Functional and molecular characterization of NOS isoforms in rat neutrophil precursor cells

Previous studies from this laboratory have demonstrated importance of neutrophil-derived nitric oxide (NO) in free radical generation, characterized nitric oxide synthase (NOS) isoforms, and have reported subcellular distribution of NOS in rat peripheral neutrophils. Maximum number of neutrophils are added per day to the circulation from bone marrow, thus neutrophils might add substantial amount of NO in the bone marrow. NO generating ability and NOS isoforms characteristics in bone marrow neutrophil precursor cells is, however, still unexplored. This study was, therefore, undertaken to investigate NO generation ability and the molecular/biochemical characteristics of NOS isoforms in neutrophil precursor cells. The neutrophil precursors were separated on Percoll density gradient and characterized by Giemsa staining, CD markers, and by their size and granularity at various stages of maturation as Bands 1, 2, and 3. Mature neutrophils were efficient in free radical generation and phagocytosis, whereas immature cells had more mitochondria and myeloperoxidase. Amount of NO augmented from immature to mature neutrophils as assessed by fluorescent probe DAF-2DA and Griess reagent. Measurement of NOS enzyme activity further confirmed the functional status of NOS in these cells. NOS isoforms were differentially expressed during neutrophil maturation as confirmed by enzyme activity, Western blotting, flowcytometry, and RT-PCR. Expression of nNOS was predominantly stable in all the stages of neutrophil maturation. iNOS expression was, however, consistently augmented during maturation, whereas eNOS expression was downregulated with neutrophil maturation. Furthermore, all NOS isoforms proteins were distributed in cytosol as well as nucleus as assessed by confocal microscopy. This study for the first time report biochemical and molecular characteristics of NOS isoforms in rat neutrophil precursor cells

Nitric oxide-mediated augmentation of neutrophil reactive oxygen and nitrogen species formation: critical use of probes

Previous reports from this laboratory and others demonstrated NO-mediated biphasic modulation of NADPH oxidase and attenuation of neutrophil reactive oxygen species generation, whereas recently we reported augmentation in DCF fluorescence following NO treatment. These discrepancies seem to be due to utilization of different probes/methods to assess effect of NO on reactive oxygen and nitrogen species (ROS/RNS, reactive species) generation. This study aims to look into this and evaluate NO-mediated enzymatic reactive species formation by using multiple probes, human neutrophils/HL60 cells and various interventions. Addition of NO donor, SNP or SNAP (100 nM-1 mM) to PMNs suspension, exhibited a concentration- and time-dependent augmentation in DCF fluorescence, but reduced DHE fluorescence. Collective generation of reactive species was confirmed by enhanced DMPO-nitrone adduct, dityrosine and rhodamine-123 and quenching of scopoletin. NO also enhanced bacterial killing, without altering phagocytosis. Addition of NO to HL-60 cells lacking functional NADPH oxidase enhanced reactive species formation, indicating importance of other enzyme(s) too. NO-dependent ROS/RNS generation was substantially reduced by NADPH oxidase inhibitor (DPI), MPO inhibitor (ABAH), or NOS inhibitor (7-NI). However, 7-NI reduced MPO activity, warranting reappraisal of those reports, which implied NOS in reactive species formation. The results obtained demonstrated NO-mediated reactive species augmentation in human PMNs. Furthermore, superoxide scavenging by NO seems to be the key process in the decrease of DHE fluorescence and suggest usefulness of DCF as the most appropriate probe to measure the NO-mediated modulation of reactive oxygen species in particular in various pathological situations

Atorvastatin protects against ischemia-reperfusion injury in fructose-induced insulin resistant rats

Purpose: High fructose (HFr) intake is known to cause insulin resistance syndrome (IRS), however its effect against acute coronary events remains elusive. The present study was undertaken to evaluate the effect of HFr (60%) diet on myocardial ischemia-reperfusion (MI-RP) injury and its modulation by atorvastatin treatment. Methods: Wistar rats kept on HFr/chow feeding for 10 weeks, received atorvastatin (30 mg/kg, per oral) or vehicle for two additional weeks followed by MI-RP injury. Results MI-RP injury was significantly augmented in HFr fed rats, as evident by the increase in infarct size (IS, 65±5% vs. 43±7%) and activities of cardiac injury biomarkers [serum lactate dehydrogenase (LDH, 698±57 vs. 444±26 U/L), creatinine kinase (CK-MB, 584±58 vs. 435±28 U/L) and tissue myeloperoxidase (MPO, 235±15 vs. 101±11 µM/min/100 mg tissue)]. Insulin resistance (plasma glucose, 64±5 vs. 100±5 mg/dl; AUC<SUB>0-120 min</SUB>, p&lt;0.05), MI-RP injury (IS 20±5%, LDH 292±28 U/L, CK-MB 257±13 U/L, MPO 95±5 µM/min/100 mg tissue) and triglyceride (TG) level were significantly reduced, while myocardial Akt, p-Akt, eNOS, p-eNOS and iNOS protein expression were significantly enhanced following atorvastatin treatment in comparison to HFr fed rats. Oxidative stress marker, malondialdehyde and circulating levels of inflammatory cytokines (CRP, IL-6, IFN-γ and TNF) were significantly reduced, while total nitrite content in the tissue and plasma was significantly augmented in atorvastatin treated rats. Atorvastatin also ameliorated endothelial dysfunction and significantly enhanced aortic Akt and eNOS protein expression. Conclusion: Atorvastatin conferred significant protection against MI-RP injury and alleviated HFr induced IRS possibly by increasing NOS expression through Akt dependent pathway

CD14 inhibition improves survival and attenuates thrombo-inflammation and cardiopulmonary dysfunction in a baboon model of Escherichia coli sepsis

Background During sepsis, gram‐negative bacteria induce robust inflammation primarily via lipopolysacharride (LPS) signaling through TLR4, a process that involves the glycosylphosphatidylinositol (GPI)‐anchored receptor CD14 transferring LPS to the Toll‐like receptor 4/myeloid differentiation factor 2 (TLR4/MD‐2) complex. Sepsis also triggers the onset of disseminated intravascular coagulation and consumptive coagulopathy. Objectives We investigated the effect of CD14 blockade on sepsis‐induced coagulopathy, inflammation, organ dysfunction, and mortality. Methods We used a baboon model of lethal Escherichia (E) coli sepsis to study two experimental groups (n = 5): (a) E coli challenge; (b) E coli challenge plus anti‐CD14 (23G4) inhibitory antibody administered as an intravenous bolus 30 minutes before the E coli. Results Following anti‐CD14 treatment, two animals reached the 7‐day end‐point survivor criteria, while three animals had a significantly prolonged survival as compared to the non‐treated animals that developed multiple organ failure and died within 30 hours. Anti‐CD14 reduced the activation of coagulation through inhibition of tissue factor‐dependent pathway, especially in the survivors, and enhanced the fibrinolysis due to strong inhibition of plasminogen activator inhibitor 1. The treatment prevented the robust complement activation induced by E coli, as shown by significantly decreased C3b, C5a, and sC5b‐9. Vital signs, organ function biomarkers, bacteria clearance, and leukocyte and fibrinogen consumption were all improved at varying levels. Anti‐CD14 reduced neutrophil activation, cell death, LPS levels, and pro‐inflammatory cytokines (tumor necrosis factor, interleukin (IL)‐6, IL‐1β, IL‐8, interferon gamma, monocyte chemoattractant protein‐1), more significantly in the survivors than non‐surviving animals. Conclusions Our results highlight the crosstalk between coagulation/fibrinolysis, inflammation, and complement systems and suggest a protective role of anti‐CD14 treatment in E coli sepsis

A time course study on prothrombotic parameters and their modulation by anti-platelet drugs in hyperlipidemic hamsters

The present study was undertaken to assess the chronology of major pathological events associated with high cholesterol (HC) diet and their modulation by anti-platelet drugs. Male Golden Syrian hamsters were fed HC diet up to 90 days. Plasma lipid, glucose and coagulation parameters (commercial kits), platelet activation (whole blood aggregation and static adhesion), endothelial dysfunction (aortic ring vasoreactivity), splenocyte TNF-α, IFN-γ and iNOS mRNA transcripts (RT-PCR), and ferric chloride (time to occlusion) induced thrombosis were monitored at 15, 30, 60, and 90 days after HC feeding and compared with normolipidemic hamsters. A significant increase in plasma lipid levels was observed at 15 days of HC feeding, but other parameters remain unaltered. Enhanced ADP, collagen, and thrombin-induced platelet aggregation, splenocyte TNF-α expression along with endothelial dysfunction were observed from 30 to 90 days of HC feeding. Platelet adhesion on collagen-/fibrinogen-coated surface and IFN-γ expression were augmented only after 60 days, while enhanced iNOS expression, reduction in thrombin time, and potentiation of ferric chloride-induced thrombosis was observed only at 90 days of HC feeding. Thus, pathological changes induced by HC diet depend on the duration and extent of hyperlipidemia. Moreover, hamsters treated with anti-platelet drugs aspirin (5 mg/kg) or clopidogrel (10 mg/kg) along with HC feeding exhibited reduction in platelet activation as well as subsequent changes observed in the abovementioned parameters following HC feeding. Since reduction in TNF-α was associated with reversion in endothelial dysfunction and prothrombotic state, the role of platelets is implicated in the pathological changes associated with HC feeding

Inhibition of complement C5 protects against organ failure and reduces mortality in a baboon model of Escherichia coli sepsis

Bacterial sepsis triggers robust activation of the complement system with subsequent generation of anaphylatoxins (C3a, C5a) and the terminal complement complex (TCC) that together contribute to organ failure and death. Here we tested the effect of RA101295, a 2-kDa macrocyclic peptide inhibitor of C5 cleavage, using in vitro whole-blood assays and an in vivo baboon model of Escherichia coli sepsis. RA101295 strongly inhibited E. coli-induced complement activation both in vitro and in vivo by blocking the generation of C5a and the soluble form of TCC, sC5b-9. RA101295 reduced the E. coli-induced “oxidative burst,” as well as leukocyte activation, without affecting host phagocytosis of E. coli. RA101295 treatment reduced plasma LPS content in E. coli-challenged baboons, implying reduced complement-mediated bacteriolysis, whereas treated animals showed slightly improved bacterial clearance during the bacteremic stage compared with controls. Treatment with RA101295 also improved consumptive coagulopathy and preserved endothelial anticoagulant and vascular barrier functions. RA101295 abolished sepsis-induced surges in proinflammatory cytokines and attenuated systemic circulatory and febrile responses, likely reflecting decreased systemic levels of LPS and C5a. Overall, RA101295 treatment was associated with significant organ protection and markedly reduced mortality compared with nontreated controls (four of five animals survived in a 100% lethal model). We therefore conclude that inhibition of C5 cleavage during the bacteremic stage of sepsis could be an important therapeutic approach to prevent sepsis-induced inflammation, consumptive coagulopathy, and subsequent organ failure and death

Nitric oxide dependent increase in free radical generation mediates release of extracellular traps from human neutrophils

High availability of NO at the inflammatory/infection site is noticed often with oxidative stress and neutrophil extracellular traps (NETs) contents, but role of NO remains unexplored in NETs formation. In the present study incubation of adhered human neutrophils with DETA-NONOate led to NETs release in a time and concentration dependent manner, as assessed by confocal microscopy and by measuring extracellular DNA and NET-bound elastase, which was blocked by N-acetyl cysteine, suggesting role of free radicals. A time and concentration dependent augmentation in free radical formation by NO donors was measured by using DCF-DA, NBT, and by p47 phox migration to the neutrophils membrane. NO mediated formation of NETs and free radicals was significantly attenuated by pretreatment of neutrophils with diphenyleneiodonium, a dual inhibitor of NADPH-oxidase/NO synthase (NOS), 4-aminobenzoic acid hydrazide, a myeloperoxidase inhibitor and 7-nitroindazole, a NOS inhibitor, suggesting enzymatic free radical generation. We thus provide first experimental evidence that NO by augmenting free radical formation in human neutrophils mediates NETs release

Nitric oxide donors release extracellular traps from human neutrophils by augmenting free radical generation

High availability of NO, oxidative stress and neutrophil extracellular trap (NETs) contents are often noticed at the site of inflammation/infection. Studies from this lab and others have reported NO mediated free radical generation from neutrophils; role of NO in NETs formation however remains undefined so far. The present study was therefore undertaken to explore the effect of NO donors on NET release from human neutrophils (PMNs), using confocal/scanning microscopy, measuring the extracellular DNA content and NET-bound elastase activity. Addition of NO donors (SNAP and SNP) to adhered PMNs led to a time and concentration dependent NETs release, which was blocked by N-acetyl cysteine, suggesting involvement of free radicals in NETs formation. Free radical formation by NO donors was assessed by using DCF-DA, DMPO-nitrone antibody and by p47 phox migration to the neutrophils membrane. NO mediated formation of free radicals and NETs was significantly reduced by the pretreatment of neutrophils with diphenyleneiodonium (DPI), a NADPH-oxidase inhibitor and 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase inhibitor, suggesting role of enzymatic free radical generation by NO donors. We thus demonstrate that NO by augmenting free radical formation in human neutrophils mediates NETs release