9, 2007). MIP-2 elevation in plasma is a marker of acute inflammation and was increased following LPS administration. Infusion of control HPs as the second event moderately increased MIP-2. When UVB HPs or PMA HPs were infused MIP-2 was significantly elevated compared to control HPs. Pretreatment of UVB HPs with the PKC inhibitor (RO31-8425) reduced MIP-2 elevation to the level of control platelets. In summary, UVB HPs can cause ALI in animals pretreated with LPS (separate 2010 ASH abstract as mentioned above). Changes to the platelets induced by UVB appear to be mediated by PKC since a PKC agonist (PMA) has similar effects on platelets in aggregation and activation as does UVB and PKC inhibitor partially inhibits UVB induced platelet activation. In vivo, both UVB and PMA treated HPs elevated MIP-2 plasma levels when injected after LPS and this response was prevented by treatment of platelets with a PKC inhibitor prior to UVB exposure. The UVB induced activation leads to a conformational change in GpIIb/IIIa which potentiates weak agonist induced aggregation and mediates an acute in vivo inflammatory response that may be responsible for the acute lung injury in the animal model. Understanding the underlying mechanisms of UVB exposure induced changes in platelets would be beneficial in designing methods to reduce the UVB associated ALI in an animal model and potentially in patients susceptible to TRALI by a primary sensitizing event and infused with high dose UVB exposed platelets. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures:No relevant conflicts of interest to declare.

Regulation of inflammation by Rac2 in immune complex-mediated acute lung injury

Acute lung injury (ALI) is an inflammatory disorder associated with recruitment and activation of neutrophils in lungs. Rac2, a member of the Rho GTPase subfamily, is an essential regulator of neutrophil degranulation, superoxide release, and chemotaxis. Here, we hypothesized that Rac2 is important in mediating lung injury. Using a model of IgG immune complex-mediated ALI, we showed that injury was attenuated in rac2(-/-) mice compared with wild-type (WT) mice undergoing ALI, with significant decreases in alveolar leukocyte numbers, vascular leakage, and the inflammatory mediators, myeloperoxidase (MPO) and matrix metalloproteinases (MMPs). Reduced injury in rac2(-/-) mice was not associated with diminished cytokine and chemokine production, since bronchoalveolar lavage (BAL) levels of IL-17, TNF, CCL3, CXCL1, and CXCL2 were similarly increased in WT and rac2(-/-) mice with ALI compared with sham-treated mice (no ALI). BAL levels of MMP-2 and MMP-9 were significantly decreased in the airways of rac2(-/-) mice with ALI. Immunohistochemical analysis revealed that MMP-2 and MMP-9 expression was evident in alveolar macrophages and interstitial neutrophils in WT ALI. In contrast, MMP-positive cells were less prominent in rac2(-/-) mice with ALI. Chimeric mice showed that Rac2-mediated lung injury was dependent on hematopoietic cells derived from bone marrow. We propose that lung injury in response to immune complex deposition is dependent on Rac2 in alveolar macrophages and neutrophils.

Production of C5a by ASP, a Serine Protease Released from Aeromonas sobria

Aeromonas sobria causes pus and edema at sites of infection. However, the mechanisms underlying these effects have not been elucidated. C5a, the amino-terminal fragment of the complement 5th component (C5), mimics these events. To investigate the involvement of C5a in the pathophysiology of A. sobria infection, we examined release of C5a from human C5 by a serine protease (ASP), a putative virulence factor secreted by this bacterium. C5 incubated with enzymatically active ASP induced neutrophil migration in a dose-dependent manner from an ASP concentration of 3 nM and in an incubation time-dependent manner in as little as 7 min, with neutrophil accumulation in guinea pigs at intradermal injection sites and neutrophil superoxide release. These effects on neutrophils were inhibited by a C5a-receptor antagonist. The ASP incubation mixture with C5 but not C3 elicited vascular leakage in a dose- and incubation time-dependent manner, which was inhibited by a histamine H(1)-receptor antagonist. Together with these C5a-like activities, ASP cleaved C5 to release only one C5a Ag, the m.w. of which was similar to that of C5a. Immunoblotting using an anti-C5a Ab revealed generation of a C5a-like fragment from human plasma incubated with ASP. These results suggest that ASP-elicited neutrophil migration and vascular leakage via C5a production from C5 could occur in vivo, which was supported by that ASP did not affect functions of C5a and neutrophil C5a receptor. Through C5a generation, ASP could be associated with the induction of pus and edema caused by infection with this bacterium.

Oxidative Stress, Inflammation and Neutrophil Superoxide Release in Patients with Crohn’s Disease: Distinction Between Active and Non-active Disease

Increased oxidative stress has been previously demonstrated in patients with Crohn's disease (CD). However, to date, this parameter has not been assessed in a comparative study of patients in prolonged remission and those with the active disease. We report here our study of lipid peroxidation, antioxidant and inflammation status in serum derived from 16 active CD patients, 27 clinically stable patients, and 15 healthy controls. Results The extent of lipid peroxidation was higher in CD patients than in the healthy controls, while the levels of lipid peroxides (PD) and of thiobarbituric acid-reactive substances (TBARS) were significantly (P < 0.01) higher in serum obtained from patients with active CD (22 and 30%, respectively) than in that obtained from patients in remission. An analysis of the antioxidant status revealed that the beta-carotene levels in sera derived from all CD patients - patients with active or stable CD (49.4 +/- 15 and 95.6 +/- 25 mg% beta-carotene, respectively) - were higher than that in the controls (145 +/- 40 mg%). Serum activity of glutathione peroxidase (GSH-Px) was significantly (P < 0.001) higher (by 31%) in the patients with active CD than in the control group. There was no significant difference in GSH-Px activity between patients in remission and the controls. In terms of the inflammatory status, we found significantly (P < 0.01) higher levels of C-reactive proteins (CRP) and of tumor necrosis factor alpha (TNFalpha) in patients with active CD than in CD patients in remission. There was a significant correlation between those parameters and the extent of lipid oxidation. Neutrophils, which are a potential source of oxygen-free radicals, were activated by incubation with phorbol myristate acetate (PMA). Superoxide and lysozyme release were significantly reduced in neutrophils derived from patients with active CD (by 25 and 28%, respectively) in comparison to the control group. However, stimulated neutrophils from stable patients demonstrated only a minimally non-significant lower release of superoxide and lysozyme compared to the controls. Conclusion The results obtained in this study demonstrate an enhanced inflammatory and oxidative stress and a decreased antioxidant status in patients with active CD. As the patients improved and became clinically stable, the oxidative parameters decreased, approaching normal values. As neutrophil activation was also lower in patients with active disease, neutrophil activation may represent a possible defense mechanism of the body against tissue injury.

Effects of perhexiline and nitroglycerin on vascular, neutrophil and platelet function in patients with stable angina pectoris

Perhexiline, a “metabolic” anti-anginal agent currently under investigation in management of congestive heart failure and acute coronary syndromes improves platelet nitric oxide responsiveness in patients with impaired responsiveness. The current study investigated possible interactions between perhexiline and the nitric oxide donor nitroglycerin on arterial stiffness, neutrophil superoxide release and on platelet nitric oxide responsiveness. Patients ( n = 39) with stable angina pectoris, awaiting cardiac catheterization were randomized to additional perhexiline or unchanged drug therapy; all patients received nitroglycerin infusion for 2 h. Vasomotor responses to perhexiline and combined perhexiline/nitroglycerin were examined using changes in augmentation index, measured via applanation tonometry. Neutrophil superoxide release was measured ex vivo utilizing lucigenin mediated chemiluminescence and effect of perhexiline on inhibition of platelet aggregation by sodium nitroprusside was also measured. Perhexiline alone did not affect augmentation index, neutrophil superoxide release, or ex vivo platelet sodium nitroprusside response. Nitroglycerin decreased augmentation index ( P < 0.01) and superoxide release ( P < 0.05). Magnitude of inhibition of superoxide release was significantly enhanced by perhexiline pre-treatment ( P < 0.05); however perhexiline had no effect on magnitude of vasomotor response to nitroglycerin. In conclusion, perhexiline exerts no effects on arterial stiffness and does not potentiate nitroglycerin induced dilatation. In patients with normal platelet function perhexiline does not affect platelet nitric oxide responsiveness. In vivo low dose nitroglycerin inhibits neutrophil superoxide release; this effect is potentiated by pre-treatment with perhexiline. These “anti-inflammatory” effects of nitroglycerin may contribute to utility in acute coronary syndromes and congestive heart failure.

Neutrophil-activating activity and platelet-activating factor synthesis in cytokine-stimulated endothelial cells: Reduced activity in growth-arrested cells

The reactivity of endothelial cells (ECs) to proinflammatory cytokines is critically important for the pathogenesis of vascular diseases. Here, we studied functional alterations of human ECs during culture under a confluent condition; i.e., the alterations of neutrophil-activating activity, platelet-activating factor (PAF) synthesis, and granulocyte–macrophage colony-stimulating factor (GM-CSF) production in cytokine-stimulated ECs. Human umbilical vein-derived ECs exhibited the increased activity in neutrophil activation, PAF synthesis, and GM-CSF production when stimulated by proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). The activity of cytokine-stimulated ECs to stimulate superoxide release in human neutrophils and to produce PAF declined markedly in parallel as ECs became growth-arrested during culture under a confluent condition. By contrast, GM-CSF production induced by cytokine stimulation was modestly increased, and up-regulation of intercellular adhesion molecule-1 (ICAM-1) and activation of mitogen-activated protein kinases were not altered. The neutrophil-activating activity of cytokine-stimulated ECs was dependent on PAF synthesis and GM-CSF production from ECs. These findings indicate that the reduced neutrophil-activating activity in growth-arrested ECs may be, at least in part, ascribed to down-regulation of PAF synthesis.

Glyceryl trinitrate prevents neutrophil activation but not thromboxane release following ischaemia-reperfusion injury.

The aim of this study was to determine whether glyceryl trinitrate (GTN) has a protective effect on neutrophil-mediated lung injury in a model of aortic occlusion (30 min) and reperfusion (120 min). Sprague-Dawley rats were randomized into control (n = 11), ischaemia-reperfusion (IR) (n = 12), and IR treated with GTN (2 micrograms kg-1 min-1) during reperfusion (n = 10). Myeloperoxidase (MPO) activity measured pulmonary neutrophil influx. Pulmonary endothelial permeability was measured by wet:dry weight ratio, bronchoalveolar lavage (BAL) protein and neutrophil counts. Neutrophil superoxide release was measured by flow cytometry in a further IR versus GTN experiment (n = 6 in each group). The significant increase in MPO activity produced by IR to a level of 7.99 units g-1 was prevented by GTN which reduced the level to 4.73 units g-1. The increase in pulmonary microvascular leakage after reperfusion was also prevented by GTN: BAL protein without GTN was 992 micrograms ml-1 and with GTN 579 micrograms ml-1; BAL neutrophil count without GTN was 3219 cells mm-3 and with GTN 820 cells mm-3; the wet:dry lung weight ratio without GTN was 3.8 and with GTN 3.3. Neutrophil superoxide release increased significantly after 40 min of reperfusion in the untreated IR group (P < 0.05). This increase was prevented in the GTN-treated group. GTN administration had no effect on plasma thromboxane production during revascularization. These data suggest that GTN administration during the reperfusion phase has the potential to decrease pulmonary microvascular injury.

Generation of Reactive Oxygen Species by Neutrophils and Endothelial Cell Injury in Normal and Preeclamptic Pregnancies

The aim of this study was to investigate the role of neutrophil-derived reactive oxygen species on endothelial cell dysfunction in preeclampsia. We first assessed the correlation between nitrite and superoxide anion production in normal nonpregnant (n=10), normal pregnant (n=15), and preeclamptic women (n=12). We then examined neutrophil-mediated oxygen radical damage to human umbilical vein endothelial cells in vitro. Neutrophil superoxide release was measured by cytochrome C reduction; nitrite release was measured by the modified Griess reaction, and endothelial cell injury was measured by 51Cr release. N-formyl-methionyl-leucyl-phenylalanine-stimulated superoxide release by neutrophils was significantly increased in women with preeclampsia compared with the other 2 groups. Nitrite release by neutrophils was significantly decreased in preeclampsia compared with normal pregnancy. When neutrophils were pretreated with superoxide dismutase, nitrite release by neutrophils did not differ between normal pregnancy and preeclampsia, suggesting that excess superoxide anion in preeclampsia could reduce bioavailability of nitric oxide through neutrophil autocrine function. Neutrophil-mediated endothelial cell injury was significantly greater in women with preeclampsia than in the other 2 groups. Hydrogen peroxide was important in neutrophil-mediated endothelial cell injury in preeclampsia as catalase inhibited endothelial cell injury. When neutrophils were pretreated with NG-nitro-L-arginine methyl ester, neutrophil-mediated endothelial cell injury in preeclampsia was decreased, indicating a role for peroxynitrite formation as a mechanism of endothelial cell injury. In conclusion, the modulation of neutrophils causing superoxide production to dominate over nitrite release provides a reasonable explanation for endothelial cell dysfunction in preeclampsia.

Serine protease inhibitors inhibit superoxide release and adherence in human neutrophils stimulated by granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha.

Stimulation of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor-alpha (TNF) results in increased superoxide (O2-) release and adherence. O2- release and adherence are dependent on activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Possible participation of serine proteases in GM-CSF- or TNF-induced activation of human neutrophils was explored with various serine protease inhibitors, including phenylmethylsulfonyl fluoride, L-1-tosylamido-2-phenylethyl-chloromethyl ketone and N-alpha-p-tosyl-L-lysine-chloromethyl ketone. GM-CSF- or TNF-induced O2- release and adherence were inhibited in parallel by pretreatment of neutrophils with these inhibitors. On the other hand, GM-CSF- or TNF-induced phosphorylation of ERK and p38 MAPK was unaffected by these inhibitors at the concentrations effective for the inhibition of O2- release and adherence. These findings suggest that serine proteases are involved in GM-CSF- and TNF-induced O2- release and adherence in human neutrophils and that serine proteases function downstream or independently of the activation of ERK and p38 MAPK.

Early immunological defects in comatose patients after acute brain injury.

The aim of this prospective study was to evaluate the phagocytic, humoral, and cellular arms of the immune system in comatose patients shortly after severe brain injury and to compare the findings with those reported earlier in patients in a persistent vegetative state. The study was conducted in intensive care units and immunology laboratories of university-affiliated hospitals in central Israel. The study group consisted of 14 men aged 16 to 65 years who were comatose as a result of acute brain injury due to mechanical trauma. All were studied within 72 hours of injury. Brain damage was severe in all cases (Glasgow Coma Scale score < 8). Healthy age- and sex-matched volunteers served as simultaneous controls. Infections arose in nine (75%) of the 12 patients in whom data were available; the cumulative mortality rate was 38% (five of 13 patients in whom outcome data were available). Every patient exhibited one or more defects in at least one arm of the immune system. Significant deficiencies were noted in neutrophil superoxide release, immunoglobulin (Ig)G, IgG1, IgM, C1q, C2, properdin, alternate C pathway, T cells, T helper cells, T suppressor cells, and natural killer cells. In an earlier series of patients examined by the authors months after the primary insult, these impairments were absent in most of the patients in the vegetative state. Significant deficiencies of the immune system, particularly the cellular arm, are precipitated by severe brain injury within 72 hours of the event. These impairments probably play a role in the high rate of complicating infections and multiple organ failure. Together with earlier findings, the results of this study indicate that if brain-injured patients survive these hazards, their immune system will eventually recover.