Respiratory burst activated by Escherichia coli in human neutrophils primed with different lipopolysaccharides

Endotoxic shock is a dangerous complication of infection caused by gram-negative bacteria. Searching for agents capable to block the effects of endotoxins is a fundamental scientific and medical problem. Here we studied the effects of neutrophil priming with non-toxic lipopolysaccharide from Rhodobacter capsulatus (LPS _{Rb. caps.} ) and toxic LPS from Escherichia coli (LPS _{E. coli} ) on the respiratory burst evoked in neutrophils by opsonized E. coli. We found that preincubation of neutrophils with each of the LPS increased ROS production by neutrophils as compared to non-primed neutrophils. Subsequent incubation of neutrophils with LPS _{Rb. caps.} and then with LPS _{E. coli} practically abolished the effects of both endotoxins.     

1,2-Diacylglycerol accumulation in human neutrophils does not correlate with respiratory burst activation

Measurements of the level of 1,2-diacylglycerol (1,2-DG) during activation of the respiratory burst of human neutrophils by formyl-methionyl-leucyl-phenylalanine (fMLP) in the presence of platelet-activating factor (PAF) or by opsonized particles show that a correlation between accumulation of 1,2-DG and O2 consumption does not exist. Inhibition of protein kinase C activity with staurosporine before addition of opsonized particles demonstrates that the first phase of the respiratory burst is not inhibited, whereas the second phase, which is accompanied by a rise in the content of 1,2-DG, is strongly inhibited. This study indicates that accumulation of 1,2-DG cannot be the sole signal for the initiation of the respiratory burst in human neutrophils.     

Cortical actomyosin breakage triggers shape oscillations in cells and cell fragments

Cell shape and movements rely on complex biochemical pathways that regulate actin, microtubules, and substrate adhesions. Some of these pathways act through altering the cortex contractility. Here we examined cellular systems where contractility is enhanced by disassembly of the microtubules. We found that adherent cells, when detached from their substrate, developed a membrane bulge devoid of detectable actin and myosin. A constriction ring at the base of the bulge oscillated from one side of the cell to the other. The movement was accompanied by sequential redistribution of actin and myosin to the membrane. We observed this oscillatory behavior also in cell fragments of various sizes, providing a simplified, nucleus-free system for biophysical studies. Our observations suggest a mechanism based on active gel dynamics and inspired by symmetry breaking of actin gels growing around beads. The proposed mechanism for breakage of the actomyosin cortex may be used for cell polarization.     

Akt phosphorylates p47phox and mediates respiratory burst activity in human neutrophils

Respiratory burst activity and phosphorylation of an NADPH oxidase component, p47(phox), during neutrophil stimulation are mediated by phosphatidylinositol 3-kinase (PI-3K) activation. Products of PI-3K activate several kinases, including the serine/threonine kinase Akt. The present study examined the ability of Akt to regulate neutrophil respiratory burst activity and to interact with and phosphorylate p47(phox). Inhibition of Akt activity in human neutrophils by an inhibitory peptide significantly attenuated fMLP-stimulated, but not PMA-stimulated, superoxide release. Akt inhibitory peptide also inhibited hydrogen peroxide generation stimulated by bacterial phagocytosis. A direct interaction between p47(phox) and Akt was shown by the ability of GST-p47(phox) to precipitate recombinant Akt and to precipitate Akt from neutrophil lysates. Active recombinant Akt phosphorylated recombinant p47(phox) in vitro, as shown by (32)P incorporation, by a mobility shift change detected by two-dimensional gel electrophoresis, and by immunoblotting with phospho-Akt substrate Ab. Mutation analysis indicated that 2 aa residues, Ser(304) and Ser(328), were phosphorylated by Akt. Inhibition of Akt activity also inhibited fMLP-stimulated neutrophil chemotaxis. We propose that Akt mediates PI-3K-dependent p47(phox) phosphorylation, which contributes to respiratory burst activity in human neutrophils.     

Intrapopulational heterogeneity of human neutrophils based on chemokinetic and respiratory burst reactions

The chemokinetic test (ameboid motility) and enhancement of oxygen-dependent metabolism of neutrophils (the NBT test) were considered in human blood stimulated with Staph. aureus allergen. There were three variants of stimulated cells: 1) neutrophils developing ameboid motility (13,5 +/- 1,7% cells), 2) neutrophils with activated oxygen-dependent metabolism (11.5 +/- 0.6%), 3) neutrophils positive in both the tests (2.6 +/- 0.8%). Unstimulated cells accounted for 71.6 +/- 4.1%. Considerable differences were recorded for each variant. The data obtained are regarded as evidence of dissimilar capability of neutrophils of the realization of effector functions.     

Stimulators of AMP-activated protein kinase inhibit the respiratory burst in human neutrophils

In the present study, we have examined the potential ability of 5'-AMP-activated protein kinase (AMPK) to modulate NADPH oxidase activity in human neutrophils. AMPK activated with either 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or with 5'-AMP significantly attenuated both phorbol 12-myristate 13-acetate (PMA) and formyl methionyl leucyl phenylalanine-stimulated superoxide anion O2- release by human neutrophils, consistently with a reduced translocation to the cell membrane and phosphorylation of a cytosolic component of NADPH oxidase, namely p47phox. AMPK was found to be present in human neutrophils and to become phosphorylated in response to either AICAR or other stimulators of its enzyme activity. Furthermore, AICAR also strongly reduced PMA-dependent H2O2 release, and induced the phosphorylation of c-jun N-terminal kinase 1 (p46), p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. Present data demonstrate for the first time that the activation of AMPK, in states of low cellular energy charge (such as under high levels of 5'-AMP) or other signals, could be a factor contributing to reduce the host defense mechanisms.     

Carotenoid cleavage products modify respiratory burst and induce apoptosis of human neutrophils

Carotenoid supplementation in the treatment of diseases associated with oxidative stress has been recently questioned because of the cell damage and the increased risk of lung cancer in male smokers. Because of the complex role of neutrophils in lung diseases, we investigated whether carotenoid derivatives could affect respiratory burst and apoptosis of human neutrophils purified from peripheral blood. Stimulation of superoxide production was induced by nanomolar and micromolar concentrations of carotenoid cleavage products with aliphatic chains of different length, but not by carotenoids lacking the carbonyl moiety. The stimulatory effect of carotenoid cleavage products was observed in cells activated by phorbol myristate acetate (PMA), while a slight inhibition of superoxide production was noticed with cells activated by the chemotactic tripeptide N-formyl-Met-Leu-Phe (f-MLP). At higher concentrations, carotenoid cleavage products inhibited superoxide production in the presence of both PMA and f-MLP. In the presence of 20 microM carotenoid cleavage products, inhibition of superoxide production was accompanied by DNA fragmentation and increased level of intracellular caspase-3 activity.     

Spontaneous and chemoattractant-induced oscillations of cytosolic free calcium in single adherent human neutrophils

Studies with fluorescent Ca2+ indicators in large populations of neutrophils in suspension reveal a stable base line followed by a rapid agonist-induced elevation of cytosolic free calcium, [Ca2+]i, concomitant with other parameters of cellular activation. To study the role of adhesion in cell activation, we monitored [Ca2+]i in single neutrophils adhered to albumin-coated or fibronectin-coated glass coverslips before and after stimulation with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Human neutrophils loaded with 2 microM fura 2/AM were allowed to adhere to coverslips for 15-20 min at 37 degrees C. [Ca2+]i was monitored with a dual excitation microfluorimeter with a time resolution of 200 ms. Statistical analysis was performed using an algorithm allowing to detect significant [Ca2+]i peaks. 54% of the cells showed spontaneous [Ca2+]i oscillations. The amplitude of these [Ca2+]i peaks averaged 77 +/- 10 nM above basal levels (mean value of 110 +/- 20 nM), and their mean duration was 28 +/- 5 s; periods of [Ca2+]i bursts could last up to 15 min. In "silent" cells exhibiting a stable [Ca2+]i base line without spontaneous oscillations, low concentrations of fMLP (10(-10)-10(-9) M) could induce sustained [Ca2+]i oscillations. By contrast, higher agonist concentrations (10(-6) M) induced a single [Ca2+]i transient followed by a stable base line. 47% of the cells showing spontaneous [Ca2+]i oscillations did not respond to fMLP. Spontaneous [Ca2+]i oscillations depended on the continuous presence of extracellular Ca2+. Therefore: (i) spontaneous oscillations of [Ca2+]i occur in neutrophils adherent to various substrata; (ii) these oscillations do not preclude and can be dissociated from the response to fMLP; (iii) neutrophil functions might be controlled by [Ca2+]i oscillations rather than by sustained alterations of [Ca2+]i.     

Mitochondrial permeability transition pore is involved in oxidative burst and NETosis of human neutrophils

Neutrophils release neutrophil extracellular traps (NETs) in response to numerous pathogenic microbes as the last suicidal resource (NETosis) in the fight against infection. Apart from the host defense function, NETs play an essential role in the pathogenesis of various autoimmune and inflammatory diseases. Therefore, understanding the molecular mechanisms of NETosis is important for regulating aberrant NET release. The initiation of NETosis after the recognition of pathogens by specific receptors is mediated by an increase in intracellular Ca²⁺ concentration, therefore, the use of Ca²⁺ ionophore A23187 can be considered a semi-physiological model of NETosis. Induction of NETosis by various stimuli depends on reactive oxygen species (ROS) produced by NADPH oxidase, however, NETosis induced by Ca²⁺ ionophores was suggested to be mediated by ROS produced in mitochondria (mtROS).Using the mitochondria-targeted antioxidant SkQ1 and specific inhibitors of NADPH oxidase, we showed that both sources of ROS, mitochondria and NADPH oxidase, are involved in NETosis induced by A23187 in human neutrophils. In support of the critical role of mtROS, SkQ1-sensitive NETosis was demonstrated to be induced by A23187 in neutrophils from patients with chronic granulomatous disease (CGD). We assume that Ca²⁺-triggered mtROS production contributes to NETosis either directly (CGD neutrophils) or by stimulating NADPH oxidase. The opening of the mitochondrial permeability transition pore (mPTP) in neutrophils treated by A23187 was revealed using the electron transmission microscopy as a swelling of the mitochondrial matrix. Using specific inhibitors, we demonstrated that the mPTP is involved in mtROS production, NETosis, and the oxidative burst induced by A23187.     

Caffeic acid derivatives isolated from the aerial parts of Galinsoga parviflora and their effect on inhibiting oxidative burst in human neutrophils

Four new caffeoyl -glucaric and -altraric acid derivatives along with eleven known compounds were isolated from aerial parts of Galinsonga parviflora. Their structures were elucidated by high-resolution spectroscopic studies. The four new compounds were determined as being 2,3,4,5-tetracaffeoylglucaric acid (1), 2,4,5-tricaffeoylglucaric acid (2), 2,3,4- or 3,4,5-tricaffeoylaltraric acid (3) and 2,3(4,5)-dicaffeoylaltraric acid (4). A reliable criterion for the determination of the linkage position of caffeic acids moieties in glucaric acid derivatives has been proposed, on the basis of detailed analysis of the respective J-couplings, including substitution and solvent influence on the observed values. All hexaric acids derivatives appeared as inhibitors of reactive oxygen species production by stimulated neutrophils.     

Hydrocortisone inhibits the respiratory burst oxidase from human neutrophils in whole-cell and cell-free systems

The effects of hydrocortisone on the respiratory burst oxidase (NADPH oxidase, EC 1.6.99.6) from human neutrophils in both whole-cell and full soluble (cell-free) systems were investigated. In the whole-cell system, hydrocortisone inhibited the generation of superoxide by neutrophils exposed to phorbol myristate acetate, suggesting that steroids inhibit the bactericidal capacity of the body in an acute inflammatory phase. Hydrocortisone, which was added to the cuvette after the addition of NADPH and before the addition of sodium dodecyl sulfate, in a cell-free system, was found to inhibit the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate. The concentration of hydrocortisone required for 50% inhibition of oxidase was 40 microM. Its inhibition was dose- and time-dependent in the cell-free system. However, hydrocortisone did not alter the Km of the oxidase for NADPH. These results suggest that steroids inhibit the reconstitution of NADPH oxidase by sodium dodecyl sulfate in the cell-free system, and that they do not alter the affinity to NADPH of the oxidase.     

Respiratory burst in activated neutrophils is directly correlated to the intracellular level of protein kinase C.

The production of superoxide anion in human and rat neutrophils is directly correlated to the level of protein kinase C. Such correlation has been established on a comparative basis by analysis of neutrophils from normal and hypertensive subjects, characterized by an increased amount of protein kinase C, and of neutrophils from normal and genetically hypertensive rats characterized by low amounts of the kinase. Protein kinase C activity in all these different populations of neutrophils is modulated by specific inhibitors in an identical dose-dependent fashion which results in a linearly correlated decrease in O2- production. Taken together, these results provide a direct demonstration that in neutrophils the intracellular level of protein kinase C represents one of the determinants of the rate and extent of O2- production.     

Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils

Neutrophils play a central role in the innate immune response and a critical role in bacterial killing. Most studies of neutrophil function have been conducted under conditions of ambient oxygen, but inflamed sites where neutrophils operate may be extremely hypoxic. Previous studies indicate that neutrophils sense and respond to hypoxia via the ubiquitous prolyl hydroxylase/hypoxia-inducible factor pathway and that this can signal for enhanced survival. In the current study, human neutrophils were shown to upregulate hypoxia-inducible factor (HIF)-1α-dependent gene expression under hypoxic incubation conditions (3 kPa), with a consequent substantial delay in the onset of apoptosis. Despite this, polarization and chemotactic responsiveness to IL-8 and fMLP were entirely unaffected by hypoxia. Similarly, hypoxia did not diminish the ability of neutrophils to phagocytose serum-opsonized heat-killed streptococci. Of the secretory functions examined, IL-8 generation was preserved and elastase release was enhanced by hypoxia. Hypoxia did, however, cause a major reduction in respiratory burst activity induced both by the soluble agonist fMLP and by ingestion of opsonized zymosan, without affecting expression of the NADPH oxidase subunits. Critically, this reduction in respiratory burst activity under hypoxia was associated with a significant defect in the killing of Staphylococcus aureus. In contrast, killing of Escherichia coli, which is predominantly oxidase independent, was fully preserved under hypoxia. In conclusion, these studies suggest that although the NADPH oxidase-dependent bacterial killing mechanism may be compromised by hypoxia, neutrophils overall appear extremely well adapted to operate successfully under severely hypoxic conditions.     

Laminin promotes the oxidative burst in human neutrophils via increased chemoattractant receptor expression

The extracellular matrix component, laminin, enhances the chemotactic responsiveness of polymorphonuclear leukocytes (PMN) in vitro, and low doses of chemoattractant substances augment the expression of PMN cell surface receptors for laminin. This study determined whether laminin acts in concert with chemoattractants to activate PMN. Laminin (5 to 100 micrograms/ml) stimulated lysozyme release and superoxide production in response to the chemoattractant, FMLP by as much as 69%. These results could be explained by changes in cell surface chemoattractant receptor expression in that incubation of normal PMN with laminin (5 to 75 micrograms/ml) increased the binding of 19 nM FML[3H]P by 35 to 80%. This corresponded to as much as a 2.5-fold increase in the number of chemoattractant receptors/cells which had a lower average affinity. Laminin did not change the number or affinity of FML[3H]P receptors present on organelle-depleted PMN cytoplasts, and the laminin-induced increase in FML[3H]P receptors expressed on PMN from a patient with a specific granule deficiency was only 11 to 21% of that seen in normal PMN. These findings suggest that chemoattractants augment the expression of laminin receptors which mediate PMN attachment to basement membranes, followed by laminin-induced increases in the expression of cryptic chemoattractant receptors contained in intracellular granules, with resultant augmentation of the oxidative burst.     

Non-specific effects of 4-chloro-m-cresol may cause calcium flux and respiratory burst in human neutrophils

We examined the effects of 4-chloro-m-cresol (4-CmC, a potent and specific activator of ryanodine receptors) on Ca(2+)-release/influx and respiratory burst in freshly isolated human PMN as well as HL60 cells. 4-CmC induces Ca(2+) store-depletion in a dose-dependent manner at concentrations between 400muM and 3mM, however no dose-dependent effect on Ca(2+)-influx was found. 4-CmC depleted Ca(2+) stores that were shared with the GPC agonists such as fMLP and PAF, and therefore 4-CmC presumably depletes Ca(2+) from ER. Since the authentic ligand for RyR is cyclic ADP-ribose (cADPR), we assessed the functional relevance of RyR in PMN by studying the presence and function of membrane-bound ADP-ribosyl cyclase (CD38) in PMN. First, expression of CD38 was confirmed by RT-PCR using cDNA from HL60 cells. Second, PMN from trauma patients showed significantly enhanced CD38 expression than those from healthy volunteers. In addition, although no chemotaxis effect was detected by 4-CmC, it stimulated respiratory burst in PMN in a dose-dependent manner. Our findings suggest that RyRs exist in human PMN and that RyR pathway may play an active role in inflammatory PMN calcium signaling. 8-Br-cADPR and cyclic 3-deaza-ADP did not have inhibitory effects either on 4-CmC-induced Ca(2+) store-depletion or on respiratory burst, on the other hand, PLC inhibitor, U73122, completely attenuated both 4-CmC-induced Ca(2+) store-depletion and respiratory burst. Although it has been used as a specific activator of RyR, 4-CmC has non-specific effects which cause Ca(2+) store-depletion and respiratory burst at least in human PMN.     

Interactions of F1 fractions from different strains of shape Paracoccidioides brasiliensis with human complement and with human neutrophils

The aim of our study was to investigate differences that might exist in the activation of the human complement system by F1 fractions from four different isolates of P. brasiliensis. Isolates HC and 18 (virulent), 265 (low virulence), and 9 (intermediate virulence, attenuated) were used; before the experiments, the virulence of isolates HC and 18 was recovered by in vivo passage in guinea pigs. The four isolates of the fungus were processed for purification of F1 fractions and the activation of the human complement system was studied by a kinetic method of hemolytic activity measurement. The incubation of F1 fractions in normal human serum resulted in different degrees of inhibition of the classical and alternative pathways. The F1 fraction from the low virulence isolate was more efficient than the F1 fraction from the virulent isolates (HC and 18). Previous absorption of sera with F1 fractions completely abolished classical pathway activation. Using zymosan, instead of F1, in the absorption process caused the same phenomenon, suggesting that natural or nonspecific antibodies are responsible for the classical pathway activation. The alternative pathway activation did not depend on these antibodies, but was enhanced by their presence. On the other hand, F1 fractions from virulent isolates were more active in the stimulation of neutrophil chemiluminescence compared with the F1 fraction from the low virulence isolate. Whole P. brasiliensis yeast cells (WYC) from two distinct strains, 18 and 265, showed the same patterns of response of those observed with the F1 fractions in the functions tested. These differences in the behavior of the F1 fractions as well as WYC in relation to human complement activation and consequently to neutrophil stimulation may correlate with the virulence of individual isolates and may contribute to the understanding of the inflammatory response generation and maintenance processes in paracoccidioidomycosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oxidative burst and 1H-NMR-detectable mobile lipids in neutrophils

Neutrophils exposed to pro-inflammatory substances have an increased capability to respond to a membrane receptor-binding stimulus triggering an oxidative burst. One other considered sign of neutrophil activation is the appearance of a high-resolution 'H-NMR spectrum probably resulting from a rearrangement of neutral lipids in the membrane. The relationship between these two events is here studied. Neutrophils were recovered from a subcutaneous fungal infection using two murine lineages that respond differently to infection. It was concluded that rearrangement of lipids in the membrane, observable by NMR, appears also in neutrophils that do not exhibit the classical sign of activation, that is the increased expression of membrane receptors.     

Relationship between pH, sodium, and shape changes in chemotactic-factor-stimulated human neutrophils

The relationship between the chemotactic-factor-elicited changes in the intracellular pH and the shape of human neutrophils was investigated using simultaneous measurements of both parameters. The results demonstrate first that fMet-Leu-Phe and leukotriene B4 elicit qualitatively similar pH and shape change responses from the neutrophils. A relationship between the chemoattractant-elicited decrease in cytoplasmic pH and the shape changes is indicated by several findings including: 1) the similarities in the time courses of the two responses, 2) the ability of propionic acid to induce a transient and pertussis-toxin-sensitive shape change response, and 3) the ability of the calcium ionophore A23187 to similarly induce both responses under conditions when the degranulation is minimized. On the other hand, several other results indicate that the drop in pH is not a sufficient condition for the chemotactic-factor-stimulated shape changes. These include: 1) the ability of pertussis toxin to inhibit the shape changes induced by propionic acid and by A23187 without affecting the drop in pH, and 2) the observation that the drop in pH induced by propionic acid persists significantly longer than the shape change. Increasing the cytoplasmic pH by adding ammonium chloride was also found to cause shape changes in the neutrophils. The response to the base differs in two important aspects from that caused by propionic acid: it is pertussis-toxin-insensitive, and it is long-lived. Chemotactic factors have been found to induce a shape change under conditions when the internal pH was artificially increased or decreased, indicating that it is not the absolute cytoplasmic pH that represents the internal signalling parameter. The results are discussed in terms of the activation of the cytoskeletal network of the neutrophils by chemotactic factors.