To determine the functional activity of neutrophils emigrating to the sites of ongoing human allergic inflammatory reactions, we assessed the intensity of their intracellular oxidative metabolism by measuring the intracellular oxidation by H2O2 of the
dye,
2',7'-dichlorofluorescein diacetate (
DCFH-DA), to fluorescent
2',7'-dichlorofluorescein. The intensity of intracellular fluorescence was measured by flow cytometry. In 13 atopic subjects, two denuded skin
blister bases were challenged for 5 hours with pollen
antigen and two
blister bases with
buffer. Both the level of
histamine and mean number of cells recovered at
antigen sites were greater than at
buffer sites. Oxidation of
DCFH-DA by neutrophils recovered from both
antigen and
buffer sites was significantly greater than that of autologous peripheral blood neutrophils (PBNs), reflecting an increase in the spontaneous generation H2O2 of cells recovered from the sites of
inflammation.
DCFH-DA oxidation by cells recovered from sites of
antigen incubation was significantly greater than cells from
buffer site incubations. Neutrophils from both
antigen and
buffer sites could be stimulated further in vitro by
phorbol myristate acetate to the same level of H2O2 generation as
phorbol myristate acetate-stimulated PBNs. In five subjects studied, the noncellular component of chamber fluids collected at 1 and 5 hours stimulated PBNs to increase their oxidative metabolism by 42% to 62%; however, this increased level of intracellular H2O2 was still much less than the spontaneous H2O2 generation observed in cells recovered from sites of allergic
inflammation. The increased oxidative metabolism of neutrophils in human allergic and inflammatory skin reactions has important pathophysiologic implications for the role of these cells in inflammatory responses.