Evidence that the superoxide-generating system of human leukocytes is associated with the cell surface.

485

A B S T R A C T Normal and cytochalasin B-treated human granulocytes have been studied to determine some of the interrelationships between phagocytosisinduced respiration and superoxide and hydrogen peroxide formation and release into the extracellular medium by intact cells. By using the scopoletin fluorescent assay to continuously monitor extracellular hydrogen peroxide concentrations during contact of cells with opsonized staphylococci, it was demonstrated that the superoxide scavengers ferricytochrome c and nitroblue tetrazolium significantly reduced the amount of H202 released with time from normal cells but did not abolish it. This inhibitory effect was reversed by the simultaneous addition of superoxide dismutase (SOD), whereas the addition of SOD alone increased the amount of detectable H202 in the medium. The addition of sodium azide markedly inhibited myeloperoxidase-H202-dependent protein iodination and more than doubled H202 release, including the residual amount remaining after exposure of the cells to ferricytochrome c, suggesting its origin from an intracellular pool shared by several pathways for H202 catabolism. When cells were pretreated with cytochalasin B and opsonized bacteria added, reduced oxygen consumption was observed, but this was in parallel to a reduction in specific binding of organisms to the cells when compared to normal. Under the influence of inhibited phagosome formation by cytochalasin B, the cells released an increased amount of superoxide and peroxide into the extracellular medium relative to oxygen consumption, and all detectable peroxide release could be inhibited by the addition of ferricytochrome c. Decreased H202 production in the presence of this compound could not be ascribed to diminished bacterial binding, decreased oxidase activity, or increased H202 catabolism and was reversed by the simultaneous addition of SOD. Furthermore, SOD and ferricytochrome c had similar effects on another H202-dependent reaction, protein iodination, in both normal and cytochalasin B cells. When oxygen consumption, 02., and H202 release were compared in the presence of azide under identical incubation conditions, the molar relationships for normal cells were 1.00:0.34:0.51 and for cytochalasin B-treated cells 1.00:0.99:0.40, respectively. Nonopsonized, or opsonized but disrupted, bacteria did not stimulate any of these metabolic functions.The results indicate that with normal cells approximately 50% of H202 released during phagocytosis is derived directly from 02: by dismutation, the remainder appearing from an (intra)cellular source shared by azide-inhibitable heme enzymes. With cytochalasin B treatment the evidence is consistent with the derivation of all H202 from an 02: precursor which is released from the cell surface. Furthermore, when activated by phagocytic particle

Section: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 73%

H2O2 Release from Human Granulocytes during Phagocytosis

Root¹,

Metcalf²

1977

485

“…Superoxide dismutase may be present in phagocytic vacuoles, because it has been localized to the cytoplasm of neutrophils (52), and may then play a significant role in catalyzing the formation of hydrogen peroxide from superoxide (49). However, in addition to the phagocytic vacuole, superoxide production appears to take place on the outer surface of the cell membrane (52,53). In fact, superoxide may be produced by neutrophils after exposure to certain stimuli in the absence of phagocytosis (54).…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of Attachment of Neutrophils to Candida albicans Pseudohyphae in the Absence of Serum, and of Subsequent Damage to Pseudohyphae by Microbicidal Processes of Neutrophils In Vitro

Daimond¹,

Krzesicki²

1978

121

A B S T R A C T Mechanisms were studied that might explain the attachment and damage to Candida albicans pseudohyphae by neutrophils in the absence of serum. Attachment of neutrophils to pseudo hyphae was inhibited by Candida mannans (1-10 mg/ml), but not by mannose, dextran, chitin, conconavalin A, or highly charged polyamino acids. Contact was also inhibited by pretreatment of Candida before incubation with neutrophils with chymotrypsin, but not trypsin or several inhibitors of proteases. Similar results were obtained with pretreatment of neutrophils, except that trypsin was inhibitory. When pseudohyphae were killed with ultraviolet light, proteinpolysaccharide complexes of mol wt <10,000 were released which appeared to bind to the surfaces of neutrophils and inhibit contact between neutrophils and Candida, as well as other fungi.Damage to Candida by neutrophils was inhibited by agents known to act on neutrophil oxidative microbicidal mechanisms, including sodium cyanide, sodium azide, catalase, superoxide dismutase, and 1, 4 diazobicyclo (2, 2, 2) octane, a singlet oxygen quencher. Neutrophils from a patient with chronic granulomatous disease did not damage Candida at all. However, the hydroxyl radical scavengers mannitol and benzoate were not inhibitory. Cationic proteins and lactoferrin also did not appear to play a major role in this system. Low concentrations of lysozyme which Preliminary findings were presented in part at the Annual

“…Other reagents were the best grade commercially available, and were used without further purification. Cytochalasin B was stored at -70°C as a stock solution in dimethylsulfoxide (50 mg/ml); cytochrome c and diethyldithiocarbamate were also stored at -70°C. Solutions of inhibitors were prepared fresh each day.…”

Section: Introductionmentioning

confidence: 99%

Termination of the Respiratory Burst in Human Neutrophils

Jandl¹,

André-Schwartz²,

Borges-Dubois³

et al. 1978

159

A B S T R A C T Recent evidence has suggested that a particulate 02-forming system is responsible for the respiratory burst in activated neutrophils. The respiratory burst is normally a transient event, lasting only 30-60 min. To investigate the mechanism by which the btrst is terminated, we examined the 02-forming activity of neutrophil particles as a function of time in the presence and absence of agents known to affect the function of intact cells. Measurements of the°2-forming capacity of the particles against time of exposure of neutrophils to opsonized zymosan, a potent stimulating agent, revealed a rapid fall in activity when exposure was continued beyond 3 min. Exposure to zymosan under conditions in which the myeloperoxidase system was inactive (i.e., in the presence of myeloperoxidase inhibitors, or in the absence of oxygen) resulted in a substantial increase in the initial 02-forming activity of particles from the zymosan-treated cells, but did not prevent the sharp fall in activity seen when zymosan exposure exceeded 10 min. The fall in activity was, however, prevented when activation took place in the presence of cytochalasin B (1.5 ,ugml), an agent thought to act largely by paralyzing the neutrophil through an interaction with its microfilament network.We conclude from these findings that the termination of the respiratory burst results at least in part from the inactivation of the particulate 02-forming system. This inactivation involves at least two processes which probably act simultaneously. One is the destruction of the system through the action of myeloperoxidase. The other appears to require active cell motility and is independent of oxygen. The current view holds that the 02-forming system of the neutrophil is located in the plasma membrane. It may be that the second process involves the internalization and degradation of this membrane-bound system.