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Secretion of neutrophil granules is dependent on calcium, but at the same time this process is regulated differently for each type of granules. We attempted to find calcium-regulated proteins that selectively translocate from the cytosol to the membranes of the neutrophil granules. An in vitro calcium-dependent translocation assay was designed by mixing cytosol with different neutrophil organelles isolated by subcellular fractionation. Immunoblotting using an anti-cytosol antiserum revealed a cytosolic protein of 42 kD that selectively binds to the specific granules of human neutrophils. It was neither associated with the azurophil granules nor with the secretory vesicles/plasma membrane. The protein was translocated at a calcium concentration of 100 micromol/L and binding was further increased by 1 mmol/L calcium. The 42-kD protein was partially purified from neutrophil cytosol by using its affinity for specific granules and by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partly purified protein allowed the 42-kD band to be excised and subjected to tryptic peptide mapping. Peptides from three peaks were N-terminally sequenced. Searching among known proteins, each one of the amino acid sequences was found to share sequence similarity to annexin XI.

When neutrophils and macrophages phagocytose a prey, e.g., complement (C3b)-opsonized yeast particles, the oxygen radical generating NADPH-oxidase is activated. In neutrophils, most of the production of oxygen metabolites occurred in an intracellular compartment, possibly in the phagolysosome. In contrast, no intracellular production could be detected in human macrophages. In these cells, the subcellular localization of the superoxide-generating NADPH-oxidase and associated cytochrome b was assessed in intact cells with indirect immunofluorescence and confocal laser scanning microscopy, and with subcellular fractionation, using centrifugation on Percoll density gradients. A dual localization of the cytochrome b as well as the dormant NADPH-oxidase activity in neutrophils was in agreement with earlier immunocytochemical, biochemical, and subcellular fractionation studies. Furthermore, most of the activity was recovered from the specific granules, whereas only a small fraction was retained in the plasma membrane. In contrast, the cytochrome b/NADPH-oxidase activity in macrophages localized primarily in the plasma membrane fraction. We suggest that the macrophages are incapable of producing reactive oxygen species intraphagosomally, due to an absence of a granule-localized pool of the membrane components of the NADPH-oxidase.

Using slot blot, we show that myeloperoxidase (MPO), a constituent of azurophil granules of neutrophil polymorphonuclear leukocytes, can be measured quantitatively using a commercially available chemiluminescence kit, originally developed for detection of specific proteins on Western blots. MPO is determined through its ability to catalyze the oxidation of luminol, resulting in the emission of light which is recorded on a photographic film. The sensitivity of the method is high and allows MPO from less than 100 cells to be detected. This method was used to determine MPO in exudate fluid and in neutrophil fractions following disintegration and subcellular fractionation of the postnuclear supernatant on two-layer Percoll gradients.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of circulating anti-neutrophil cytoplasm antibodies (ANCA), which are defined by a diffuse, granular staining of the cytoplasm of alcohol-fixed human neutrophils by indirect immunofluorescence (IIF). Detection of antineutrophil cytoplasm antibodies has a high sensitivity and specificity for active Wegener's granulomatosis (WG) and reflects the effect of treatment. In the present enzyme-linked assay, immunoplates were coated with the cytoplasmic alpha fraction of neutrophils obtained from apparently healthy human donors by nitrogen bomb cavitation and subsequent Percoll gradient centrifugation. Alkaline phosphatase-labelled anti-human IgG was used as a secondary antibody. Diluted sera from 70 patients with WG and 16 patients with other diseases with anti-myeloperoxidase antibodies (anti-MPO) were examined. It is concluded that the ELISA accurately detects IIF ANCA positive patients with WG, is helpful in detecting WG patients in remission, is not influenced by the presence of anti-MPO and may help in detecting ANCA in cases with granulocyte-specific anti-nuclear antibodies since this IIF pattern obscures the IIF ANCA patterns. The ELISA with titration can be carried out in 3.5 h whereas a rapid test just to detect ANCA can be performed in 30 min.