Cytokines Induced Neutrophil Extracellular Traps Formation: Implication for the Inflammatory Disease Condition

Neutrophils (PMNs) and cytokines have a critical role to play in host defense and systemic inflammatory response syndrome (SIRS). Neutrophil extracellular traps (NETs) have been shown to extracellularly kill pathogens, and inflammatory potential of NETs has been shown. Microbial killing inside the phagosomes or by NETs is mediated by reactive oxygen and nitrogen species (ROS/RNS). The present study was undertaken to assess circulating NETs contents and frequency of NETs generation by isolated PMNs from SIRS patients. These patients displayed significant augmentation in the circulating myeloperoxidase (MPO) activity and DNA content, while PMA stimulated PMNs from these patients, generated more free radicals and NETs. Plasma obtained from SIRS patients, if added to the PMNs isolated from healthy subjects, enhanced NETs release and free radical formation. Expressions of inflammatory cytokines (IL-1β, TNFα and IL-8) in the PMNs as well as their circulating levels were significantly augmented in SIRS subjects. Treatment of neutrophils from healthy subjects with TNFα, IL-1β, or IL-8 enhanced free radicals generation and NETs formation, which was mediated through the activation of NADPH oxidase and MPO. Pre-incubation of plasma from SIRS with TNFα, IL-1β, or IL-8 antibodies reduced the NETs release. Role of IL-1β, TNFα and IL-8 thus seems to be involved in the enhanced release of NETs in SIRS subjects.     

Silibinin induced-autophagic and apoptotic death is associated with an increase in reactive oxygen and nitrogen species in HeLa cells

Abstract

Silibinin, as the major active constituent of silymarin, has its various biological effects. Here, we investigated the inhibitory effects of silibinin on HeLa cell growth in relation to autophagy and apoptosis induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. Silibinin dose and time-dependently decreased cell growth cultured in medium containing 10% fetal bovine serum or in serum free media (SFM) with an IC₅₀ of approximately 80–100 and 40–60 μM at 24 h, respectively. Silibinin induced autophagy at 12 h, confirmed by monodansylcadervarine (MDC) staining and up-regulation of beclin-1, and induced apoptosis at 24 h, detected by observation of apoptotic bodies and activation of caspase-3. 3-methyladenine (3-MA) inhibited silibinin-induced autophagy and attenuated the silibinin's inhibitory effect on cell viability, suggesting that autophagy enhanced silibinin-induced cell death. Silibinin increased ROS levels at 12 h, and ROS scavenger, N-acetylcysteine (NAC), significantly reversed the cytotoxicity of silibinin through inhibiting both autophagy and apoptosis. Specific antioxidants were applied and results indicated that hydroxyl radical (·OH) was the major ROS induced by silibinin, and OH scavenger glutathione (GSH) inhibited apoptosis and autophagy. Silibinin also generated RNS production in the cells at 12 h. High concentration of N omega-nitro-l-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor attenuated the cytotoxicity of silibinin by decreasing ROS levels, leading to down-regulation of apoptosis. Silibinin also could interrupt the respiring functions of mitochondria, leading to ROS production and oxidative damage.     

Regulation of globular adiponectin-induced apoptosis by reactive oxygen/nitrogen species in RAW264 macrophages

Adiponectin, produced predominantly by differentiating adipocytes, is a protein hormone with antidiabetic and immunosuppressive properties. Here, we report evidence that treatment with globular adiponectin (gAd) induces apoptosis in murine macrophage-like RAW264 cells through the generation of reactive oxygen and/or nitrogen species (ROS/RNS). Treatment with gAd induced apoptosis and enhanced the activities of caspase-3 and -9, but not caspase-8. The gAd stimulation increased ROS generation and significantly reduced the ratio of NADPH to total NADP. Pretreatment with diphenyleneiodonium or apocynin reduced ROS and apoptosis in gAd-treated cells. In addition, transfection with p47(phox)- or gp91(phox)-specific small interfering RNA (siRNA) partially reduced ROS and apoptosis in response to gAd treatment. These results suggest that the administration of gAd induces apoptosis after ROS generation involving activation of NADPH oxidases. The gAd stimulation increased the release of NO into the culture medium, the activity of nitric oxide synthase (NOS), and the expression of inducible NOS (iNOS) mRNA in RAW264 cells. l-NAME reduced gAd-induced apoptotic cell death. In addition, transfection with an iNOS-specific siRNA markedly reduced the generation of NO and the population of apoptotic cells. Taken together, these results demonstrate that the gAd-induced apoptotic process in RAW264 cells involves ROS and RNS, which are generated by NADPH oxidases and iNOS, respectively.     

IGF2BP1: a novel binding protein of p38 MAPK

Maneb (MB) and paraquat (PQ) provoke oxidative stress-mediated cell damage. Role of xanthine oxidase (XO) in oxidative stress and its association with nitric oxide (NO)/NO synthase (NOS) have been widely reported. While inducible NOS (iNOS) is implicated in MB+PQ-induced toxicity in rat polymorphonuclear leukocytes (PMNs), role of XO and its alliance with iNOS have not yet been established. The study investigated the role of XO in MB+PQ-induced oxidative stress in rat PMNs and its regulation by iNOS and inflammatory cytokines. MB+PQ-augmented reactive oxygen species (ROS), superoxide, nitro-tyrosine, lipid peroxidation (LPO), and nitrite levels along with the catalytic activity of iNOS, superoxide dismutase (SOD), and XO. XO inhibitor, allopurinol (AP), alleviated MB+PQ-induced changes except nitrite content and iNOS activity. Conversely, an iNOS inhibitor, aminoguanidine, mitigated MB+PQ-induced LPO, nitrite, iNOS, and nitro-tyrosine levels; however, no change was observed in ROS, SOD, and XO. Nuclear factor-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), tumor necrosis factor-alpha (TNF-α) inhibitor, pentoxyfylline, and an anti-inflammatory agent, dexamethasone, attenuated MB+PQ-induced increase in XO, superoxide, and ROS with parallel reduction in the expression of interferon-gamma (IFN-γ), TNF-α, and interleukin-1β (IL-1β) in rat PMNs. Exogenous IFN-γ, TNF-α, and IL-1β enhanced superoxide, ROS, and XO in the PMNs of control and MB+PQ-treated rats; however, IFN- γ was found to be the most potent inducer. Moreover, AP ameliorated cytokine-induced free radical generation and restored XO activity towards normalcy. The results thus demonstrate that XO mediates oxidative stress in MB+PQ-treated rat PMNs via iNOS-independent but cytokine (predominantly IFN-γ)-dependent mechanism.     

Silibinin induced-autophagic and apoptotic death is associated with an increase in reactive oxygen and nitrogen species in HeLa cells

Silibinin, as the major active constituent of silymarin, has its various biological effects. Here, we investigated the inhibitory effects of silibinin on HeLa cell growth in relation to autophagy and apoptosis induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. Silibinin dose and time-dependently decreased cell growth cultured in medium containing 10% fetal bovine serum or in serum free media (SFM) with an IC(50) of approximately 80-100 and 40-60 μM at 24 h, respectively. Silibinin induced autophagy at 12 h, confirmed by monodansylcadervarine (MDC) staining and up-regulation of beclin-1, and induced apoptosis at 24 h, detected by observation of apoptotic bodies and activation of caspase-3. 3-methyladenine (3-MA) inhibited silibinin-induced autophagy and attenuated the silibinin's inhibitory effect on cell viability, suggesting that autophagy enhanced silibinin-induced cell death. Silibinin increased ROS levels at 12 h, and ROS scavenger, N-acetylcysteine (NAC), significantly reversed the cytotoxicity of silibinin through inhibiting both autophagy and apoptosis. Specific antioxidants were applied and results indicated that hydroxyl radical (·OH) was the major ROS induced by silibinin, and OH scavenger glutathione (GSH) inhibited apoptosis and autophagy. Silibinin also generated RNS production in the cells at 12 h. High concentration of N omega-nitro-l-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor attenuated the cytotoxicity of silibinin by decreasing ROS levels, leading to down-regulation of apoptosis. Silibinin also could interrupt the respiring functions of mitochondria, leading to ROS production and oxidative damage.     

TGF-β1–ROS–ATM–CREB signaling axis in macrophage mediated migration of human breast cancer MCF7 cells

Macrophages in the tumor microenvironment play an important role in tumor cell survival. They influence the tumor cell to proliferate, invade into surrounding normal tissues and metastasize to local and distant sites. In this study, we evaluated the effect of conditioned medium from monocytes and macrophages on growth and migration of breast cancer cells. Macrophage conditioned medium (MϕCM) containing elevated levels of cytokines TNF-α, IL-1β and IL-6 had a differential effect on non-invasive (MCF7) and highly invasive (MDA-MB-231) breast cancer cell lines. MϕCM induced the secretion of TGF-β1 in MCF7 cells. This was associated with apoptosis in a fraction of cells and generation of reactive oxygen and nitrogen species (ROS and RNS) and DNA damage in the remaining cells. This, in turn, increased expression of cAMP response element binding protein (CREB) and vimentin resulting in migration of cells. These effects were inhibited by neutralization of TNF-α, IL-1β and IL-6, inhibition of ROS and RNS, DNA damage and siRNA mediated knockdown of ATM. In contrast, MDA-MB-231 cells which had higher basal levels of pCREB were not affected by MϕCM. In summary, we have found that pro-inflammatory cytokines secreted by macrophages induce TGF-β1 in tumor cells, which activate pCREB signaling, epithelial–mesenchymal-transition (EMT) responses and enhanced migration.     

Silibinin induces the generation of nitric oxide in human breast cancer MCF-7 cells

Abstract

Increasing research has concentrated on the anti-tumour efficacy of silibinin, a flavonolignan that is clinically used as an hepatoprotectant. However, previous work has found that silibinin-induced apoptosis is accompanied by protective superoxide (O₂??) generation in MCF-7 cells. This study further reports the formation of reactive nitrogen species (RNS) in the same system. It finds that silibinin induces nitric oxide (?NO) generation in a time- and concentration-dependent manner. Moreover, the results support that there exists an inter-regulation pattern between RNS and reactive oxygen species (ROS) generation. In addition, silibinin is also found to induce RNS and ROS generation in the isolated populations of mouse peripheral blood mononuclear cells (PBMCs) and a simple in vivo model of Caenorhabditis elegans.     

Atmospheric Pressure Room Temperature Plasma Jets Facilitate Oxidative and Nitrative Stress and Lead to Endoplasmic Reticulum Stress Dependent Apoptosis in HepG2 Cells

Atmospheric pressure room temperature plasma jets (APRTP-Js) that can emit a mixture of different active species have recently found entry in various medical applications. Apoptosis is a key event in APRTP-Js-induced cellular toxicity, but the exact biological mechanisms underlying remain elusive. Here, we explored the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in APRTP-Js-induced apoptosis using in vitro model of HepG2 cells. We found that APRTP-Js facilitated the accumulation of ROS and RNS in cells, which resulted in the compromised cellular antioxidant defense system, as evidenced by the inactivation of cellular antioxidants including glutathione (GSH), superoxide dismutase (SOD) and catalase. Nitrotyrosine and protein carbonyl content analysis indicated that APRTP-Js treatment caused nitrative and oxidative injury of cells. Meanwhile, intracellular calcium homeostasis was disturbed along with the alteration in the expressions of GRP78, CHOP and pro-caspase12. These effects accumulated and eventually culminated into the cellular dysfunction and endoplasmic reticulum stress (ER stress)-mediated apoptosis. The apoptosis could be markedly attenuated by N-acetylcysteine (NAC, a free radical scavenger), which confirmed the involvement of oxidative and nitrative stress in the process leading to HepG2 cell apoptosis by APRTP-Js treatment.     

T cells expanded in presence of IL-15 exhibit increased antioxidant capacity and innate effector molecules

Persistence of effector cytotoxic T lymphocytes (CTLs) during an immunological response is critical for successfully controlling a viral infection or tumor growth. Various cytokines are known to play an important part in regulating the immune response. The IL-2 family of cytokines that includes IL-2 and IL-15 are known to function as growth and survival factors for antigen-experienced T cells. IL-2 and IL-15 possess similar properties, including the ability to induce T cell proliferation. Whereas long-term IL-2 exposure has been shown to promote apoptosis and limit CD8(+) memory T cell survival and proliferation, it is widely believed that IL-15 can inhibit apoptosis and helps maintain a memory CD8(+) T-cell population. However, mechanisms for superior outcomes for IL-15 as compared to IL-2 are still under investigation. Our data shows that human T cells cultured in the presence of IL-15 exhibit increased expression of anti-oxidant molecules glutathione reductase (GSR), thioredoxin reductase 1 (TXNDR1), peroxiredoxin (PRDX) and superoxide dismutase (SOD). An increased expression of cell-surface thiols, intracellular glutathione, and thioredoxins was also noted in IL-15 cultured T cells. Additionally, IL-15 cultured T cells showed an increase in cytolytic effector molecules. Apart from increased level of Granzyme A and Granzyme B, IL-15 cultured T cells exhibited increased accumulation of reactive oxygen (ROS) and reactive nitrogen species (RNS) as compared to IL-2 cultured T cells. Overall, this study suggests that T cells cultured in IL-15 show increased persistence not only due to levels of anti-apoptotic proteins, but also due to increased anti-oxidant levels, which is complimented by increased cytolytic effector functions.     

Dynamic determination of Ox-LDL-induced oxidative/nitrosative stress in single macrophage by using fluorescent probes

Increased oxidative/nitrosative stress, resulting from generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears to play an important role in the inflammatory responses to atherosclerosis. By using MitoTracker Orange CM-H(2)TMRos, CM-H(2)DCFDA (DCF-DA), Dihydrorhodamine 123 (DHR123), DAF-FM, Dihydroethidium (DHE) and JC-1 alone or in all combinations of red and green probes, the present study was designed to monitor the ROS and RNS generation in acute exposure of single monocyte U937-derived macrophage to oxidized low density lipoprotein (Ox-LDL). Acute Ox-LDL (100 microg/ml) treatment increased time-dependently production of intracellular nitric oxide (NO), superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)), and decreased mitochondrial membrane potential (Deltapsi) in single cell. Pretreatment of aminoguanidine (an inhibitor of inducible nitric oxide synthase (iNOS), 10 microM) and vitamin C (an antioxidant agent, 100 microM) for 2h, reduced significantly the Ox-LDL-induced increase of NO and O2*-, and vitamin C completely inhibited increase of intracellular NO and O2*-. In contrast to aminoguanidine, Vitamin C pretreatment significantly prevented Ox-LDL-induced overproduction of NO and O2*- (P<0.01), indicating that antioxidant may be more effective in therapeutic application than iNOS inhibitor in dysfunction of ROS/RNS. By demonstrating a complex imbalance of ROS/RNS via fluorescent probes in acute exposure of single cell to Ox-LDL, oxidative/nitrosative stress might be more detected in the early atherosclerotic lesions.     

The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H₂O₂, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2''-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H₂O₂ and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.     

Advanced glycosylated end products-mediated activation of polymorphonuclear neutrophils in diabetes mellitus and associated oxidative stress

Two important consequences of hyperglycemia in diabetes are development of oxidative stress and formation of advanced glycation end products (AGE) which are known to be associated with diabetic complications. Relationship between AGE formation and development of oxidative stress (OS) is yet to be established. In the present study, the involvement of AGE in PMN-mediated ROS generation and the associated OS were investigated in type 2 diabetic mellitus (DM) patients. We assessed OS parameters (serum MDA, FRAP and GSH), PMN oxidative functions (respiratory burst and superoxide production) and total serum AGE in 90 subjects divided equally in three groups--control group, Group I consisting of type 2 diabetic patients without microvascular complications and Group II consisting of type 2 diabetic patients with microvascular complications. PMNs isolated from both groups (I and II) exhibited higher level of respiratory burst (RB) and produced increased amount of superoxide anion as compared to the controls. The increase was more pronounced in diabetes with complications, as compared to those without. Serum malondialdehyde (MDA) level was elevated, whereas glutathione (GSH) and ferric reducing ability of plasma (FRAP) levels were significantly reduced in diabetes as compared to the controls, suggesting the presence of oxidative stress in DM. A positive correlation between PMN oxidative function and OS parameters suggested the involvement of PMN in the development of OS in DM. Serum AGE level was also elevated in diabetic groups as compared to the controls. Further, the positive correlation between serum AGE level and PMN oxidative function suggested the involvement of AGE in increased RB and generation of reactive oxygen species (ROS) by resting diabetic PMN. The results of the study indicate that AGE-PMN interaction possibly upregulates NADPH oxidase, leading to enhanced ROS generation and thus contributes to the pathogenesis in diabetes.     

The effect of the supernatants obtained fromSporothrix schenkii andCandida albicans culture on the generation of reactive oxygen species by polymorphonuclear leukocytes

The effect of the supernatants obtained from the liquid culture medium ofSporothrix schenkii andCandida albicans on the generation of superoxide anion (O ₂ ^– and hydroxyl radicals OH_., the elements of reactive oxygen species (ROS), and chemilimunescence (CL), a measure of several ROS, by polymorphonuclear leukocytes (PMNs) was examined. In our study, it was shown that the supernatant ofS. schenkii increased all types of ROS generation examined and CL, while that ofC. albicans increased OH_. generation and CL. The effect of the supernatants ofS. schenkii on OH_. generation and CL and that ofC. albicans on CL were most remarkable when the supernatant obtained 8 weeks after the inoculation was used. The supernatant ofS. schenkii was shown to be a much more potent stimulant than the supernatant ofC. albicans. This ROS-stimulating effect of the supernatant ofS. schenkii was heat stable but not dialyzable. These findings suggest the possible role of ROS produced by infiltrated PMNs in the inflammatory skin lesions induced byS. schenkii.     

Mitochondria transmit apoptosis signalling in cardiomyocyte-like cells and isolated hearts exposed to experimental ischemia-reperfusion injury

Ischemia-reperfusion (I/R) is a condition leading to serious complications due to death of cardiac myocytes. We used the cardiomyocyte-like cell line H9c2 to study the mechanism underlying cell damage. Exposure of the cells to simulated I/R lead to their apoptosis. Over-expression of Bcl-2 and Bcl-x(L) protected the cells from apoptosis while over-expression of Bax sensitized them to programmed cell death induction. Mitochondria-targeted coenzyme Q (mitoQ) and superoxide dismutase both inhibited accumulation of reactive oxygen species (ROS) and apoptosis induction. Notably, mtDNA-deficient cells responded to I/R by decreased ROS generation and apoptosis. Using both in situ and in vivo approaches, it was found that apoptosis occurred during reperfusion following ischemia, and recovery was enhanced when hearts from mice were supplemented with mitoQ. In conclusion, I/R results in apoptosis in cultured cardiac myocytes and heart tissue largely via generation of mitochondria-derived superoxide, with ensuing apoptosis during the reperfusion phase.     

Constitutive Activation of Epidermal Growth Factor Receptor Promotes Tumorigenesis of Cr(VI)-transformed Cells through Decreased Reactive Oxygen Species and Apoptosis Resistance Development

Hexavalent chromium (Cr(VI)) compounds are well-established lung carcinogens. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor that regulates cell survival, tumor invasion, and angiogenesis. Our results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) is able to cause malignant cell transformation. These transformed cells exhibit apoptosis resistance with reduced poly ADP-ribose polymerase cleavage (C-PARP) and Bax expression and enhanced expressions of Bcl-2 and Bcl-xL. These transformed cells also exhibit reduced capacity of reactive oxygen species (ROS) generation along with elevated expression of antioxidant manganese superoxide dismutase 2 (SOD2). The expression of this antioxidant was also elevated in lung tumor tissue from a worker exposed to Cr(VI) for 19 years. EGFR was activated in Cr(VI)-transformed BEAS-2B cells, lung tissue from animals exposed to Cr(VI) particles, and human lung tumor tissue. Further study indicates that constitutive activation of EGFR in Cr(VI)-transformed cells was due to increased binding to its ligand amphiregulin (AREG). Inhibition of EGFR or AREG increased Bax expression and reduced Bcl-2 expression, resulting in reduced apoptosis resistance. Furthermore, inhibition of AREG or EGFR restored capacity of ROS generation and decreased SOD2 expression. PI3K/AKT was activated, which depended on EGFR in Cr(VI)-transformed BEAS-2B cells. Inhibition of PI3K/AKT increased ROS generation and reduced SOD2 expression, resulting in reduced apoptosis resistance with commitment increase in Bax expression and reduction of Bcl-2 expression. Xenograft mouse tumor study further demonstrates the essential role of EGFR in tumorigenesis of Cr(VI)-transformed cells. In summary, the present study suggests that ligand-dependent constitutive activation of EGFR causes reduced ROS generation and increased antioxidant expression, leading to development of apoptosis resistance, contributing to Cr(VI)-induced tumorigenesis.     

Delayed cardioprotection with isoflurane: role of reactive oxygen and nitrogen

We determined whether isoflurane can confer delayed cardioprotection in the adult rat by triggering increased production of reactive oxygen (ROS) and nitrogen species (RNS). Our objectives were to determine 1) the concentration of isoflurane that confers delayed cardioprotection in the adult rat, 2) the role of ROS and RNS in the induction of delayed cardioprotection, and 3) the cellular sources of ROS and RNS responsible for induction of delayed cardioprotection by isoflurane. Male Sprague-Dawley rats at 8 wk of age (n = 8 rats/group) were exposed to 0.5%, 0.8%, 1%, and 2% (vol/vol) isoflurane-100% oxygen for 2 h. Isoflurane conferred delayed cardioprotection 24 h later at a concentration of 0.8% (vol/vol). Administration of manganese (III) tetrakis (4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide scavenger (15 mg/kg ip), or N(G)-nitro-L-arginine methyl ester (L-NAME), a general nitric oxide synthase inhibitor (15 mg/kg ip), 15 min before isoflurane treatment abolished the delayed cardioprotective effects of isoflurane. MnTBAP and L-NAME had no effect on delayed cardioprotection in untreated hearts. Perfusion of isolated hearts with hydroethidine, a fluorescent probe for superoxide, after isoflurane treatment resulted in a twofold increase in ethidine staining of isoflurane-treated hearts compared with untreated controls, which was attenuated by myxothiazol, an inhibitor of the mitochondrial electron transport chain (0.2 mg/kg ip) and L-NAME (15 mg/kg ip). Nitrite and nitrate content in isoflurane-treated hearts was 1.5-fold higher than in untreated hearts, whereas myocardial reduced glutathione levels were decreased by 13% in 0.8% but not in 1.0% isoflurane-treated hearts. We conclude that isoflurane confers delayed cardioprotection in the adult rat, triggered by ROS and RNS.     

Cinnabarinic acid generated from 3-hydroxyanthranilic acid strongly induces apoptosis in thymocytes through the generation of reactive oxygen species and the induction of caspase

3-Hydroxyanthranilic acid (3HAA) is one of the tryptophan metabolites along the kynurenine pathway and induces apoptosis in T cells. We investigated the mechanism of 3HAA-induced apoptosis in mouse thymocytes. The optimal concentration of 3HAA for apoptosis induction was 300-500 microM. The induction of apoptosis by a suboptimal concentration (100 microM) of 3HAA was enhanced by superoxide dismutase (SOD) as well as MnCl2 and further promoted in the presence of catalase. The 3HAA-mediated generation of intracellular reactive oxygen species (ROS) was enhanced by SOD or MnCl2 and inhibited by catalase. Corresponding to apoptosis induction, the generation of cinnabarinic acid (CA) through the oxidation of 3HAA was enhanced by SOD or MnCl2 in the presence of catalase. The synthesized CA possessed more than 10 times higher apoptosis-inducing activity than 3HAA. The intracellular ROS generation was induced by CA within 15 min and decreased to the control levels within 4 h, whereas the 3HAA-induced ROS generation increased gradually up to 4 h. Corresponding to ROS generation, the mitochondrial membrane potential was downregulated within 15 min and retained by the CA treatment. Apoptosis induction by 3HAA or CA was dependent on caspases, and caspase-3 was much more strongly activated by CA than 3HAA. In conclusion, the CA generated from 3HAA possesses a strong apoptosis-inducing activity in thymocytes through ROS generation, the loss of mitochondrial membrane potential, and caspase activation.     

The role of oxidative stress in lung injury induced by cigarette smoke

Oxidative stress is a damaging process resulting from an imbalance between excessive generation of oxidant compounds and insufficient antioxidant defence mechanisms. Oxidative stress plays a crucial role in the initiation and progression of cigarette smoke-induced lung injury, deterioration in lung functions, and development of chronic obstructive pulmonary disease (COPD). In smokers and in patients with COPD, the increased oxidant burden derives from cigarette smoke per se, and from activated inflammatory cells releasing enhanced amounts of reactive oxygen and nitrogen species (ROS, RNS, respectively). Although mild oxidative stress resulting from cigarette smoking leads to the upregulation of the antioxidative enzymes synthesis in the lungs, high levels of ROS and RNS observed in patients with COPD overwhelm the antioxidant enzymes capacities, resulting in oxidant-mediated lung injury and cell death. In addition, depletion of antioxidative systems in the systemic circulation was consistently observed in such patients. The imbalance between the generation of ROS/RNS and antioxidant capacities — the state of “oxidative stress” — is one of the major pathophysiologic hallmarks in the development of COPD. Detrimental effects of oxidative stress include impairment of membrane functions, inactivation of membrane-bound receptors and enzymes, and increased tissue permeability. In addition, oxidative stress aggravates the inflammatory processes in the lungs, and contributes to the worsening of the protease-antiprotease imbalance. Several markers of oxidative stress, such as increases in lipid peroxidation products and reductions in glutathione peroxidase activity, have been shown to be related to the reductions in pulmonary functions. In the present article we review the current knowledge about the vicious cycle of cigarette smoking, oxidative stress, and inflammation in the pathogenesis of COPD.     

Circulating and airway neutrophils in cystic fibrosis display different TLR expression and responsiveness to interleukin-10

We compared blood neutrophils (PMNs) collected from healthy subjects with PMNs derived from either blood or airways collected from the same cystic fibrosis (CF) patients. When compared to healthy blood PMNs, CF blood PMNs expressed enhanced level of CD64, a marker of neutrophil activation, and lower level of Toll-like receptor-2 (TLR2). CF airway PMNs expressed enhanced level of TLR4. Interleukin-8 (IL-8) production by CF blood PMNs could be enhanced upon addition of lipopolysaccharide or peptidoglycan, and this production was inhibited by recombinant human IL-10. In contrast, CF airway PMNs released spontaneously high level of IL-8 that was neither further enhanced by microbial activators nor inhibited by recombinant human IL-10. The levels of IL-10 receptors were similar in all types of neutrophils. These data further demonstrate that circulating PMNs from CF patients display a distinct pattern of surface markers, including TLRs, as compared to PMNs from healthy donors, and that airways PMNs from CF patients are primed and resistant to anti-inflammatory signals delivered by IL-10.