Activity and activation of the granulocyte superoxide-generating system

Abstract

Phagocytic cells generate superoxide (02) as a part of the respiratory burst of phagocytosis. We studied human granulocyte #{176}2 production in vitro in response to phorbol myristate acetate, a soluble fatty acid ester that stimulates 02 generation. The properties of this compound permit the adaptation of a continuous assay of 0 production to the study of the activity and activation (i.e., the change from the resting to the active state) of the 02-generating system. The two processes, measured respectively as the rate of 02 production and the lag time for its attainment, respond differently to manipulations involving temperature. pH, phorbol myristate acetate concentration, N-ethyl maleimide inhibition. and energy metabolism. Changes in pH profoundly influence the rate without changing the lag time. Increases in temperature above 3TC depress the rate but continue to shorten the lag time. Thus. the activation and activity of the 02-generating system appear to have different pH and temperature dependency. In addition. they have different dose-response curves for phorbol myristate acetate stimulation: at high concentrations the rate reaches a plateau but the lag time continues to shorten. Activity of the human 02-generating system is sensitive to metabolic inhibitors. such as N-ethyl maleimide. 2-deoxyglucose, and the combination of 2-deoxyglucose with dinitrophenol or cyanide. The rate of activation (lag time) is unaffected by these compounds. These findings show that the activity of the human 02-generating system and its activation are separable processes. Furthermore. comparison of the above characteristics of human peripheral blood and guinea pig peritoneal exudate granulocytes reveals several important differences. Hospital Medical ('enter and Sidne8' Farber ('ancer institute.

Decrease in CuZn‐superoxide dismutase mRNA level during differentiation of human monocytic and promyelotic leukemia cells

“…On the other hand, myeloperoxidase activity is reported to be less in mature granulocytes than in immature HL-60 cells [29]. Differentiated U937 or HL-60 cells produce active oxygens [9,30], and the authors initially expected that CuZn-SOD mRNA would increase during differentiation as a protective measure against the cytotoxic effects of active oxygens. The a ±, standard deviation from triplicate assays decrease in CuZn-SOD mRNA followed by that of its activity, as observed in the present communication, may be biologically significant for the efficient production of active oxygens.…”

“…The expression of the proto-oncogene c-myc is diminished during the differentiation of HL-60 or U937 cells [4][5][6][7], whereas that of c-sis and c-fms genes increases during TPA-induced differentiation [8]. Besides these phenotypic changes, differentiated HL-60 and U937 cells produce superoxide anions [9].…”

Identification of a second class of IgG Fc receptors on human neutrophils. A 40 kilodalton molecule also found on eosinophils.

“…Superoxide Generation Assay. Continuous measurement of superoxide-dependent cytochrome C reduction (21,22) was performed in a Varian DMS 100 double-beam spectrophotometer at 37°C. In the standard assay, both sample and reference cuvettes contained 2 × 106 granulocytes and 100 nmol ferricytochrome C in 0.95 ml KRP buffer.…”

Effect of nitric oxide on staphylococcal killing and interactive effect with superoxide

“…The amount of superoxide was evaluated by the reduction of ferricytochrome c determined spectrophotometrically at 550 nm by using the micromolar extinction coefficient 0.021 (1). In some studies the kinetic assay described by Newberger et al was used (40). Here, the superoxide-generating system at 37ЊC was placed in a 5-ml tube and vortexed vigorously before being transferred to a 1-ml cuvette in the carrier of a Beckman Du-62 spectrophotometer.…”