Rita Cramer

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.

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.

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.

Stimulation of the respiration of polymorphonuclear leucocytes by phospholipase C

Abstract Polymorphonuclear leucocytes treated in vitro with phospholipase C exhibit a marked increase of the respiration, which is insensitive to KCN, and of the catabolism of glucose through the hexose monophosphate shunt. These changes are similar to those occuring in phagocytosing polymorphonuclear leucocytes. Also treatment with phospholipase C markedly inhibits glycolysis which, on the contrary, is stimulated in phagocytosing leucocytes. The relevance of these findings to the biochemical mechanism of phagocytosis is discussed.

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.

NADPH oxidizing activity in rabbit polymorphonuclear leukocytes: Localization in azurophilic granules

Abstract Rabbit polymorphonuclear leukocyte granules were submitted to zonal fractionation through a discontinuous sucrose gradient. Distribution of azurophilic and specific granules, enzymatically characterized by peroxidase and alkaline phosphatase respectively, was as reported by others. NADPH oxidizing activity was associated with azurophilic granules. 3-Amino-1H-1, 2,4-triazole stimulated NADPH oxidation by azurophilic granules and inhibited peroxidase. Relationships between peroxidase and NADPH oxidizing activity are discussed.

Mode of activation of granule-bound NADPH oxidase in leucocytes during phagocytosis

Abstract Activation of a granule-bound NADPH oxidase is responsible for the stimulation of oxygen uptake, H 2 O 2 production and hexose monophosphate activity, concomitant with phagocytosis. The oxidase activity of isolated granules is virtually unaffected by the presence of Triton X-100 in the assay medium, and solubilization of this enzyme by a cationic detergent does not produce any rise in its activity. On the other hand, the affinity of the oxidase for NADPH is increased several-fold when the cell passes from the resting to the phagocytizing state. We conclude that the activation of NADPH oxidase by phagocytosis is not due to a loss of latency of the enzyme but to a change of its kinetic properties.

A simple and rapid method for isolation of eosinophilic granulocytes from human blood.

A simple and rapid method for the purification of morphologically and functionally intact eosinophils from human blood of both normal and eosinophilic subjects is described. The method is based on a single centrifugation of total blood leukocytes suspended in Percoll with specific gravity 1.0853 g/ml, after erythrocyte removal by dextran sedimentation. The peculiarity of this isolation technique is the maintenance of strictly physiological values of pH and osmolality throughout the entire procedure. Moreover, the cells are not subjected to measures aimed at changing the physical properties of either neutrophils or eosinophils. Because of such characteristics, this isolation method could be usefully exploited for comparative studies of normal and eosinophilic nor‐modense eosinophils and of neutrophils and eosinophils from the same noneosinophilic subject.

Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis II. Presence of the NADPH2 oxidizing activity in a myeloperoxidase-deficient subject

1. The biochemical properties of leucocytes from a myeloperoxidase-deficient subject were compared with those of leucocytes from healthy subjects. 2. Myleoperoxidase-deficient leucocytes responded to phagocytosis of heat-killed bacteria with increased respiration, increased oxidation of glucose through the hexose monophosphate shunt and increased production of H2O2 as normal leucocytes do. 3. The ability of granules isolated from myeloperoxidase-deficient leucocytes to oxidize nicotinamide coenzymes was comparable to that of granules isolated from normal leucocytes. 4. The results argue against the hypothesis that oxidation of NADPH2 in leucocytes is performed by myeloperoxidase.