Mechanism of the luminol-dependent chemiluminescence of human neutrophils.

Abstract

We examined the chemiluminescent response of normal and myeloperoxidase-deficient neutrophils in the presence and absence of luminol. Myeloperoxidase deficiency was total as assessed by spectrophotometric assay with o-dianisidine, by inability of the ceils to fix 125l to opsonized zymosan, and by inability of a cell sonicate to decarboxylate 1-(14C)-alanine to 14CO2 in the presence of hydrogen peroxide and chloride. The myeloperoxidase-deficient cells showed significantly greater oxygen consumption and glucose oxidation than control cells upon stimulation. When chemiluminescence was measured with opsonized zymosan in a nonenhanced system, the response of the myeloperoxidase-deficient cells was 25 to 50% of normal. In the luminol-enhanced system, however, the myeloperoxidase-deficient cells showed virtually no chemiluminescence in response to either opsonized zymosan or phorbol myristate acetate as stimulus. This was further investigated with normal cells. In the presence of azide, normal cells showed an attenuated chemiluminescent response with opsonized zymosan (~50% of normal) but virtually no chemiluminescence when measured in the luminol system. In contrast, superoxide dismutase inhibited the luminol-free chemiluminescence but did not affect the luminol-enhanced system. These results suggested that phagocytosis-induced chemiluminescence in the absence of luminol depended, at least in part, on the interaction of O2– with the particle whereas the luminol-enhanced chemiluminescence was absolutely dependent upon a myeloperoxidase-mediated reaction, likely via generation of hypochlorous acid. Chemiluminescence was measurable in a cellfree system consisting of a granule fraction from control cells, H2O2, CI–, and luminol. Omission of any one component abolished the response as did the addition of azide or cyanide; a granule fraction from a myeloperoxidase-deficient patient could not be substituted for the normal granule fraction. The plausibility of the involvement of hypochlorous acid was further demonstrated in a model system. When reagent hypochlorous acid was added to luminol at physiologic pH, a very rapid burst of chemiluminescence was observed.