Role of macrophage oxidative burst in the action of anthrax lethal toxin.

BACKGROUND: Major symptoms and death from systemic Bacillus anthracis infections are mediated by the action of the pathogen's lethal toxin on host macrophages. High levels of the toxin are cytolytic to macrophages, whereas low levels stimulate these cells to produce cytokines (interleukin-1 beta and tumor necrosis factor-alpha), which induce systemic shock and death. MATERIALS AND METHODS: Experiments were performed to assess the possibility that the oxidative burst may be involved in one or both of lethal toxin's effects on macrophages. Toximediated cell lysis, superoxide anion and cytokine production were measured. Effects of antioxidants and macrophage mutations were examined. RESULTS: RAW264.7 murine macrophages treated with high levels of toxin released large amounts of superoxide anion, beginning at about 1 hr, which correlates with the onset of cytolysis. Cytolysis could be blocked with various exogenous antioxidants or with N-acetyl-L-cysteine and methionine, which promote production of the endogenous antioxidant, glutathione. Mutant murine macrophage lines deficient in production of reactive oxygen intermediates (ROIs) were relatively insensitive to the lytic effects of the toxin, whereas a line with increased oxidative burst potential showed elevated sensitivity. Also, cultured blood monocyte-derived macrophages from a patient with Chronic Granulomatous Disease, a disorder in which the phagocyte's oxidative burst is disabled, were totally resistant to toxin, in contrast to control monocytes. CONCLUSIONS: These results imply that the cytolytic effect of the toxin is mediated by ROIs. Additionally, cytokine production and consequent pathologies showed partial dependence on macrophage ROIs. Antioxidants moderately inhibited toxin-induced cytokine production in vitro, and BALB/c mice pretreated with N-acetyl-L-cysteine or mepacrine showed partial protection against lethal toxin. Thus ROIs are involved in both the cytolytic action of anthrax lethal toxin and the overall pathologic process in vivo.     

Genetic control of neutrophil superoxide production in diabetes-resistant ALR/Lt mice

The neutrophil oxidative burst reaction differentiates ALR/Lt mice, known for an unusual systemic elevation of antioxidant defenses, from ALS/Lt mice, a related strain known for reduced ability to withstand oxidative stress. Neutrophils from marrow of ALS mice produced a normal neutrophil oxidative burst following phorbol ester stimulation. In contrast, ALR mice exhibited a markedly suppressed superoxide burst. F1 progeny from reciprocal outcrosses between ALR and ALS mice exhibited an intermediate burst level, higher than ALR but significantly lower than ALS. To elucidate the genetic basis for this strain difference, F1 mice were backcrossed to ALS mice, and marrow neutrophils isolated from the progeny were phenotyped for oxidative burst capacity. A genome-wide sweep using polymorphic markers distinguishing the two parental strains was performed to map the trait. A 1:1 phenotypic distribution was observed, and a locus (Suppressor of superoxide production, Susp) controlling this phenotype was mapped to Chromosome 3 near D3Mit241 at 33.1 cM. This locus probably represents an important regulatory element in the overall ALR strain resistance to oxidative stress, since diminished ability to mount a neutrophil burst in backcross segregants correlated with elevated hepatic superoxide dismutase 1 (SOD1) activity, an ALR strain characteristic.     

Effect of a short-term diet and exercise intervention on oxidative stress, inflammation, MMP-9, and monocyte chemotactic activity in men with metabolic syndrome factors.

The present study was designed to examine the effects of lifestyle modification on key contributing factors to atherogenesis, including oxidative stress, inflammation, chemotaxis, and cell adhesion. Obese men (n = 31), 15 of whom had metabolic syndrome, were placed on a high-fiber, low-fat diet in a 3-wk residential program where food was provided ad libitum and daily aerobic exercise was performed. In each subject, pre- and postintervention fasting blood was drawn for circulating levels of serum lipids, glucose and insulin (for estimation of insulin sensitivity), oxidative stress-generating enzyme myeloperoxidase and marker 8-isoprostaglandin F2alpha, the inflammatory protein C-reactive protein, soluble ICAM-1 as an indicator of endothelial activation, sP-selectin as a marker of platelet activation, the chemokine macrophage inflammatory protein-1alpha, and total matrix metalloproteinase-9. Using subject sera and human aortic endothelial cell culture systems, we measured VCAM-1 cell surface abundance and monocyte chemotactic protein-1, nitric oxide, superoxide, and hydrogen peroxide production in vitro by fluorometric detection. Also determined in vitro was serum-induced, monocyte adhesion and monocyte chemotactic activity. After 3 wk, significant reductions (P < 0.05) in body mass index, all serum lipids and lipid ratios, fasting glucose, insulin, homeostasis model assessment for insulin resistance, myeloperoxidase, 8-isoprostaglandin F2alpha, C-reactive protein, soluble ICAM-1, soluble P-selectin, macrophage inflammatory protein-1alpha, and matrix metalloproteinase-9 were noted. In vitro, serum-stimulated cellular VCAM-1 expression, monocyte chemotactic protein-1 production, and fluorometric detection of superoxide and hydrogen peroxide production decreased, whereas a concomitant increase in NO production was noted (all P < 0.01). Additionally, both monocyte adhesion (P < 0.05) and MCA (P < 0.01) decreased. Nine of 15 were no longer positive for metabolic syndrome postintervention. Intensive lifestyle modification may ameliorate novel coronary artery disease risk factors in men with metabolic syndrome factors before reversal of obesity.     

The effects of 3-methylcholanthrene on macrophage respiratory burst and biotransformation activities in the common carp (Cyprinus carpio L.)

The sensitivity of phagocytic cell function as a bioindicator of pollution stress by polycyclic aromatic hydrocarbons was evaluated in the common carp (Cyprinus carpio L). The time course response of the head-kidney macrophage respiratory burst was measured 1, 2, 3, 5 and 7 days after intraperitoneal injection of a prototypical Cyp 1A inducer (3-methylcholanthrene). This immune activity was compared to the rate of induction of total cytochrome P450, ethoxyresorufin O-deethylase activity (EROD) and glutathione S-transferase activity (GST) in the liver and head-kidney. 3-methylcholanthrene (40 mg kg(-1)) caused a rapid increase in the macrophage respiratory burst. This response was maximal at day 3 post exposure and coincided with maximum induction of cytochrome P450 and EROD activity in liver and head-kidney. Moreover, alpha-naphtoflavone, which functions as both an Ah receptor antagonist and an inhibitor of cytochrome P450 1A activity, reversed the 3-methylcholanthrene induction of immune and enzymatic parameters measured, suggesting metabolic processes. Taken together these results suggest that the induction of macrophage oxidative function may be an equally sensitive marker of exposure to polycyclic aromatic hydrocarbon as the induction of biotransformation activities and confirm that responses mediated by the Ah receptor are similar, if not identical, to those of mammals.     

The use of acetylated cytochrome c in detecting superoxide anion production in rabbit alveolar macrophages

Polymorphonuclear phagocytes have been shown to undergo marked alteration in oxidative metabolism during phagocytosis. These alterations, collectively known as the "respiratory burst", include increased glucose oxidation through the hexose monophosphate shunt (1), increased oxygen consumption (1), and increased superoxide (O-2)3 (2) and H2O2 production (3). Similar metabolic events have also been shown to occur in the rabbit alveolar macrophage (AM). There is consistent evidence that the macrophage undergoes increased oxygen consumption (4-6) and hexose monophosphate shunt activity (4-9) upon phagocytosis. There are conflicting data, however, concerning the ability of the macrophage to produce O-2. Some studies suggest that macrophages are incapable of producing measurable amounts of O-2 upon phagocytosis (7, 10-12). Other studies, however, suggest that macrophages are indeed capable of producing substantial amounts of O-2 during phagocytosis (8, 13-15). This study was designed to resolve the discrepancies in the literature concerning O-2 production in macrophages.     

A microfluidic device to establish concentration gradients using reagent density differences

Microfabrication has become widely utilized to generate controlled microenvironments that establish chemical concentration gradients for a variety of engineering and life science applications. To establish microfluidic flow, the majority of existing devices rely upon additional facilities, equipment, and excessive reagent supplies, which together limit device portability as well as constrain device usage to individuals trained in technological disciplines. The current work presents our laboratory-developed bridged μLane system, which is a stand-alone device that runs via conventional pipette loading and can operate for several days without need of external machinery or additional reagent volumes. The bridged μLane is a two-layer polydimethylsiloxane microfluidic device that is able to establish controlled chemical concentration gradients over time by relying solely upon differences in reagent densities. Fluorescently labeled Dextran was used to validate the design and operation of the bridged μLane by evaluating experimentally measured transport properties within the microsystem in conjunction with numerical simulations and established mathematical transport models. Results demonstrate how the bridged μLane system was used to generate spatial concentration gradients that resulted in an experimentally measured Dextran diffusivity of (0.82 ± 0.01) × 10(-6) cm(2)/s.     

Infection of differentiated U937 cells by Salmonella typhimurium: Absence of correlation between oxidative burst and antimicrobial defence

Abstract The human histiocytic lymphoma cell line U937 can be induced to differentiate along the monocyte/macrophage pathway by either phorbol myristate acetate (PMA) or by the combination of retinoïc acid (RA) and 1,25-dihydroxyvitamin D3 (VD). U937 cells treated with either PMA or RA/VD were able to phagocytose Salmonella typhimurium in the presence of non-immune human serum. However, only cells differentiated by RA/VD were capable of developing an oxidative metabolic burst in response to infection. Since the oxidative burst is considered to be a potent antimicrobial mechanism, we investigated its effect on S. typhimurium . The oxidative burst failed to affect either the viability or the multiplication of S. typhimurium suggesting that if plays only a minor role in the host defence against S. typhimurium .     

Chronic Household Air Pollution Exposure Is Associated with Impaired Alveolar Macrophage Function in Malawian Non-Smokers

Background

Household air pollution in low income countries is an important cause of mortality from respiratory infection. We hypothesised that chronic smoke exposure is detrimental to alveolar macrophage function, causing failure of innate immunity. We report the relationship between macrophage function and prior smoke exposure in healthy Malawians.

Methods

Healthy subjects exposed daily to cooking smoke at home volunteered for bronchoalveolar lavage. Alveolar macrophage particulate content was measured as a known correlate of smoke exposure. Phagocytosis and intraphagosomal function (oxidative burst and proteolysis) were measured by a flow cytometric assay. Cytokine responses in macrophages were compared following re-exposure in vitro to wood smoke, before and after glutathione depletion.

Results

Volunteers had a range of alveolar macrophage particulate loading. The macrophage capacity for phagosomal oxidative burst was negatively associated with alveolar macrophage particulate content (n = 29, r² = 0.16, p = 0.033), but phagocytosis per se and proteolytic function were unaffected. High particulate content was associated with lower baseline CXCL8 release (ratio 0.51, CI 0.29–0.89) and lower final concentrations on re-exposure to smoke in vitro (ratio 0.58, CI 0.34–0.97). Glutathione depletion augmented CXCL8 responses by 1.49x (CI 1.02–2.17) compared with wood smoke alone. This response was specific to smoke as macrophages response to LPS were not modulated by glutathione.

Conclusion

Chronic smoke exposure is associated with reduced human macrophage oxidative burst, and dampened inflammatory cytokine responses. These are critical processes in lung defence against infection and likely to underpin the relationship between air pollution and pneumonia.     

Tetrahydrophthalazine derivative "sodium nucleinate" exert its anti-inflammatory effects through inhibition of oxidative burst in human monocytes

We described the use of a new chemical substance Sodium nucleinate (SN) as an immunomodulatory substance exhibiting antiinflammatory properties. Sodium nucleinate (SN) registrated in Russian Federation as Tamerit, is 2-amino-1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt dihydrate, derivative of well known chemical substance luminol. To comprehend the mechanisms of SN immunomodulatory activity, we examined the SN modulation of the oxidative burst responses of whole blood human monocytes and polimorphonuclear cells (PMC) stimulated with phorbol 12-myristate 13-acetate (PMA) or E. coli suspension in vitro. SN did not inhibit the proportion of neutrophils and monocytes phagocytosing E. coli. Oxidative burst responses of monocytes stimulated with PMA were strongly inhibited at SN concentration ranging from 10-500 mg/ml, less efficient inhibitor was SN in E. coli stimulated monocytes (inhibition range was from 50-500 mg/ml SN). SN inhibited PMC oxidative burst only in range 100-500 mg/ml SN. In conclusion, we found SN as an efficient inhibitor of oxidative burst in monocytes. Since ROS generation in monocytes/macrophages has been found to be important for LPS-driven production of several proinflammatory cytokines, SN may exsert its antiinflammatory effects through monocyte/macrophage oxidative burst inhibition.     

Regulation of macrophage physiology by L-arginine: role of the oxidative L-arginine deiminase pathway

The L-arginine content of the extracellular fluid in sites of predominant macrophage infiltration is reduced below plasma levels due to the activity of macrophage-derived arginase. Investigation of the effects of altered L-arginine availability on macrophage physiology reveals that culture of rat peritoneal macrophages in media containing L-arginine in the concentrations present in inflammatory lesions (less than 0.1 mM) enhances activation-associated functions. In contrast, culture in the higher L-arginine concentrations found in standard tissue culture media (0.4 to 1.2 mM) suppresses most macrophage functions (superoxide production, phagocytosis, and protein synthesis). An exception is the tumor cytotoxicity of Corynebacterium parvum-elicited macrophages which is enhanced by culture in supraphysiologic concentrations of L-arginine. Work reported here investigated the mechanisms for these L-arginine-dependent effects and, more specifically, the role of the recently described oxidative L-arginine deiminase pathway in the regulation of macrophage physiology. Overnight culture of resident or C. parvum-elicited peritoneal macrophages in media containing increasing concentrations of L-arginine (6 microM to 1 mM) resulted in: inhibition of electron transport chain activity (resident and C. parvum-elicited macrophages), increased lactate production (resident macrophages), and decreased ATP content (resident and C. parvum-elicited macrophages). In line with these findings, viability was markedly decreased after 2 days of culture when the initial L-arginine concentration was greater than or equal to 0.1 mM. As shown before, increasing media concentrations of L-arginine were associated with suppression of superoxide production and cytotoxicity in resident macrophages, and with reduced superoxide production and increased cytotoxicity in C. parvum-elicited macrophages. All L-arginine-dependent metabolic and functional alterations, as well as the loss of viability, were prevented by NG-monomethyl-L-arginine, a specific inhibitor of the oxidative L-arginine deiminase pathway. These results demonstrate that flux of L-arginine through the oxidative L-arginine deiminase pathway results in the inhibition of oxidative metabolism in rat macrophages. This metabolic inhibition may, through alterations in the macrophage high energy phosphate stores, mediate the suppression of cell functions and result ultimately in cell death.     

Identification of the bacterial superoxide dismutase (SodM) as plant-inducible elicitor of an oxidative burst reaction in tobacco cell suspension cultures

Three of the most abundant proteins (OmpW, MopB and SodM) of the extracellular proteome of Xanthomonas campestris pv. campestris were analysed in a luminol-based oxidative burst assay to identify novel pathogen-associated molecular patterns (PAMP). Tobacco cell suspension cultures were used as a model system to monitor elicitor induced plant defence reaction. The candidate proteins were isolated from two-dimensional gels prior to application to the oxidative burst assay. The superoxide dismutase (SodM) was the only isolated protein that could elicit a notable hydrogen peroxide (H2O2) production in tobacco cell cultures indicating the initiation of plant defence. An alignment of the SodM sequences from X. campestris pv. campestris and Escherichia coli revealed 55.7% identity and 29% of the sequence were substitutions for amino acids with similar physico-chemical properties. By using a commercially available purified E. coli derived SodM preparation, it was possible to show that the amino acid sequence of this protein is responsible for the elicitation of an oxidative burst reaction in the tobacco cell culture model. This suggests that the bacterial superoxide dismutase is a novel pathogen-associated molecular pattern. The minimal elicitor active sequence, however, is still elusive.     

Oxidant Trade-Offs in Immunity: An Experimental Test in a Lizard

Immune system functioning and maintenance entails costs which may limit investment into other processes such as reproduction. Yet, the proximate mechanisms and ‘currencies’ mediating the costs of immune responses remain elusive. In vertebrates, up-regulation of the innate immune system is associated with rapid phagocytic production of pro-oxidant molecules (so-called ‘oxidative burst’ responses). Oxidative burst responses are intended to eliminate pathogens but may also constitute an immunopathological risk as they may induce oxidative damage to self cells. To minimize the risk of infection and, at the same time, damage to self, oxidative burst activity must be carefully balanced. The current levels of pro- and antioxidants (i.e. the individual oxidative state) is likely to be a critical factor affecting this balance, but this has not yet been evaluated. Here, we perform an experiment on wild-caught painted dragon lizards (Ctenophorus pictus) to examine how the strength of immune-stimulated oxidative burst responses of phagocytes in whole blood relates to individual oxidative status under control conditions and during an in vivo immune challenge with Escherichia coli lipopolysaccharide (LPS). Under control conditions, oxidative burst responses were not predicted by the oxidative status of the lizards. LPS-injected individuals showed a strong increase in pro-oxidant levels and a strong decrease in antioxidant levels compared to control individuals demonstrating a shift in the pro-/antioxidant balance. Oxidative burst responses in LPS-injected lizards were positively related to post-challenge extracellular pro-oxidants (reflecting the level of cell activation) and negatively related to pre-challenge levels of mitochondrial superoxide (suggesting an immunoregulatory effect of this pro-oxidant). LPS-challenged males had higher oxidative burst responses than females, and in females oxidative burst responses seemed to depend more strongly on antioxidant status than in males. Our results confirm the idea that oxidative state may constrain the activity of the innate immune system. These constraints may have important consequences for the way selection acts on pro-oxidant generating processes.     

Macrophage cell line RAW264.7 but not P-388D1 is an appropriate in vitro-model for studying oxidative burst as well as cytokine production in context of fatty acid enrichment

Oxidative burst and cytokines synthesis by macrophages is a crucial point for successful pathogen defense. However, macrophage cell lines commonly used in inflammatory research differ in their responses to external stimuli. Thus, there is the necessity to carefully characterize the cells before experimental usage. In this study we investigated the applicability of two widely-used macrophage cell lines, RAW264.7 and P-388D1, for studying oxidative burst and cytokine synthesis. Cells were tested for NADPH oxidase activity, iNOS-mRNA levels, and the release of NO, TNF-α, IL-6 and IL-10. Stimulation of RAW264.7 triggered oxidative burst as well as synthesis of TNF-α, IL-6 and IL-10. In contrast, following stimulation P-388D1 produced TNF-α and IL-6 only. Our findings confirm the relevance of cell line selection for reliability of in vitro-experiments. Moreover, the results approve RAW264.7 cells to be a suitable model to investigate the modulation capability of macrophages e.g. in context of fatty acid supplementation.     

Activation-induced depletion of protein kinase C alpha provokes desensitization of monocytes/macrophages in sepsis

Sepsis accounts for the majority of fatal casualties in critically ill patients, because extensive research failed to significantly improve appropriate therapy strategies. Thus, understanding molecular mechanisms initiating the septic phenotype is important. Symptoms of septic disease are often associated with monocyte/macrophage desensitization. In this study, we provide evidence that a desensitized cellular phenotype is characterized by an attenuated oxidative burst. Inhibition of the oxidative burst and depletion of protein kinase C alpha (PKC alpha) were correlated in septic patients. To prove that PKC alpha down-regulation indeed attenuated the oxidative burst, we set up a cell culture model to mimic desensitized monocytes/macrophages. We show that LPS/IFN-gamma-treatment of RAW264.7 and U937 cells lowered PKC alpha expression and went on to confirm these data in primary human monocyte-derived macrophages. To establish a role of PKC alpha in cellular desensitization, we overexpressed PKC alpha in RAW264.7 and U937 cells and tested for phorbolester-elicited superoxide formation following LPS/IFN-gamma-pretreatment. Inhibition of the oxidative burst, i.e., cellular desensitization, was clearly reversed in cells overexpressing PKC alpha, pointing to PKC alpha as the major transmitter in eliciting the oxidative burst in monocytes/macrophages. However, PKC alpha inactivation by transfecting a catalytically inactive PKC alpha mutant attenuated superoxide formation. We suggest that depletion of PKC alpha in monocytes from septic patients contributes to cellular desensitization, giving rise to clinical symptoms of sepsis.     

Rac-dependent feedforward autoactivation of NOX2 leads to oxidative burst

NADPH oxidase 2 (NOX2) produces the superoxide anion radical (O₂⁻), which has functions in both cell signaling and immune defense. NOX2 is a multimeric-protein complex consisting of several protein subunits including the GTPase Rac. NOX2 uniquely facilitates an oxidative burst, which is described by initially slow O₂⁻ production, which increases over time. The NOX2 oxidative burst is considered critical to immune defense because it enables expedited O₂⁻ production in response to infections. However, the mechanism of the initiation and progression of this oxidative burst and its implications for regulation of NOX2 have not been clarified. In this study, we show that the NOX2 oxidative burst is a result of autoactivation of NOX2 coupled with the redox function of Rac. NOX2 autoactivation begins when active Rac triggers NOX2 activation and the subsequent production of O₂⁻, which in turn activates redox-sensitive Rac. This activated Rac further activates NOX2, amplifying the feedforward cycle and resulting in a NOX2-mediated oxidative burst. Using mutagenesis-based kinetic and cell analyses, we show that enzymatic activation of Rac is exclusively responsible for production of the active Rac trigger that initiates NOX2 autoactivation, whereas redox-mediated Rac activation is the main driving force of NOX2 autoactivation and contributes to generation of ∼98% of the active NOX2 in cells. The results of this study provide insight into the regulation of NOX2 function, which could be used to develop therapeutics to control immune responses associated with dysregulated NOX2 oxidative bursts.     

Biochemical and functional characterisation of macrophage stimulating factors secreted by mitogen-induced goldfish kidney leucocytes

Mitogen-stimulated goldfish kidney leucocytes secrete a number of different macrophage activation factors (MAF) that induce profound physiological changes in macrophages. MAF produced by goldfish kidney leucocytes was characterised using fast performance liquid chromatography (FPLC) and bioassays that measured MAF-induced respiratory burst (RB) and nitric oxide (NO) responses of activated macrophages. Mitogen-induced fish kidney leucocyte supernatants were fractionated using gel permeation FPLC (GP-FPLC) and the ability of different fractions to induce NO or RB measured. A MAF of M(r) 50 kD, that induced a potent nitric oxide response in both a long-term goldfish macrophage cell line (GMCL) and in in vitro-derived fish kidney macrophages (IVDKM) was identified. The GP-FPLC partially purified 50 kD MAF activity occasionally induced significantly higher nitric oxide production than that of the crude MAF preparations. This increase in the NO-inducing activity was due to segregation of the 50 kD MAF from a novel macrophage deactivating molecule of M(r) 10-12 kD present in crude MAF preparations. This 10-12 kD molecule was shown to inhibit nitric oxide production in cytokine-activated goldfish macrophages. Mitogen-induced fish kidney leucocyte supernatants contained two distinct MAFs that induced the respiratory burst in GMCL and IVDKM: the 50 kD and 30 kD proteins. The partially purified 30 kD MAF primed goldfish macrophage for increased RB activity after only 6 h of treatment, and continued to augment the RB activity after 24 h of stimulation. In contrast, the GP-FPLC partially purified 50 kD molecule also primed the RB after only 6 h of stimulation, but subsequently deprimed the RB after 24 h of stimulation, an effect similar to that observed for crude MAF preparations. The 50 kD MAF activity was further purified using chromatofocusing FPLC (C-FPLC) using basic pH gradients and was shown to consist of two distinct NO-inducing molecules (> pI 9.3). Mitogen-stimulated fish kidney leucocytes secrete several factors that profoundly affect the anti-microbial responses of teleost macrophages and which undoubtedly are responsible for regulating teleost macrophage function in vivo.     

Independent stimulation of motility and the oxidative metabolic burst of human polymorphonuclear leukocytes.

It has recently been suggested that a single stimulus to the membrane of the polymorphonuclear neutrophil leukocyte (PMN) produces a sequential, stereotyped response involving motility, degranulation, and the oxidative metabolic burst, and conversely, that the chemotactic response is dependent upon the stimulation of the hexose monophosphate shunt (HMPS). We have used small, synthetic substances, known to cause either increased motility or the metabolic burst, to examine whether these events can be stimulated independently. Phorbol myristate acetate (PMA) is a surface active agent that causes marked stimulation of iodination, superoxide production, chemiluminescence, and the HMPS. Such stimulation by PMA did not alter random or directional motility of PMN in the chemotaxis-under-agarose assay. Also, preincubation of PMN with PMA did not deplete their energy source for chemotaxis as demonstrated by a normal chemotactic response to zymosan activated serum. N-formylmethionyl peptides (f-met-phe, f-met-leu-phe) caused a dose-related stimulation of random and directional motility of PMN, but only a very slight stimulation of the HMPS, protein iodination, superoxide production, or chemiluminescence, and this minimal response occurred at more than 1000 times the concentration needed for stimulation of motility. These results indicate that stimulation of motility in the metabolic burst may involve separate events at the membrane of the PMN and that the events are not necessarily interdependent.     

Leishmania-macrophage interactions: insights into the redox biology

Leishmaniasis is a neglected tropical disease that affects about 350 million individuals worldwide. The protozoan parasite has a relatively simple life cycle with two principal stages: the flagellated mobile promastigote living in the gut of the sandfly vector and the intracellular amastigote within phagolysosomal vesicles of the vertebrate host macrophage. This review presents a state-of-the-art overview of the redox biology at the parasite-macrophage interface. Although Leishmania species are susceptible in vitro to exogenous superoxide radical, hydrogen peroxide, nitric oxide, and peroxynitrite, they manage to survive the endogenous oxidative burst during phagocytosis and the subsequent elevated nitric oxide production in the macrophage. The parasite adopts various defense mechanisms to cope with oxidative stress: the lipophosphoglycan membrane decreases superoxide radical production by inhibiting NADPH oxidase assembly and the parasite also protects itself by expressing antioxidant enzymes and proteins. Some of these enzymes could be considered potential drug targets because they are not expressed in mammals. In respect to antileishmanial therapy, the effects of current drugs on parasite-macrophage redox biology and its involvement in the development of drug resistance and treatment failure are presented.     

The neutrophil respiratory burst. Responses to fatty acids, N-formylmethionylleucylphenylalanine and phorbol ester suggest divergent signalling mechanisms.

The oxygen-dependent respiratory burst is a key neutrophil function required for the killing of bacteria. However, despite intensive investigation, the molecular events which initiate the respiratory burst remain unclear. Recent reports have suggested the agonist-induced hydrolysis of cellular phosphatidylcholine (PtdCho) by phospholipase D may be an essential requirement for initiating or mediating the respiratory burst. We have investigated the effects of the chemotactic peptide N-formylmethionylleucylphenylalanine (fMLF), the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and the polyunsaturated fatty acids arachidonic [20:4 (n-6)] and docosahexaenoic [22:6 (n-3)] acids in light of this hypothesis. Ethanol-inhibited superoxide production in response to 20:4, 22:6 and fMLF, in a dose-dependent fashion, suggesting an involvement of phospholipase D. The phosphatidic-acid phosphohydrolase inhibitor DL-propranolol completely inhibited superoxide production induced by both 20:4 and 22:6, and partially inhibited the response to TPA. In contrast, superoxide production in response to fMLF was increased by propranolol. fMLF and TPA, but not the fatty acids, stimulated phospholipase D as indicated by the accumulation of phosphatidic acid and, in the presence of ethanol, phosphatidylethanol derived from PtdCho. Extracellular Ca2+ was found to be an essential requirement for fMLF-induced superoxide production. However, responses to the fatty acids were dramatically enhanced under Ca(2+)-free conditions. Responses to TPA were independent of the extracellular Ca2+ concentration. Both fatty acids and fMLF, but not TPA, mobilised Ca2+ from intracellular stores, a response insensitive to the effects of both ethanol and propranolol. These results show that, unlike fMLF and TPA, the fatty acids do not cause hydrolysis of PtdCho by phospholipase D. However, the data indirectly suggests that the fatty acids may initiate the phospholipase-D-catalysed hydrolysis of phospholipids other than PtdCho.     

Modulation of the macrophage oxidative burst by Histoplasma capsulatum

The production of reactive oxygen species by phagocytic cells is an important host defense against invading microorganisms. Because pathogens that achieve intracellular survival escape destruction by reactive oxidants, we investigated the relationship between the intracellular survival of H. capsulatum and the macrophage oxidative burst. H. capsulatum yeast failed to stimulate the release of reactive oxygen metabolites in unprimed murine macrophages despite extensive phagocytosis of the microorganisms. This effect was observed with live as well as heat-killed fungi over a wide range of yeast-to-macrophage ratios. Preincubation of murine macrophages with heat-killed H. capsulatum (but not with latex spheres), followed by incubation with unopsonized zymosan, resulted in inhibition of oxidative burst triggering without inhibition of zymosan phagocytosis. Ingestion of H. capsulatum yeast opsonized with the cognate mouse antibody resulted in significant oxidant release, suggesting that suppression of the respiratory burst may be circumvented through Fc-mediated phagocytosis.