Pierluigi Patriarca

Enzymatic basis of metabolic stimulation in leucocytes during phagocytosis: The role of activated NADPH oxidase☆

Abstract A comparison is made between the activities of KCN-insensitive NADPH- and NADH-oxidase associated with granules of guinea pig polymorphonuclear leucocytes. Both enzymes are activated when bacteria or polystyrene spherules are put in contact with the leucocytes. A rise in NADPH oxidase activity is already detectable after a few seconds from the addition of bacteria to leucocytes. The enhanced NADPH oxidase activity is due to a 10-fold decrease of Km, whose value approaches cellular NADPH concentration. Phagocytosis induces a 3-fold increase of the intracellular NADP + NADPH ratio, without affecting the NAD+ NADH ratio. In view of these results and other considerations, we conclude that the metabolic stimulation of PMN leucocytes by phagocytosis is supported by changes in kinetic properties of NADPH oxidase.

Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis I. Evidence for superoxide anion involvement in the oxidation of NADPH2

1. The oxidation of NADPH2 by leucocyte granules, as measured at acid pH in the presence of Mn-2+, was found to be inhibited by superoxide dismutase. 2. Omission of Mn-2+ markedly lowered the oxidase activity at acid pH, which was still inhibited by superoxide dismutase. 3. At alkaline pH the oxidase activity was lower than at acid pH. 4. During oxidation of NADPH2 by leucocyte granules, reduction of cytochrome c occurred which was partially inhibited by superoxide dismutase. 5. It was concluded that NADPH2 oxidation occurs through an enzymatic reaction and a nonenzymatic chain reaction. Superoxide anion (O-minus-2 and NADPH- free radical would be involved in the chain reaction. The differential sensitivity of NADPH2 oxidation to superoxide dismutase in different experimental conditions (see above 1, 2 and 3) was explained on the basis of changes in the properties of the chain reaction.

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.

Purification and properties of a cytolytic toxin in venom of the jellyfish Carybdea marsupialis

A haemolytic toxin was purified by ion-exchange chromatography and FPLC gel filtration from the nematocysts of the jellyfish Carybdea marsupialis. Sheep red cells, but not human or rabbit red cells, were susceptible to lysis by the toxin. The toxin is a protein with an apparent molecular mass of about 102-107 kDa, is heat labile, highly unstable in polar media, inactivated by reducing agents, and devoid of phospholipase activity. The experimental data speak in favour of a pore-forming mechanism of toxin action.

Biologically active polypeptides in the venom of the jellyfish Rhopilema nomadica

A tropical jellyfish, Rhopilema nomadica (Scyphozoa, Rhizostomeae) has recently invaded the eastern Mediterranean. Its painful stings have been the bane of bathers and fishermen from Egypt to Turkey. This paper reports on the presence of haemolytic activity and alpha-chymotrypsin-like serine protease activity in the venom of the R. nomadica nematocysts. In addition, the presence of phospholipase A2 activity, which has been described previously, is confirmed. Some properties of these activities are defined.

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.

Incidence of myeloperoxidase deficiency in an area of northern Italy: histochemical, biochemical and functional studies

Forty‐five subjects with a complete deficiency of myeloperoxidase were identified in an area of the region Friuli‐Venezia Giulia in north‐eastern Italy using the Hemalog D system as the screening technique. Histochemical and biochemical tests performed on the leucocytes of some of these subjects confirmed the defects shown by the Hemalog D system. The defect was of genetic origin in seven subjects. The genetic origin could be suspected in another eight subjects since more than two affected members were present in a given family. Eosinophil peroxidase, which is present in MPO deficient subjects, interfered with the guaiacol assay of MPO, and in several cases masked the genetic transmission. An assay was developed using o‐dianisidine as the electron donor which considerably reduced the interference by EPO. With this assay an autosomal recessive pattern of inheritance was found. The MPO deficient leucocytes had a higher respiratory burst than control cells and an impaired bactericidal activity, at early post‐phagocytic periods, which became comparable to that of control cells at later stages. Particle ingestion by the MPO‐deficient cells was normal.

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.