Con-A-stimulated superoxide production by granulocytes: reversible activation of NADPH oxidase

Abstract

Stimulation of granulocyte (PMN) superoxide (O2-) production by concanavalin-A (Con-A) can be monitored continuously in the spectrophotometer. Both the rate of activation and final activity of the O2--generating system is dependent on the concentration of Con-A. Alpha methylmannoside (alpha MM) can prevent Con-A, but not phorbol myristate acetate (PMA) or zymosan, induced O2- production. Alpha MM inhibits both the rate of activation and the final rate of O2- production. When alpha MM is added after the attainment of a maximal rate of O2- production with Con-A, O2- production continues for another minute before it ceases. When PMA is added to such treated cells, it restores O2- production. Although the inhibition of O2- production by alpha MM on previously activated cells requires time, most of the bound concanavalin-A is removed immediately after the addition of alpha MM. Treatment of cells with L-1-tosylamido-2-phenylethyl-chloromethyl ketone (TPCK) prevents activation of PMN by Con-A to a greater extent than it does for either PMA or zymosan. TPCK has no effect on the binding of Con-A. TPCK, when added after Con-A, will inactivate O2- production by the cells. The addition of PMA after TPCK treatment restores O2--generating activity. Membrane-enriched particles from PMN activated with Con-A, alpha MM, and PMA demonstrate that the change in O2- production seen by whole cells is due to an alteration of the activity of the NADPH oxidase. Thus, Con-A stimulation of human PMN O2- production can be prevented and reversed by the addition of either alpha MM or TPCK and that PMA can reactivate Con-A and either alpha MM- or TPCK-treated cells. The activation, inactivation, and reactivation occur as a result of changes in the plasma membrane NADPH-dependent O2-- generating enzyme.

Activation of murine polymorphonuclear neutrophils for fungicidal activity with supernatants from antigen-stimulated immune spleen cell cultures

“…Only relatively short periods (1 to 3 h) of PMN incubation with supernatants were required for enhancement of bactericidal activity, using a short challenge time (2 h). On the other hand, the relationship between factors in active supernatants reported here and the multitude of compounds reported to be capable of altering the metabolic activity of human (1,9,12,21,23,27) or rat (17) PMN is not clear at this time.…”

“…However, we speculate that T lymphocytes are essential for the initiation of this process, but additional experiments are required to determine whether they or other lymphoid cells, e.g., B cells or monocyte- on August 1, 2020 by guest http://iai.asm.org/ Downloaded from macrophages, or both, produce active factors. Furthermore, we speculate that soluble active factors elevate PMN to an enhanced metabolic state in a manner described for several activating compounds (5,9,21,27), namely, interaction with membrane receptors. As shown in this study and our previous report (8) on in vivo-activated PMN, enhanced killing by activated PMN appears linked with severalfold greater production of toxic products from oxidative metabolism after appropriate stimulation.…”

Neutrophil Erythrotoxicity Induced by Phorbol Myristate Acetate: Mechanisms Involved in Neutrophil Activation

“…The time pattern of the neutrophil respiratory response to a single stimulus is often unique and ranges from a shortlived but intense "burst" of 0 ;-production in response to FMLP [36] or concanavalin A [20] to a more sustained "respiratory marathon ," that does not decline for up to 90 min after phorbol ester stimulation [37] . To our knowledge , the time patterns of neutrophil-mediated cytotoxicity triggered by PMA have not been reported.…”

Quantification of O‐GlcNAc protein modification in neutrophils by flow cytometry

“…Previous reports indicated that the increase was short lived and that O ‐GlcNAc modification began to decrease within 10 min of stimulation (6). This difference may result from the use of cytochalasin D. Cytochalasins have been shown to increase neutrophil adhesion (8) and superoxide production in response to ligand stimulation, such as fMLF (9, 10). In this study, cytochalasin D was selected over other cytochalasins, because it does not alter cellular glucose transport (11), the major energy source of activated neutrophils.…”