Changes in Superoxide Dismutase Activity and Peroxynitrite Content in Rat Peritoneal Macrophages Exposed to He-Ne Laser Radiation

The formation of reactive oxygen and nitrogen species by rat peritoneal macrophages induced by a low-intensity He-Ne laser radiation (LR) was studied in this work. It was found that the formation of reactive oxygen species, nitric oxide, and peroxynitrite as well as changes in the activity of superoxide dismutase (SOD) depended to a large extent on the LR dose. In particular, it was found that activation of SOD at low LR doses was accompanied by nitric oxide level increase, while the level of peroxynitrite showed no significant changes. On the other hand, an enhanced LR dose inhibited the enzyme, and this was accompanied by peroxynitrite accumulation. All the measurements were carried out the day after LR treatment. The revealed regularities consequently demonstrate the existence of a deferred LR action on macrophages associated with the production of reactive oxygen and nitrogen species.     

Enhanced Mitochondrial Radical Production in Patients with Rheumatoid Arthritis Correlates with Elevated Levels of Tumor Necrosis Factor alpha in Plasma

Mitochondrial dysfunction contributes to cell damage in a number of human diseases. One significant mechanism by which mitochondria damage cells is by producing reactive oxygen species from the respiratory chain. In this study we measured the production of reactive oxygen species by leukocyte mitochondria in blood from rheumatoid arthritis patients. To do this we used the chemiluminescence of lucigenin, which is accumulated by mitochondria within cells and reacts with superoxide to form a chemiluminescent product. By using specific inhibitors we could distinguish between the production of reactive oxygen species by mitochondria and by NADPH oxidase. There was a five-fold increase in mitochondrial reactive oxygen species production in whole blood and monocytes from patients with rheumatoid arthritis, when compared to healthy subjects or patients with non-rheumatic diseases. There was no increase in mitochondrial reactive oxygen species production by neutrophils from rheumatoid arthritis patients. The enhanced mitochondrial radical production in rheumatoid arthritis patients correlated significantly with increased levels of tumor necrosis factor alpha in plasma (p<0.0001). As tumor necrosis factor alpha is known to increase mitochondrial reactive oxygen species production the elevated mitochondrial radical formation seen in rheumatoid arthritis patients may be due to activation of the mitochondrial radical production. These data suggest that elevated mitochondrial oxidative stress contributes to the pathology of rheumatoid arthritis.     

Chronic cold stress modulates the function of peritoneal macrophages in vivo

On day 7 of chronic cold stress, concentration of corticosterone in plasma was established to increase, zymosan-induced production of reactive oxygen species was activated, and IL-10 synthesis by macrophages was enhanced regardless of the opiate receptor blockade. Increase in the concentration of corticosterone was independent of the blockade of opioid receptors, whereas the zymosan-induced increase in the production of oxygen radicals and activation of the spontaneous production of IL-10 was blocked in mice by naloxone. Chronic cold stress had no effect on the production of IL-1β and TNF-α.     

Tripartite-motif protein 30 negatively regulates NLRP3 inflammasome activation by modulating reactive oxygen species production

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is critical for caspase-1 activation and the proteolytic processing of pro-IL-1β. However, the mechanism that regulates NLRP3 inflammasome activation remains unclear. In this paper, we demonstrate that tripartite-motif protein 30 (TRIM30) negatively regulates NLRP3 inflammasome activation. After stimulation with ATP, an agonist of the NLRP3 inflammasome, knockdown of TRIM30 enhanced caspase-1 activation and increased production of IL-1β in both J774 cells and bone marrow-derived macrophages. Similarly with ATP, knockdown of TRIM30 increased caspase-1 activation and IL-1β production triggered by other NLRP3 inflammasome agonists, including nigericin, monosodium urate, and silica. Production of reactive oxygen species was increased in TRIM30 knockdown cells, and its increase was required for enhanced NLRP3 inflammasome activation, because antioxidant treatment blocked excess IL-1β production. Conversely, overexpression of TRIM30 attenuated reactive oxygen species production and NLRP3 inflammasome activation. Finally, in a crystal-induced NLRP3 inflammasome-dependent peritonitis model, monosodium urate-induced neutrophil flux and IL-1β production was reduced significantly in TRIM30 transgenic mice as compared with that in their nontransgenic littermates. Taken together, our results indicate that TRIM30 is a negative regulator of NLRP3 inflammasome activation and provide insights into the role of TRIM30 in maintaining inflammatory responses.     

Calcium & ROS: Two orchestra directors for the requiem of death

Tumor necrosis factor alpha (TNF) triggers regulated necrosis of mycobacterium-infected macrophages through of mitochondrial reactive oxygen species (mitoROS) production in a RIPK1/3-dependent manner. To explain that, Roca and colleagues describe an inter-orgallenar circuit which involves the lysosomal ceramide production, mitoROS, BAX activation and RyR Ca²⁺ efflux from the endoplasmic reticulum into the mitochondrion.     

TLR-Mediated Production of Reactive Oxygen Species and Tumor Necrosis Factor Alpha by Human Peripheral Blood Neutrophils

This paper presents the study on TLR-mediated production of reactive oxygen species and tumor necrosis factor alpha by peripheral blood neutrophils in healthy donors stimulated with zymosan (TLR2/6 ligand), peptidoglycan (TLR2/1 ligand), and lipopolysaccharide (TLR4 ligand). Luminol- and lucigen-independent chemiluminescence was used to detect the production of reactive oxygen species. The concentration of tumor necrosis factor alpha was measured by enzyme immunoassay. The plots of dependence of the light sums of luminol- and lucigenin-dependent chemiluminescence on the concentration of each ligand were shaped as saturation curves. The comparison of the light sums of lucigenin-dependent chemiluminescence (the production of superoxide anion radical) and luminol-dependent chemiluminescence (the total production of reactive oxygen species) showed that the contribution of NADPH oxidase to the total TLR-mediated production of oxidants can reach 40–50%. Stimulation indices were calculated to compare the ability of TLR ligands to stimulate the production of reactive oxygen species and tumor necrosis factor alpha by neutrophils. It has been established that the activation of neutrophils with zymosan leads to higher (more than 8-fold) production of reactive oxygen species rather than production of tumor necrosis factor alpha. Unlike zymosan, lipopolysaccharide stimulated the production of tumor necrosis factor alpha to a greater extent (by more than 2 times) than the production of reactive oxygen species. Peptidoglycan takes an intermediate position between these ligands. Thus, the production of effector molecules (reactive oxygen species and tumor necrosis factor alpha) by human peripheral blood neutrophils depends on the nature of the TRL ligand.     

Mitochondria contribute to LPS-induced MAPK activation via uncoupling protein UCP2 in macrophages

The mitochondrion is a major organelle contributing to energy metabolism but also a main site of ROS (reactive oxygen species) production. LPS (lipopolysaccharide)-induced ROS signalling is a critical event in macrophage activation. In the present paper we report that part of LPS-mediated ROS signalling comes from mitochondria inside a signal amplification loop that enhances MAPK (mitogen-activated protein kinase) activation. More precisely, we have identified the inner mitochondrial membrane UCP2 (uncoupling protein 2) as a physiological brake on ROS signalling. Stimulation of murine bone marrow-derived macrophages by LPS quickly down-regulated UCP2 through the JNK (c-Jun N-terminal kinase) and p38 pathways. UCP2 down-regulation was shown to be necessary to increase mitochondrial ROS production in order to potentiate MAPK activation. Consistent with this, UCP2-deficient macrophages exhibit an enhanced inflammatory state characterized by increased nitric oxide production and elevated migration ability. Additionally, we found that the absence of UCP2 renders macrophages more resistant to nitric oxide-induced apoptosis.     

Natural antioxidants attenuate mycolactone toxicity to RAW 264.7 macrophages

Mycobacterium ulcerans produces a macrolide exotoxin, mycolactone which suppresses immune cells activity, is toxic to most cells and the key virulence factor in the pathogenesis of Buruli ulcer disease. Mycolactone is reported to mediate the production of reactive oxygen species in keratinocytes; cells that play critical role in wound healing. Increased levels of reactive oxygen species have been shown to disrupt the well-ordered process of wound repair; hence, the function of wound-healing cells such as macrophages, keratinocytes, and fibroblast could be impaired in the presence of the reactive oxygen species mediator, mycolactone. To ensure regeneration of tissues in chronic ulcers, with proper and timely healing of the wounds, natural antioxidants that can combat the effects of induced reactive oxygen species in wound-healing cells ought to be investigated. Reactive oxygen species activity was determined in mycolactone-treated RAW 264.7 macrophages and the scavenging ability of the antioxidants (ascorbic acid, gallic acid, and green tea kombucha) against mycolactone-induced reactive oxygen species (superoxide anions) was assessed using fluorescein probe (DCF-DA) and nitroblue tetrazolium dye. Cytotoxicity of the antioxidants, mycolactone, and the protective effect of the antioxidants on the cells upon treatment with mycolactone were determined using the Alamar blue assay. The expression levels of endogenous antioxidant enzyme genes (superoxide dismutase, catalase, and glutathione peroxidase) in response to mycolactone-mediated reactive oxygen species were determined using RT-qPCR. Mycolactone induced the production of reactive oxygen species in RAW 264.7 macrophages, and the resulting superoxide anions were scavenged by some of the antioxidants. The selected endogenous antioxidant enzyme genes in the macrophages were upregulated in the presence of the antioxidants and mycolactone. The exogenously supplied ascorbic acid and green tea kombucha offered moderate protection to the macrophages against the toxicity of mycolactone. We conclude that the results provide insights into alternate and adjunct therapeutic approaches in Buruli ulcer treatment, which could significantly attenuate the toxicity of the pathogenic factor; mycolactone.     

Modulation of the activation of extracellular signal-regulated kinase (ERK) and the production of inflammatory mediators by ADP-ribosylation inhibitors

ADP-ribosylation is involved in nuclear factor kappaB (NF-kappaB)-dependent gene expression induced by lipopolysaccharide in murine macrophages. Here we have investigated the mechanism by which ADP-ribosylation inhibitors block signaling pathways induced in macrophages. In RAW264.7 macrophages the inducers of NF-kappaB activate the production of reactive oxygen species and three mitogen-activated protein kinases (MAPK), the extracellular signal regulated kinase (ERK), the c-jun N-terminal kinase/stress-activated protein kinase (JNK), and p38. We demonstrate that ADP-ribosylation inhibitors specifically inhibit ERK MAPK activation and reduce the release of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha), IL-6 and nitrite.     

Modulation of the activity of sea bass (Dicentrarchus labrax) head-kidney macrophages by macrophage activating factor(s) and lipopolysaccharide

The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status.     

Heme Amplifies the Innate Immune Response to Microbial Molecules through Spleen Tyrosine Kinase (Syk)-dependent Reactive Oxygen Species Generation

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.     

Inflammation and macrophage modulation in adipose tissues

The adipose tissue is an active endocrine organ that harbours not only mature and developing adipocytes but also a wide array of immune cells, including macrophages, a key immune cell in determining metabolic functionality. With adipose tissue expansion, M1 pro‐inflammatory macrophage infiltration increases, activates other immune cells, and affects lipid trafficking and metabolism, in part via inhibiting mitochondrial function and increasing reactive oxygen species (ROS). The pro‐inflammatory cytokines produced and released interfere with insulin signalling, while inhibiting M1 macrophage activation improves systemic insulin sensitivity. In healthy adipose tissue, M2 alternative macrophages predominate and associate with enhanced lipid handling and mitochondrial function, anti‐inflammatory cytokine production, and inhibition of ROS. The sequence of events leading to macrophage infiltration and activation in adipose tissue remains incompletely understood but lipid handling of both macrophages and adipocytes appears to play a major role.     

Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages

Uncoupling protein-2 (UCP2) is a member of the inner mitochondrial membrane anion-carrier superfamily. Although mRNA for UCP2 is widely expressed, protein expression is detected in only a few cell types, including macrophages. UCP2 functions by an incompletely defined mechanism, to reduce reactive oxygen species production during mitochondrial electron transport. We observed that the abundance of UCP2 in macrophages increased rapidly in response to treatments (rotenone, antimycin A and diethyldithiocarbamate) that increased mitochondrial superoxide production, but not in response to superoxide produced outside the mitochondria or in response to H2O2. Increased UCP2 protein was not accompanied by increases in ucp2 gene expression or mRNA abundance, but was due to enhanced translational efficiency and possibly stabilization of UCP2 protein in the inner mitochondrial membrane. This was not dependent on mitochondrial membrane potential. These findings extend our understanding of the homeostatic function of UCP2 in regulating mitochondrial reactive oxygen production by identifying a feedback loop that senses mitochondrial reactive oxygen production and increases inner mitochondrial membrane UCP2 abundance and activity. Reactive oxygen species-induction of UCP2 may facilitate survival of macrophages and retention of function in widely variable tissue environments.     

Diphenyleneiodonium suppresses apoptosis in cerulein-stimulated pancreatic acinar cells

NADPH oxidase has been considered a major source of reactive oxygen species in phagocytic and non-phagocytic cells. Apoptosis linked to oxidative stress has been implicated in pancreatitis. Recently, we demonstrated that NADPH oxidase subunits Nox1, p27phox, p47phox, and p67phox are constitutively expressed in pancreatic acinar cells, which are activated by cerulein, a cholecystokinin analogue. Cerulein induces an acute and edematous form of pancreatitis. We investigated whether inhibition of NADPH oxidase by diphenyleneiodonium suppresses the production of reactive oxygen species and apoptosis by determining viable cell numbers, DNA fragmentation, TUNEL staining, caspase-3 activity, and the expression of apoptosis-inducing factor in pancreatic acinar AR42J cells stimulated with cerulein. Inhibition on NADPH oxidase by diphenyleneiodonium was assessed by the alterations in NADPH oxidase activity and translocation of the cytosolic subunits p67phox and p47phox to the membrane. Intracellular Ca2+ level was monitored to investigate the relationship between NADPH oxidase and Ca2+ in cells stimulated with cerulein. As a result, cerulein induced the activation of NADPH, increased production of reactive oxygen species, and apoptotic indices determined by the expression of apoptosis-inducing factor, caspase-3 activation, TUNEL staining, DNA fragmentation, and cell viability. Treatment with DPI inhibited cerulein-induced activation of NADPH oxidase, the production of reactive oxygen species, and apoptosis, but not the increase of intracellular Ca2+ levels in pancreatic acinar cells. These results demonstrate that the cerulein-induced increase in intracellular Ca2+ level may be an upstream event of NADPH oxidase activation. Diphenyleneiodonium, an NADPH oxidase inhibitor, inhibits the expression of apoptosis-inducing factor and caspase-3 activation, and thus apoptosis in pancreatic acinar cells.     

Imaging reactive oxygen species in asthma

Inflammatory processes in asthma are characterized by an infiltration of inflammatory cells including mononuclear phagocytes. It has been observed that mononuclear phagocytes, alveolar macrophages and blood monocytes, release higher quantities of reactive oxygen species in asthmatic patients than in healthy subjects. Chemiluminescence assays were developed to measure the superoxide anion and the other reactive oxygen species. The chemiluminescence response was first analysed with a luminometer, which made it possible to study cells in suspension before and after PMA-stimulation. Secondly a video-imaging camera was used in experiments on adherent cells before and after stimulation with PMA and/or specific stimulus IgE/anti-IgE. Both techniques showed that human alveolar macrophages, blood monocytes, PMN and lymphocytes were spontaneously primed in vivo and were more easily stimulated in asthma. Analysis of adherent cells in vitro may provide give information on the physiological condition of adherent cells in vivo.     

Vaccination of brushtail possums, Trichosurus vulpecula, with Bacille Calmette-Guerin induces T lymphocytes that reduce Mycobacterium bovis replication in alveolar macrophages via a contact-dependent/nitric oxide-independent mechanism

The permissiveness of alveolar macrophages from brushtail possums for the replication of Mycobacterium bovis was examined. Mycobacterium bovis replication was indirectly measured by assessing bacterial metabolism via the incorporation of [3-H]-uracil by bacilli released from lysed macrophages previously infected with mycobacteria. Alveolar macrophages allowed substantial replication of virulent M. bovis, in contrast to Bacille Calmette-Guerin (BCG) Pasteur, which replicated poorly. The addition of crude lymphokines enhanced the metabolic activity of phagocytosed M. bovis in possum macrophages. Possum lymphokines enhanced the ability of possum macrophages to generate reactive oxygen intermediates, measured by the reduction of nitroblue tetrazolium, which is indicative of an activation process. Similarly, the addition of recombinant possum TNF-alpha enhanced the permissiveness of alveolar macrophages for M. bovis. In contrast to mouse peritoneal macrophages, possum alveolar macrophages did not release significant levels of nitric oxide (NO) after stimulation with M. bovis and/or lymphokines. However, the uptake of virulent M. bovis by possum macrophages was associated with an enhanced ability of cells to release TNF-alpha, whereas very low levels of TNF-alpha were released after infection with BCG. The addition of a selective inhibitor of inducible NO synthase had no impact on the replication of M. bovis or BCG in possum macrophages in the presence or absence of lymphokines. Co-culturing infected possum alveolar macrophages with autologous blood mononuclear cells from BCG-vaccinated possums led to a significant decrease in the metabolic activity of intracellular M. bovis. This effect was contact dependent and NO independent and was mediated by a population of CD3+ cells. In addition, adding scavengers of reactive oxygen intermediates did not abrogate this phenomenon.     

Gold nanoparticles inhibited the receptor activator of nuclear factor-κb ligand (RANKL)-induced osteoclast formation by acting as an antioxidant

Gold nanoparticles inhibited osteoclast (OC) formation induced by the receptor activator of nuclear factor-κB ligand (RANKL) in bone marrow-derived macrophages (BMMs). This was accompanied by a decreased level of tartrate-resistant alkaline phosphatase (TRAP) and less activation of nuclear factor (NF)-κB. The nanoparticles also reduced the production of reactive oxygen species (ROS) in response to RANKL and upregulated RANKL-induced glutathione peroxidase-1 (Gpx-1), suggesting a role as an antioxidant in the BMM. The inhibitory effects on OC formation might have been due to elevated defense against oxidative stress.