Abstract
The extracellular H2O2 concentration surrounding stimulated human neutrophils was continuously quantitated with a sensitive, H2O2-detecting electrode. Following stimulation of neutrophils with phorbol myristate acetate, opsonized zymosan particles, or N-formyl-Met-Leu-Phe, the extracellular H2O2 concentration rapidly increased and maintained steady state conditions before falling to undetectable levels in a manner that was dependent on the triggering agent used. Total extracellular H2O2 accumulation for each stimulus was quantitated as the integral of the H2O2 concentration with respect to time. H2O2 accumulation in the extracellular milieu was unaffected by the addition of superoxide dismutase, whereas exogenous catalase or myeloperoxidase completely consumed the released H2O2. Analysis of H2O2 metabolism by neutrophils revealed that stimulus-dependent differences in the size of the extracellular H2O2 pool may be partially attributable to differences in hypochlorous acid generation by the H2O2, myeloperoxidase, chloride system. Finally, both the concentration of H2O2 in the extracellular space and its utilization by myeloperoxidase could be diminished in the presence of an extracellular target cell. These data indicate that the ability of a triggering agent to stimulate the neutrophil to generate H2O2 and release myeloperoxidase, coupled with the characteristics of a target cell population, control H2O2 metabolism in effector-target cell interactions.
Highlights
The extracellular HzOz concentrationsurrounding catalyzed dismutation to produce H202(16)
H202may remain in the myl-Met-Leu-Phe,the extracellular HzOz concentra- vacuole or diffuse either into the cytoplasm or outside the tion rapidly increased and maintained stsetatdeycon- cell
H202that has diffused into the cytoplasm is primarily reduced by either catalase or glutathione peroxiof the extracellular HzOzpool may be partiallyattrib- dase [16]
Summary
It is clear that nally, both the concentration of HzOzin the extracel- the ability of a phagocyte to mediate Hz02-dependentdamage lular space anitds utilizationby myeloperoxidase couldagainst an extracellular target is not dictated by the total be diminished in the presence oefxtarnacellulartarget amount of H20, produced but rather the extracellular H202 cell These data indicate thtahte ability of a triggering concentration as a function of time. Advances in polarographic techniques have led to the development of H202-sensingelectrodes that rapidly quantitate H202 concentrations while consuming only miniscule amounts for analysis Utilizing this approach, we have quantitated and examined both the characteristics of the free poolof H202 released by human neutrophils and its interaction with an extracellular target cell population.
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