F. Rossi

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.

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.

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.

Exocytosis in human polymorphonuclear leukocytes induced by A 23187 and calcium

Recently, both we [l] and Schell-Frederick 121 have found that ionophores for divalent cations can mimic the phagocytosis-associated stimulation of oxidative metabolism of polymorphonuclear leukocytes (PMNL). We now report that the ionophore for divalent cations A 23187 [3] , in the presence of Ca2’, stimulates another physiological response of PMNL to either phagocytosis or surface membrane perturbations [4] , i.e. the selective discharge by exocytosis of granular constituents. This process has been studied both by biochemically assaying the distribution of enzymes between cells and the extracellular fluid, and by following the extrusion of granules by electron microscopic analysis.

Interrelationship between oxygen consumption, superoxide anion and hydrogen peroxide formation in phagocytosing guinea pig polymorphonuclear leucocytes

The paper presents an experimental procedure for a simultaneous assay of oxygen consumption, O2- release and H2O2 accumulation at a very early stage of the respiratory burst that is induced by phagocytosis in guinea pig polymorphonuclear leucocytes. The main findings are as follows: (a) The oxygen consumption that is measurable does not correspond to all oxygen that is reduced. The relationship between the actual oxygen consumed and the amount that is reduced depends on the fate of the intermediate products O2- and H2O2. (b) O2- is measurable extracellularly by the reduction of cytochrome c. When cytochrome c oxidizes the extracellular O2-, molecular oxygen is formed. This fact is shown by a decrease of oxygen consumption. The molar ratio between the O2- detected and the oxygen given back is 1. (c) The amount of O2- released from the cells accounts for only a small part of oxygen actually reduced. (d) H2O2 is detectable only in the presence of NaN3. In this condition almost all oxygen consumed is recovered in the form of H2O2. The molar ratio O2/H2O2 is near unity. The amount of H2O2 derived from dismutation of O2- released is only an aliquot of the total H2O2 accumulated. Thus, most of H2O2 is derived from intracellular sources. (e) In the absence of inhibitors of H2O2 degrading reactions, no detectable accumulation of peroxide occurs. Under these conditions, the main part of H2O2 formed is degraded in almost equal amount by catalase and myeloperoxidase, while only a small aliquot is degraded by NaN3 insensitive reactions.SummaryThe paper presents an experimental procedure for a simultaneous assay of oxygen consumption, O2− release and H2O2 accumulation at a very early stage of the respiratory burst that is induced by phagocytosis in guinea pig polymorphonuclear leucocytes. The main findings are as follows:(a)The oxygen consumption that is measurable does not correspond to all oxygen that is reduced. The relationship between the actual oxygen consumed and the amount that is reduced depends on the fate of the intermediate products O2− and H2O2.(b)O2− is measurable extracellularly by the reduction of cytochrome c. When cytochrome c oxidizes the extracellular O2−, molecular oxygen is formed. This fact is shown by a decrease of oxygen consumption. The molar ratio between the O2− detected and the oxygen given back is 1.(c)The amount of O2− released from the cells accounts for only a small part of oxygen actually reduced.(d)H2O2 is detectable only in the presence of NaN3. In this condition almost all oxygen consumed is recovered in the form of H2O2. The molar ratio O2/H2O2 is near unity. The amount of H2O2 derived from dismutation of O2−1 released is only an aliquot of the total H2O2 accumulated. Thus, most of H2O2 is derived from intracellular sources.(e)In the absence of inhibitors of H2O2 degrading reactions, no detectable accumulation of peroxide occurs. Under these conditions, the main part of H2O2 formed is degraded in almost equal amount by catalase and myeloperoxidase, while only a small aliquot is degraded by NaN3 insensitive reactions.

Perturbation of leukocyte metabolism by nonphagocytosable concanavalin A-coupled beads

Concanavalin A (ConA) has become an interesting probe for the study of the mechanism of metabolic stimulation of leukocytes concomitant with phagocytosis [l-4] . The binding of this lectin to polymorphonuclear leukocytes (PMNL) and macrophages leads to a perturbation of their oxidative metabolism, i.e. enhanced respiration and glucose oxidation, similar to that detecta,ble when the challenge is performed with phagocytosable matter. Although we have gained much evidence that ConA is exerting its effect by promoting a rearrangement of the molecular organization of the phagocyte surface [ 1,3] , the possibility of a contribution to the stimulation by pynocytosed lectin could not entirely be excluded. To rule out this possibility we have exposed leucocytes to ConA covalently coupled to nonphagocytosable beads. Andersson et al. [5] and Ono et al. [6] have shown that this lectin displays mitogenic activity also when bound to an insoluble carrier.

Superoxide dismutase in leukocytes

1 ] or NBT [ 141 in the xanthine-xanthine oxidase reaction. The reduction of NBT and cytochrome c was followed at 560 and 550 nm respectively. One unit of SOD activity is defined as the amount of enzyme that causes a decrease of the optical density from 0.025 to 0.0125 in 1 min using 1 cm light path cuvette. Disc gel elect ophoresis was performed according to the method of Davis [ 161. The protein bands were stained using Coomassie blue. Superoxide dismutase activity was detected on the gels according to the method of Beauchamp and Fridovich [ 141. Catalase activity was measured as oxygen liberated from HZOz, produced by glucose-glucose oxidase reaction, upon addition of the catalase-containing sample [ 171. Hemoglobin was assayed by the cyanmethemoglobin method [ 181. Cytocbrome c, NBT and glucose oxidase were obtained from Sigma. Xanthine oxidase was obtained from Boehringer.

Studies on the NADPH oxidizing activity in polymorphonuclear leucocytes: The mode of association with the granule membrane the relationship to myeloperoxidase and the interference of hemoglobin with NADPH oxidase determination

Abstract Phospholipase C-treated polymorphonuclear leucocytes were used to study the properties of NADPH oxidase activity of stimulated polymorphonuclear leucocytes. A comparison of the effects of phospholipase C treatment of whole leucocytes on the NADPH oxidase activity with other granule enzymes showed that the activities of β-glucuronidase and acid phosphatase were un-affected, whereas the NADPH oxidase activity was stimulated 4-fold and myeloperoxidase was inhibited about 30%. The distribution of NADPH oxidase activity among subcellular fractions of polymorphonuclear leucocyte homogenates was unaffected by phospholipase C whereas the other enzymes were released into the medium in soluble form; β-glucuronidase > acid phosphatase and myeloperoxidase. A number of solubilizing agents and procedures were tested for their ability to release NADPH oxidase activity from granules of phospholipase C-stimulated polymorphonuclear leucocytes. All procedures used caused appreciable release of granule protein but no release of NADPH oxidase activity. Most of the procedures used strongly inhibited the oxidase activity. These results indicate that the enzyme is tightly bound to granule structures and that the integrity of these structures is required for activity. Some of the solubilizing agents used (KCI, guanidium chloride) were very effective in solubilizing myeloperoxidase. The differential response of myeloperoxidase and NADPH oxidase to treatment with phospholipase C or solubilizing procedures suggests that the two activities are not due to the same enzyme. However, definite conclusion cannot be drawn because of the complex nature of myeloperoxidase. It was found necessary to lyse any erythrocytes present as contaminants of polymorphonuclear leucocytes preparations, since hemoglobin was converted to methemoglobin during the NADPH oxidase assay and methemoglobin exhibits appreciable NADPH oxidase activity.

Stimulation of the respiration of polymorphonuclear leucocytes by antileucocyte antibodies

Gli anticorpi antileucociti provocano una marcata stimolazione della respirazione, rotenone, Antimicina A e cianuro insensibile, ed una aumentata attività del ciclo degli esosomonofosfati nei leucociti di cavia.