Burst In Human Monocytes Research Articles

IFNγ regulates NAD+ metabolism to promote the respiratory burst in human monocytes

AbstractInterferon γ (IFNγ) is an essential and pleiotropic activator of human monocytes, but little is known about the changes in cellular metabolism required for IFNγ-induced activation. We sought to elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. We found that IFNγ increased oxygen consumption rates (OCR) in monocytes, indicative of reactive oxygen species generation by both mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Transcriptional profiling revealed that this oxidative phenotype was driven by IFNγ-induced reprogramming of NAD+ metabolism, which is dependent on nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. Consistent with this pathway, monocytes from patients with gain-of-function mutations in STAT1 demonstrated higher-than-normal OCR, whereas chemical or genetic disruption of mitochondrial complex I (rotenone treatment or Leigh syndrome patient monocytes) or NADPH oxidase (diphenyleneiodonium treatment or chronic granulomatous disease [CGD] patient monocytes) reduced OCR. Interestingly, inhibition of NAMPT in healthy monocytes completely abrogated the IFNγ-induced oxygen consumption, comparable to levels observed in CGD monocytes. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for IFNγ activation of human monocytes.

HIV-1 gp120 envelope glycoprotein determinants for cytokine burst in human monocytes.

The first step of HIV infection involves the interaction of the gp120 envelope glycoprotein to its receptor CD4, mainly expressed on CD4+ T cells. Besides its role on HIV-1 entry, the gp120 has been shown to be involved in the production of IL-1, IL-6, CCL20 and other innate response cytokines by bystander, uninfected CD4+ T cells and monocytes. However, the gp120 determinants involved in these functions are not completely understood. Whether signalling leading to cytokine production is due to CD4 or other receptors is still unclear. Enhanced chemokine receptor binding and subsequent clustering receptors may lead to cytokine production. By using a comprehensive panel of gp120 mutants, here we show that CD4 binding is mandatory for cytokine outburst in monocytes. Our data suggest that targeting monocytes in HIV-infected patients might decrease systemic inflammation and the potential tissue injury associated with the production of inflammatory cytokines. Understanding how gp120 mediates a cytokine burst in monocytes might help develop new approaches to improve the chronic inflammation that persists in these patients despite effective suppression of viremia by antiretroviral therapy.

Nutritionally relevant concentrations of resveratrol and hydroxytyrosol mitigate oxidative burst of human granulocytes and monocytes and the production of pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophages

The health benefits of bio-active phenolic compounds have been largely investigated in vitro at concentrations which exceed those reachable in vivo. We investigated and compared the anti-inflammatory effects of resveratrol, hydroxytyrosol and oleuropein at physiologically relevant concentrations by using in vitro models of inflammation.Human granulocytes and monocytes were stimulated with phorbol myristate acetate (PMA) and the ability of resveratrol, hydroxytyrosol and oleuropein to inhibit the oxidative burst and CD11b expression was measured. Nitric oxide (NO), prostaglandin E2 (PGE2) levels, COX-2, iNOS, TNFα, IL-1β and miR-146a expression and activation of the transcription factor Nrf2 were evaluated in macrophages RAW 264.7 stimulated with LPS (1μg/ml) for 18h, exposed to resveratrol, hydroxytyrosol and oleuropein (5 and 10μM). Synergistic effects were explored as well, together with the levels of PGE2, COX-2 and IL-1β expression in macrophages after 6h of LPS stimulation. PGE2 and COX-2 expression were also assessed on human monocytes.All the tested compounds inhibited granulocytes oxidative burst in a concentration dependent manner and CD11b expression was also significantly counteracted by resveratrol and hydroxytyrosol. The measurement of oxidative burst in human monocytes produced similar effects being resveratrol more active. Hydroxytyrosol and resveratrol inhibited the production of NO and PGE2 but did not reduce iNOS, TNFα or IL-1β gene expression in LPS-stimulated RAW 264.7 for 18h. Resveratrol slightly decreased COX-2 expression after 18h but not after 6h, but reduced PGE2 levels after 6h. Resveratrol and hydroxytyrosol 10μM induced NRf2 nuclear translocation and reduced miR-146a expression in LPS treated RAW 264.7.Overall, we reported an anti-inflammatory effect of resveratrol and hydroxytyrosol at low, nutritionally relevant concentrations, involving the inhibition of granulocytes and monocytes activation, the modulation of miR-146a expression and the activation of Nrf2. A regular dietary intake of resveratrol and hydroxytyrosol may be a useful complementary strategy to control inflammatory diseases.

Pneumocystis carinii-induced activation of the respiratory burst in human monocytes and macrophages.

Human monocytes and monocyte-derived macrophages were studied for their ability to phagocytose Pneumocystis carinii and produce superoxide (O2-) during the process. One x 10(6) freshly isolated monocytes, incubated with 0.1-3.75 x 10(6) P. carinii cysts, increased O2- production in a dose-related way. Antibodies were essential for the process since opsonized, but not unopsonized, pneumocysts induced O2- production significantly above the response obtained by lung tissue from rats (10.7 and 4.9 versus 3.0 fmol/cell per 90 min). The difference between pneumocysts opsonized in untreated versus complement-depleted serum was not significant (10.7 versus 12.6 fmol/cell per 90 min). Monocyte-derived macrophages also activated the respiratory burst when stimulated with pneumocysts, and this effect could be significantly increased, from 4.2 to 8.8 fmol/cell per 90 min, when cells were primed with interferon-gamma (IFN-gamma). Cells primed with IL-3 also increased O2- production, though to a lesser extent. In contrast, granulocyte-macrophage colony-stimulating factor (GM-CSF) had only a small effect on the respiratory burst in cells stimulated with P. carinii. Priming with IFN-gamma increased the rate of phagocytosis in macrophages. After incubation for 90 min or more, however, the percentage of cells with phagocytic vacuoles was only slightly higher in IFN-gamma-primed cells. When examined by electron microscopy (EM), most vacuoles contained partially or totally degraded pneumocysts. In conclusion, we have demonstrated the ability of monocytes and monocyte-derived macrophages to ingest and degrade pneumocysts, activating the respiratory burst during the process.

C-terminal fragment of α1-antitrypsin activates human monocytes to a pro-inflammatory state through interactions with the CD36 scavenger receptor and LDL receptor

Monocyte scavenger receptor, CD36 has been implicated in the pathogenesis of atherosclerosis as a major oxidised LDL receptor mediating lipid accumulation and foam cell formation. Previously, we found that treatment of monocyte cultures with the carboxyl terminal fragment of α1-antitrypsin (C-36) increases lipid binding and uptake, induces LDL receptor mRNA and CD36 receptor protein expression, and also significantly increases production of pro-inflammatory molecules. To assess the role of the CD36 receptor in proatherogenic monocyte activation by the C-36 fragment, we tested whether specific anti-CD36 receptor antibodies would block the effects of C-36 on monocyte activation. We find that pre-incubation of cells with anti-LDL and anti-CD36 receptor antibodies (10 μg/ml) blocks binding of 125I-C-36 by about 50%. Similarly, cells pre-incubated with oxidised LDL or native LDL at concentrations from 2.5 to 10 μg/ml showed a loss of 125I-C-36 binding (up to 49 and 57%) and uptake (up to 47 and 59.8%), respectively. In parallel experiments, monocytes were first incubated for 1 or 6 h with anti-CD36 antibodies (10 μg/ml) prior to adding C-36 peptide. Anti-CD36 antibodies suppressed C-36-induced production of gelatinase B, monocyte chemoattractant protein-1, interleukin-6 and cellular oxygen consumption to control levels, whereas levels of TNFα were unaffected. In contrast, saturation of LDL receptors with excess of anti-LDL (20 μg/ml) significantly inhibited C-36 induced TNFα levels. Results indicate that the C-36 peptide binds to both LDL and CD36 scavenger receptors which involves selective upregulation of pro-inflammatory molecules and activation of the respiratory burst in human monocytes. This also supports important roles for CD36 and LDL receptors in atherogenesis and suggests that blockade of CD36 receptor can be protective in pro-inflammatory activation of human monocytes.

Nonstructural 3 Protein of Hepatitis C Virus Triggers an Oxidative Burst in Human Monocytes via Activation of NADPH Oxidase

It has been shown that oxidative stress occurs in chronic hepatitis C. Release of reactive oxygen species (ROS) from sequestered phagocytes and activated resident macrophages represents the predominant component of oxidative stress in the liver. However, little is known about the ability of the monocyte to produce ROS in response to protein of hepatitis C virus. In this study, we investigated the ROS production in human monocytes stimulated by several viral proteins of hepatitis C virus. Human monocytes from healthy blood donors were incubated with recombinant viral protein: Core, NS3, NS4, and NS5. ROS production was measured by chemiluminescence. Only NS3 triggered ROS production in human monocytes. Generated ROS were mainly the anion superoxide. NS3 also induced a rapid and transient increase in intracellular calcium concentration measured by a video digital microscopy technique. By using different metabolic inhibitors, we showed that ROS production requires calcium influx, tyrosine kinases, and the stress-activated protein kinase, p38. The study of p47(PHOX) phosphorylation and translocation showed that NADPH oxidase was activated and involved in ROS production induced by NS3. In a second experiment, NS3 inhibited the oxidative burst induced by phorbol 12-myristate 13-acetate. These results indicate that NS3 activates NADPH oxidase and modulates ROS production, which may be involved in the natural history of hepatitis C infection.

Non-structural 3 protein of hepatitis C virus triggers an oxidative burst in human monocytes via activation of NADPH oxidase

Non-structural 3 protein of hepatitis C virus triggers an oxidative burst in human monocytes via activation of NADPH oxidase

Non-structural 3 protein of hepatitis C virus triggers an oxidative burst in human monocytes via activation of NADPH oxidase

Non-structural 3 protein of hepatitis C virus triggers an oxidative burst in human monocytes via activation of NADPH oxidase

The phagocytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione.

During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20-30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by gamma-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 microM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.

Protein S-thiolation and dethiolation during the respiratory burst in human monocytes. A reversible post-translational modification with potential for buffering the effects of oxidant stress.

Stimulation of the respiratory burst in mouse macrophages or human neutrophils results in the formation of disulfide bonds between low m.w. thiols and sulfhydryl groups on specific cytosolic proteins (S-thiolation). S-thiolation is reversible in certain chemical systems. The aim of the present study was to analyze the dynamic nature of this process in human monocytes under physiologic conditions. We report here that the extent of S-thiolation and the rate of respiratory burst stimulated by opsonized zymosan or phorbol diester increased for 10 to 20 min and then declined (dethiolation) in close association. Individual proteins underwent S-thiolation and dethiolation at different rates. H2O--appeared particularly effective in mediating S-thiolation, based on inhibition of S-thiolation by added catalase and accentuation by azide, which inhibits cellular catalase. S-thiolation did not occur in stimulated monocytes from patients with chronic granulomatous disease. The addition of H2O2 to monocytes or lymphocytes induced rapid S-thiolation (1 to 3 min); a subsequent dethiolation returned most of the proteins to baseline by 15 to 30 min. At 0 degrees C and after addition of 1,3-bis-(2-chloroethyl)-1-nitrosourea, there was effective S-thiolation on exposure to H2O2, but dethiolation was inhibited, suggesting a possible role for glutathione (GSH)/thioredoxin reductase systems in this process. GSH was determined to be the most abundant low m.w. thiol bound to S-thiolated proteins, but gamma-glutamylcysteine and cysteine were also bound. The time of maximal reduction in cytosolic GSH during the respiratory burst (10 min) coincided with the time at which protein-bound GSH was highest. S-thiolation-dethiolation represents a reversible post-translational modification that could protect cellular proteins from irreversible oxidative damage.

Simultaneous evaluation of phagocytosis and FcγR-mediated oxidative burst in human monocytes by a simple flow cytometry method

In this study we describe a fast, simple, and objective flow cytometry method to quantify simultaneously phagocytosis and FcγR-mediated oxidative burst in human monocytes. Peripheral blood monocytes isolated by elutriation were sequentially incubated with 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) and sheep erythrocytes opsonized with rabbit IgG (SRBC-IgG), and then analysed by flow cytometry. Two parameters were studied simultaneously in the same cell population: forward scatter modifications (grade of phagocytosis) and fluorescence intensity of DCFH oxidation (index of oxidative burst). We found a statistically significant correlation ( r = 0.96274) between the forward scatter increase and the amount of phagocytosis as determined by light microscopy. We also found a significant correlation between the oxidative burst and phagocytosis studied both microscopically ( r = 0.7714) and by flow cytometry ( r = 0.78056). As the presence of DCFH did not impair monocyte phagocytosis, we conclude that this simple method could be a useful tool in functional studies of monocytes.

Suppression of superoxide release from human monocytes by somatostatin-related peptides

Somatostatin and octreotide share with vasoactive intestinal peptide the property of having an inhibitory effect on leukocyte functions. While there are studies reporting the inhibitory effect of the latter on respiratory burst in human monocytes, no such reports are available about similar inhibitory effects of the former. The aim of the present study was to investigate such effects of somatostatin and octreotide on human monocytes. Release of superoxide anion from monocytes was measured by superoxide dismutase-inhibitable reduction of cytochrome c in vitro. Somatostatin 1–14, somatostatin 1–28 and octreotide inhibited release of superoxide anion from stimulated monocytes. Formylpeptide-stimulated reduction of cytochrome c was inhibited by 1 μmol/l of octreotide and somatostatin 1–14 by about 50% and 35%, respectively. The effect was dose-dependent with half-maximal effective peptide concentrations at about 10 nmol/l. Somatostatin 1–28, which is the major form found in circulating plasma, also antagonized formylpeptide-stimulated respiratory burst activity; when directly compared to the effect of 1 μmol/l of somatostatin 1–14, somatostatin 1–28 was significantly more active ( P<0.05). Our observations suggest that somatostatin-related peptides have a regulatory role in oxygen radical metabolism and a mediator role in the neuro-immune axis.

Recombinant human MCP-1/JE induces chemotaxis, calcium flux, and the respiratory burst in human monocytes

The JE gene was first described as a platelet-derived growth factor (PDGF)-inducible gene in mouse 3T3 cells. The human homologue of JE encodes a protein whose predicted amino acid sequence is identical to that of the monocyte chemoattractant MCP-1 (also called MCAF and SMC- CF), which belongs to a recently identified family of small secretory proteins with cytokine properties. We purified recombinant human MCP- 1/JE (hMCP-1/JE) produced in COS cells and demonstrated that it is chemotactic for human monocytes with a specific activity similar to natural MCP-1. In addition, pure recombinant hMCP-1/JE stimulates monocytes, inducing an increase in cytosolic free calcium and the respiratory burst, but is completely inactive on human neutrophils. These results help to define functionally a well-known growth factor- inducible gene and a member of a new family of cytokines.

Stimulation of oxidative burst in human monocytes by lipoteichoic acids

Lipoteichoic acid isolated from Streptococcus faecalis or Streptococcus pyogenes caused direct activation of the respiratory burst in human peripheral blood monocytes. This activity appears to be related to the ability of lipoteichoic acid to bind to the monocyte membrane and trigger the polarization of receptors (capping).

Effect of VIP on the respiratory burst in human monocytes ex vivo during prolonged strain and energy deficiency

VIP-stimulated cyclic AMP production and VIP effect on the production of reactive oxygen compounds in human monocytes activated by serum opsonized zymosan (respiratory burst) were studied during a ranger training course lasting for five days with almost continuous physical activity, and deficiency of sleep and energy. Respiratory burst was inhibited and cyclic AMP production was stimulated by VIP on all days. Maximum cyclic AMP production stimulated by VIP (0.1 μM) on the day of control was 148.6% of basal, and 255.3%, 213.8%, 218.9% and 198.7% on Days 1, 2, 3 and 5. Maximum inhibition was observed 20 min after addition of the peptide on the day of control, after 5 min on Days 1, 2 and 3, and after 10 min on Day 5. Inhibition at the 5-min time point was 33.1% on the day of control, and 34.7%, 53.6%, 53.3% and 36.2% on the different days during the training course. The observed increment in VIP effect adds to prior reported data about increased VIP secretion during the training course, and may indicate enhanced physiological significance of VIP during stress.

Vasoactive intestinal peptide inhibits the respiratory burst in human monocytes by a cyclic AMP-mediated mechanism

The neuropeptide vasoactive intestinal peptide (VIP) was shown to inhibit the production of reactive oxygen compounds (respiratory burst) in monocytes activated by serum opsonized zymosan. Reactive oxygen compounds are of importance for host defence against micro-organisms and cancer, but normal tissues are also susceptible to damage from these reactive substances. Maximum inhibition of respiratory burst was 40% by 0.1 μM VIP (ID 100), while ID 50 for the VIP effect was 0.36 nM VIP. PHM-27, closely related to VIP on the basis of the amino acid sequence, inhibited the respiratory burst with much lower potency ( ID 50 = 60 nM , ID 100 = 1 μ M ). Secretin, related to VIP and PHM-27, produced no effect on the respiratory burst in monocytes. VIP was also shown to stimulate the cyclic AMP production in monocytes in a dose dependent manner. IBMX and forskolin, as well as the cyclic AMP analogue butyryl cyclic AMP were shown to produce an inhibition of the respiratory burst. In conclusion, this study showed that VIP inhibited the respiratory burst in monocytes by a cyclic AMP-mediated mechanism, and serves to establish still another role for VIP as a mediator in the neuro-immune axis.

Activation of the oxidative burst in human monocytes is associated with inhibition of methionine-dependent methylation of neutral lipids and phospholipids.

Chemotaxis and generation of the oxidative burst by phagocytes are among the biological functions thought to require methylation reaction(s) for their expression. The present study investigated the effect of different stimuli of the oxidative burst on lipid methylation by human elutriated monocytes as measured by methyl group incorporation from [methyl-3H]methionine into both phospholipid and neutral lipid extracts. Normal monocytes, incubated at 37 degrees C for 1 h with 2 microM methionine, incorporated 10.2-fmol/10(6) cells and 73.6-fmol/10(6) cells of methyl groups into neutral lipids and phospholipids, respectively. Stimulators of the respiratory burst, such as the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate, and the calcium ionophore, A23187, decreased the incorporation of methyl groups into both neutral lipids and phospholipids in a similar manner. Increasing the concentration of methionine in the medium reversed or attenuated the inhibition achieved at lower levels. An inverse relationship existed between the degree of methylation and the extent of stimulation of the oxidative burst, measured as superoxide anion (O-2) release. Stimulated monocytes oxidized methionine to methionine sulfoxide (which cannot act as a methyl-donor), and this was dependent on activation of the respiratory burst. Elimination of the accumulated methionine sulfoxide by replacement of the medium or by prevention of extracellular methionine oxidation by catalase did not effectively restore the normal level of methylation in stimulated cells, and the reduced methylation was not primarily related to a defective methionine uptake by stimulated monocytes. These data suggest that intracellular events related to activation of the respiratory burst are responsible for the decreased lipid methylation in stimulated cells, possibly by their leading to intracellular formation of methionine sulfoxide and by their limiting the availability of methyl-donor. This mechanism may be of potential relevance for the expression of biological functions where methionine-dependent reactions are involved.

Measurement of the respiratory burst in human monocytes and polymorphonuclear leukocytes by nitro blue tetrazolium reduction and chemiluminescence

We compared measurements of chemiluminescence (CL) assessing the rate of production of oxygen intermediates at a given instance, and of nitro blue tetrazolium (NBT) reduction detecting total superoxide produced during the assay period, for the assessment of the respiratory burst of both human monocytes (Mø) and polymorphonuclear leukocytes (PMN). In the CL experiments, opsonized and non-opsonized zymosan was used to stimulate peripheral blood Mø and PMN. Opsonized zymosan yielded an earlier and 2-fold higher peak response than non-opsonized zymosan. The stimulatory action of zymosan in CL varied with the time of incubation and depended on the concentration of cells and zymosan. No light generation was observed in the absence of viable cells. In contrast to NBT reduction, incubation of PMN with dextran sulfate did not result in increased light generation but rather in a quenching of the response in CL. Opsonized zymosan also yielded a higher level on NBT reduction than non-opsonized zymosan. Comparing NBT reduction with CL in 21 healthy individuals, with 2 × 10 5 Mø and 2.5 × 10 5 PMN for NBT and 5 × 10 5 PMN for CL tests, we found that NBT brings about spontaneous oxidative metabolism, possibly reflecting the intracellular compartment, the chemiluminescent response of resting cells being only marginal. The data suggest higher sensitivity of the CL method. These results provide useful comparative data for 2-established methods used to document the respiratory burst in phagocytic cells.

Human β 1h globulin and C3b stimulate the respiratory burst in human monocytes

Human β 1h globulin and C3b stimulate the respiratory burst in human monocytes