Renzo Menegazzi

Killing by neutrophil extracellular traps: fact or folklore?

Neutrophil 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 because 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 because they are released and recovered in cell medium by incubation with DNase. It is concluded that NETs entrap but do not kill microbes.

TNF-Induced Shedding of TNF Receptors in Human Polymorphonuclear Leukocytes: Role of the 55-kDa TNF Receptor and Involvement of a Membrane-Bound and Non-Matrix Metalloproteinase

A down-modulation of both the 55-kDa (TNF-R55) and the 75-kDa (TNF-R75) TNF receptors is observed in neutrophils exposed to a variety of stimuli. Proteolytic cleavage of the extracellular region of both receptors (shedding) and, with TNF, internalization of TNF-R55 and shedding of TNF-R75 are the proposed mechanisms. We have characterized the TNF-induced shedding of TNF receptors in neutrophils and determined the nature of the involved proteinase. Neutrophils exposed to TNF release both TNF receptors. A release of TNF receptors comparable to that observed with TNF was induced with TNF-R55-specific reagents (mAbs and a mutant of TNF) but not with the corresponding TNF-R75-specific reagents. A hydroxamic acid compound (KB8301) almost completely inhibited shedding of TNF-R55 and to a lesser degree shedding of TNF-R75. KB8301 also inhibited FMLP-induced shedding to a similar extent. Shedding was also inhibited by 1,10-phenanthroline, but this effect was considered nonspecific as the compound, at variance with KB8301, almost completely inhibited TNF and FMLP-induced PMN activation. Diisopropylfluorophosphate partially inhibited shedding of TNF-R75, suggesting the contribution of a serine proteinase to the release of this receptor. Shedding activity was not affected by matrix metalloproteinases inhibitors nor was it released in the supernatants of FMLP-stimulated neutrophils. These results suggest that TNF induces release of its receptors, that such a release is mediated via TNF-R55, and that a membrane-bound and non-matrix metalloproteinase is involved in the process. The possibility that ADAM-17, which we show to be expressed in neutrophils, might be the involved proteinase is discussed.

Common methodology is inadequate for studies on the microbicidal activity of neutrophils

Microbicidal 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 tretment. 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 ∼60% and ∼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 Single-Step, Sensitive Flow Cytofluorometric Assay for the Simultaneous Assessment of Membrane-Bound and Ingested Candida albicans in Phagocytosing Neutrophils

Distinguishing ingested particles from those attached to the cell surface is an essential requirement when performing quantitative studies of phagocytosis. In the present report, we describe a simple, sensitive and reliable flow cytofluorometric method that achieves this goal in a Candida albicans‐human neutrophils (PMN) system.

A new, one-step assay on whole cell suspensions for peroxidase secretion by human neutrophils and eosinophils

The degranulation of neutrophils and eosinophils is frequently monitored by assaying myeloperoxidase (MPO) and eosinophil peroxidase (EPO) activity in the cell‐free supernatant of degranulating cells, after removal of the cells by centrifugation. This procedure leads to underestimation of the extent of degranulation, since both peroxidases tend to stick to cell surfaces, to test tube walls, and to particulate stimuli used to elicit degranulation, because of their highly cationic nature. In this paper we describe a method for assaying MPO and EPO secretion in whole cell suspensions that avoids separation of the cells from the incubation medium. The least toxic and thus safest among the sensitive peroxidase substrates, 3,3′,5,5′‐tetramethylbenzidine (TMB), was employed for peroxidase assay. The method we describe here is applied to the detection of peroxidase release by neutrophil and eosinophil cell suspensions incubated in either polypropylene test tubes or flat‐ bottomed microtiter plate wells. Because of the omission of the centrifugation step, the TMB method offers two major advantages over the currently used techniques: (1) higher estimates of degranulation, which permits the use of a smaller number of cells (in the microassay version, 150,000 neutrophils and 50,000 eosinophils) and smaller amounts of the secretagogues, and (2) rapidity, since the degranulation assay can be performed immediately on completion of the cell incubation with the secretagogue.

Uptake of human eosinophil peroxidase and myeloperoxidase by cells involved in the inflammatory process

We have recently shown that human neutrophils bind and internalize human eosinophil peroxidase (EPO) but not myeloperoxidase (MPO). In the present work, we studied the interactions of human EPO and MPO with other cells that may be involved in the inflammatory process, i.e., lymphocytes, monocytes, platelets, fibroblasts, and endothelial cells. The results indicate that EPO is bound by all the cell types considered, but is efficiently internalized only by lymphocytes, monocytes, and endothelial cells. Conversely, MPO binds appreciably only to fibroblasts and endothelial cells, although with a lower affinity than EPO, but its internalization by any of the cell types studied is hardly detectable. Furthermore, both peroxidases bind strongly to collagen fibers, whereas only EPO binds to elastin. The results suggest that EPO, owing to its high cytophilia, exerts its biological activity close to the site at which it is released from the eosinophil.

A simple reliable assay for myeloperoxidase activity in mixed neutrophil-eosinophil cell suspensions: Application to detection of myeloperoxidase deficiency

Quantitation of myeloperoxidase (MPO) activity by guaiacol peroxidation (GP) assay is profoundly affected by the peroxidase present in eosinophils (EPO) that contaminate the granulocyte suspensions. Inclusion of 3-amino-1,2,4-triazole (AMT) in the GP assay permits quantitation of MPO activity in mixed neutrophil-eosinophil suspension because of the differential inhibition of EPO and MPO by AMT. Results show that: (1) the peroxidase activity of eosinophil-free granulocyte suspensions is not appreciably affected by AMT; (2) in the presence of AMT the peroxidase activities of granulocyte preparations containing different numbers of eosinophils are similar on a neutrophil basis, regardless of the number of eosinophils and correspond with the activity of eosinophil-free granulocyte suspensions; (3) AMT almost completely inhibits the activity of partially purified EPO, only slightly affecting the catalytic activity of partially purified MPO; (4) AMT completely inhibits the residual peroxidase activity of granulocyte suspensions from MPO-deficient subjects contributed by contaminating eosinophils. The GP assay in the presence of AMT was used to study the pattern of hereditary transmission of MPO deficiency. The genealogy derived on the basis of this assay was compatible with an autosomal recessive inheritance, in agreement with previously reported results, while no definite pattern of inheritance could be established by use of the GP assay without AMT. We suggest that the GP assay supplemented with AMT is the method of choice for detection of MPO deficiency, particularly partial deficiency.

Chloride Movements in Human Neutrophils during Phagocytosis: Characterization and Relationship to Granule Release

Chloride 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.

Role of Intracellular Chloride in the Reversible Activation of Neutrophil β2 Integrins: A Lesson from TNF Stimulation

The process of β2 integrin activation, which enhances the interaction of these heterodimers with ligands, plays a crucial role in the adherence-dependent neutrophilic polymorphonuclear leukocytes’ (PMN) responses to TNF. Our previous observation, showing that a marked decrease of the high basal Cl− content (Cl−i) is an essential step in the TNF-induced activation of PMN, stimulated this study, which investigates the role of alterations of Cl−i in the activation of β2 integrins triggered by TNF. Here we show that TNF enhances the expression of activation-specific neoepitopes of β2 integrins, namely, epitope 24, a unique epitope present on all three leukocyte integrin α subunits, and epitope CBRM1/5, localized to the I domain on the α-chain of Mac-1 (CD11bCD18). Moreover, we demonstrate that the conformational changes underlying the expression of the neoepitopes are dependent on a drop in Cl−i because 1) inhibition of Cl−i decrease is invariably accompanied by inhibition of β2 integrin activation, 2) Cl−i decrease induced by means other than agonist stimulation, i.e., by placing PMN in Cl−-free buffers, activates β2 integrins, and 3) restoration of the original Cl−i levels is accompanied by deactivation of β2 integrins. We also show that Cl−i decrease is required for TNF-induced cytoplasmic alkalinization, but such a rise in pHi does not seem to be relevant for β2 integrin activation. The results of our study emphasize the role of Cl− as a new PMN “second messenger.”

Role of myeloperoxidase in respiratory burst of human polymorphonuclear leukocytes. Studies with myeloperoxidase-deficient subjects.

A comparative study of the respiratory burst [monitored as superoxide (O2−) production] of normal and myeloperoxidase (MPO) -deficient poiymorphonuclear leukocytes (PMNs) was carried out on 11 MPO-deficient subjects that represent the largest sample of this kind ever studied. The rate of O2− production by isolated PMNs and whole blood from normal and MPO-deficient subjects was comparable during the initial 30–40 min of incubation with serum-treated zymosan (STZ). Afterwards, the amount of O2− produced became progressively higher in MPO-deficient cells at least until 120 min incubation with STZ. On the contrary the rate of O2− production by both cell types in response to 4-β-phorbol-12-myristate-13-acetate (PMA) was the same. The PMNs of four MPO-deficient subjects were tested for their ingestion ability by counting the number of ingested particles on toluidine blue-stained sections of epoxy-embedded PMN suspensions. Both cell types ingested STZ particles at a comparable rate at early postphagocytic times, whereas on prolonged incubation MPO-deflcient PMNs ingested more STZ particles than normal PMNs. These results suggest that the ingestion capacity of normal cells may undergo a more rapid deterioration than that of MPO-deficient cells during incubation with STZ. Evidence for a higher deterioration of normal PMNs with respect to MPO-deficient PMNs was obtained also from studies on the effect of storage on O2− generation. After standing at melting ice temperature for 3 h, normal PMNs produced less O2− than MPO-deficient PMNs in response to PMA, and the difference in O2− production by the two cell types in response to STZ was evident at earlier postphagocytic periods than with freshly isolated cells. Taken all together these results suggest that normal PMNs and MPO-deficient PMNs do not intrinsically differ in O2− generating potential and that the difference in the respiratory burst observed during phagocytosis may be accounted for by a more marked deterioration, in normal PMNs, of one or more functions related to the respiratory burst.