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Glycosylation improves the priming effect exerted by recombinant human granulocyte colony-stimulating factor (lenograstim) on human neutrophil superoxide production.
No full textThe role of glycosylation in modulating the activity of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) on polymorphonuclear leukocytes (PMNs) was investigated. We addressed this study by comparing the effects of lenograstim (glycosylated rHuG-CSF) and its deglycosylated counterpart on superoxide production by PMNs on fibronectin. When the triggering activity of the cytokine was evaluated, no O2- release was elicited from neutrophils treated with either preparation of rHuG-CSF. Instead, a clear potentiation of both fMLP- and TNF-induced respiratory burst was produced by preincubating the cells with rHuG-CSF. Such effect was found to be significantly increased when glycosylated versus deglycosylated preparation was used, leading to the conclusion that the sugar moiety of the molecule could be of importance in improving the priming activity exerted by rHuG-CSF on PMN metabolic response
Killing by neutrophil extracellular traps: fact or folklore?
No full textNeutrophil extracellular traps (NETs) are DNA structures released by dying neutrophils and claimed to constitute a new microbicidal mechanism. Killing by NET-forming cells is ascribed to these structures since it is prevented by preincubation with DNase, which has been shown to dismantle NETs, before addition of the target microorganisms. Curiously, the possibility that the microorganisms ensnared in NETs are alive has not been considered. Using Staphylococcus aureus and Candida albicans blastospores we demonstrate that the microorganisms captured by NETs and thought to be killed are alive, since they are released and recovered in cell medium by incubation with DNase. It is concluded that NETs entrap but do not kill microbes.
Submitted July 1, 2011.
Accepted December 17, 201
Chloride movements in human neutrophils during phagocytosis: Characterization and Relationship to Granule Release
No full textChloride ion efflux is an early event occurring after exposure of human neutrophils to several soluble agonists. Under these circumstances, a rapid and reversible fall in the high basal intracellular chloride (Cl–i) levels is observed. This event is thought to play a crucial role in the modulation of several critical neutrophil responses including activation and up-regulation of adhesion molecules, cell attachment and spreading, cytoplasmic alkalinization, and activation of the respiratory burst. At present, however, no data are available on chloride ion movements during neutrophil phagocytosis. In this study, we provide evidence that phagocytosis of Candida albicans opsonized with either whole serum, complement-derived opsonins, or purified human IgG elicits an early and long-lasting Cl– efflux accompanied by a marked, irreversible loss of Cl–i. Simultaneous assessment of Cl– efflux and phagocytosis in cytochalasin D-treated neutrophils indicated that Cl– efflux occurs without particle ingestion. These results suggest that engagement of immune receptors is sufficient to promote chloride ion movements. Several structurally unrelated chloride channel blockers inhibited phagocytosis-induced Cl– efflux as well as the release of azurophilic—but not specific—granules. It implicates that different neutrophil secretory compartments display distinct sensitivity to Cl–i modifications. Intriguingly, inhibitors of Cl– exchange inhibited cytosolic Ca2+ elevation, whereas Cl– efflux was not impaired in Ca2+-depleted neutrophils. We also show that FcγR(s)- and CR3/CR1-mediated Cl– efflux appears to be dependent on protein tyrosine phosphorylation but independent of PI3K and phospholipase C activation
Evidence that TNF-induced respiratory burst of adherent PMN is mediated by integrin alpha(L)beta(2).
No full textPolymorphonuclear leukocytes (PMN) respond to tumor necrosis factor (TNF) with a respiratory burst (RB) only after adherence to surfaces coated with extracellular matrix proteins such as fibronectin and fibrinogen (permissive substrates) but not with others such as laminin or collagen (nonpermissive substrates). As PMN adherence to both types of surfaces is dependent on beta(2) integrins, we investigated the molecular basis of the different metabolic response to TNF. In particular, we evaluated the relative role of each beta(2) integrin (alpha(L)beta(2), alpha(M)beta(2), and alpha(X)beta(2)) in adherence and O(2)(-) production of PMN residing on fibronectin- and laminin-coated surfaces, which were considered as models of permissive and nonpermissive surfaces, respectively. By using alpha chain-specific monoclonal antibodies (mAb), we show that alpha(M)beta(2) and alpha(X)beta(2) mediate adherence to fibronectin and laminin; alpha(L)beta(2) is not involved in adherence to laminin and has only a minimal contribution in adherence to fibronectin. Furthermore, production of O(2)(-) in response to TNF was induced by immobilized anti-alpha(L)beta(2) but not anti-alpha(M)beta(2) or anti-alpha(X)beta(2) mAb. A strong correlation was also found between expression of alpha(L)beta(2) and TNF-induced RB on fibronectin. Lastly, PMN responded to TNF on laminin with a RB after the inclusion of alpha(L)-specific mAb in the laminin coat. Thus, we conclude that TNF-induced RB by PMN residing on fibronectin is mediated by alpha(L)beta(2) and that alpha(M)beta(2) and alpha(X)beta(2) are likely to play an ancillary role to the signaling activity of alpha(L)beta(2) by facilitating its recruitment to sites of adherence. The nonpermissiveness of laminin appears to be a consequence of its inability to act as a ligand for alpha(L)beta(2
Common methodology is inadequate for studies on the microbicidal activity of neutrophils.
No full textMicrobicidal activity of neutrophils is usually measured by colony-counting techniques after cell lysis in distilled water. While studying the effect of the reduced nicotinamide adenine dinucleotide phosphate-oxidase inhibitor diphenyleneiodonium (DPI) on the staphylocidal activity of neutrophils, we obtained inconsistent results: various degrees of inhibition in some experiments and no effect in others. The lysis step, i.e., dilution of neutrophils in distilled water, was the source of error. Cell-associated microorganisms were not dispersed effectively by this treatment. We overcame this problem by using water at pH 11 for cell lysis. Under these conditions, killing was inhibited completely and reproducibly by DPI. Here, we show that cell lysis in distilled water is incomplete and leads to an overestimate of microbial killing. This hinders identification of partial defects and makes complete defects appear as partial. We found that DPI-treated neutrophils and chronic granulomatous disease neutrophils were completely defective in killing of Staphylococcus aureus and Candida albicans and partially defective in killing of Escherichia coli after lysis with water pH 11, whereas after lysis in distilled water, killing of S. aureus and C. albicans was approximately 60% and approximately 70% of control killing, respectively, and killing of E. coli was normal. Likewise, killing of S. aureus by myeloperoxidase-deficient neutrophils was severely impaired after lysis in water pH 11 but appeared normal after lysis in distilled water. As most studies about neutrophil microbicidal activity have been performed using distilled water, our findings indicate that previous data about killing defects and the effects of agents that modulate microbicidal activity of neutrophils should be re-evaluated
A novel point mutation in the CYBB gene promoter leading to a rare X minus chronic granulomatous disease variant--impact on the microbicidal activity of neutrophils.
No full textThis article reports an atypical and extremely rare case of X-linked CGD in an Italian family characterized by a low expression of gp91phox (X91- CGD). A novel point mutation in the CYBB gene's promoter (insertion of a T at position -54T to -56T) appeared to prevent the full expression of this gene in the patient's neutrophils and correlated with a residual oxidase activity in the whole cells population. The expression and functional activity of the oxidase in eosinophils appeared to be almost normal. Gel shift assays indicated that the mutation led to decreased interactions with DNA-binding proteins. The total O2- production in the patient's granulocytes (5-7% of normal) supported no microbicidal power after 45 min and 60 min of contact with S. aureus and C. albicans, respectively. Despite this residual oxidase activity, the patients suffered from severe and life-threatening infections. It was concluded that in these X91- CGD neutrophils, the O2- production per se was not sufficient to protect the patient against severe infections
Triggering of chloride ion efflux from human neutrophils as a novel function of leukocytes β2 integrins: relationship with spreading and activation of the respiratory burst.
No full textActivation of neutrophil (NEU) and eosinophil (EOS) respiratory burst by tumor necrosis factor-α on biological surfaces.
No full textIntracellular shunting of O2− contributes to charge compensation and preservation of neutrophil respiratory burst in the absence of voltage-gated proton channel activity
No full textProton efflux via voltage-gated proton channels (Hv1) is considered to mediate the charge compensation necessary to preserve NADPH oxidase activity during the respiratory burst. Using the Hv1 inhibitor Zn(2+), we found that the PMA-induced respiratory burst of human neutrophils is inhibited when assessed as extracellular production of O2(-) and H2O2, in accordance with literature studies, but, surprisingly, unaffected when measured as oxygen consumption or total (extracellular plus intracellular) H2O2 production. Furthermore, we show that inhibiting Hv1 with Zn(2+) results in an increased production of intracellular ROS. Similar results, i.e. decreased extracellular and increased intracellular ROS production, were obtained using a human granulocyte-like cell line with severely impaired Hv1 expression. Acidic extracellular pH, which dampens proton efflux, also augmented intracellular production of H2O2. Zinc caused an increase in the rate but not in the extent of depolarization and cytosolic acidification indicating that mechanisms other than proton efflux take part in charge compensation. Our results suggest a hitherto unpredicted mechanism of charge compensation whereby, in the absence of proton efflux, part of O2(-) generated within gp91(phox) in the plasma membrane is shunted intracellularly down electrochemical gradient to dampen excessive depolarization. This would preserve NADPH oxidase activity under conditions such as the inflammatory exudate in which the acidic pH hinders charge compensation by proton efflux
Measurement of phagosomal pH of normal and CGD-like human neutrophils by dual fluorescence flow cytometry.
No full textBACKGROUND:
Phagosomal pH is thought to play an important role in the antimicrobial activity of polymorphonuclear leukocytes (PMNs). In this study, we set up a method for a rapid and accurate measurement of phagosomal pH in PMNs with the use of Candida albicans doubly labeled with a pH-insensitive and a pH-sensitive probe and flow cytometry.
METHODS:
Heat-killed, serum-opsonized C. albicans were doubly labeled with fluorescein, a pH-sensitive probe, and rhodamine, a pH-insensitive probe, and incubated with human PMNs. Flow cytometric readings of PMN-associated Candida were then taken, and the intraphagosomal pH was calculated on the basis of the ratio of fluorescein:rhodamine fluorescence by using a calibration curve obtained after equilibration of phagosomal pH with different external pH values after addition of digitonin.
RESULTS:
A rapid rise in phagosomal pH, which reached pH 7.8, was observed 2 min after initiation of phagocytosis and progressively declined to pH 6.9 after 15 min. Such a rise was not observed in PMNs with defective microbicidal activity (deficient in nicotinamide adenine dinucleotide phosphate oxidase), where phagosomal pH dropped to pH 6.6, 2 min after phagocytosis. The abnormal initial acidification in PMNs deficient in nicotinamide adenine dinucleotide phosphate oxidase was prevented by using lysosomotropic weak bases or the vacuolar-type H(+) pump inhibitor concanamycin A.
CONCLUSIONS:
Phagosomal pH of PMNs can be easily and accurately measured by dual fluorescence flow cytometry. The method can be applied to assess phagosomal pH in PMNs with defective microbicidal activity and to monitor the outcome of pharmacologic interventions aimed at correcting its abnormalities
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