We have demonstrated that human fibroblasts can release O2-. radicals by an NADPH oxidase system that appears to be functionally similar to the phagocytic system. Further analysis of these systems, however, with respect to the low-potential b-type cytochromes involved suggests that these two O2-.-generating systems are not structurally identical. Immunoblot analysis of fibroblast membranes with six different antibodies directed against both subunits of human neutrophil cytochrome b-558 indicated that the b-type cytochrome molecules involved in these systems were not identical. None of these anti-(neutrophil cytochrome b) antibodies recognized a similar cytochrome in fibroblast membranes, suggesting that the two cytochrome species are immunologically distinct. In addition, fibroblasts obtained from a patient suffering from X-linked chronic granulomatous disease (CGD) had a normal cytochrome b-558 content compared with control fibroblast membranes, whereas the cytochrome b-558 concentration in polymorphonuclear leucocytes (PMNs) from this patient was decreased to 10% of that found in PMNs from healthy controls. Likewise, the stimulated O2-. release in PMNs from this patient was less than 10% of that in control PMNs, whereas the fibroblasts showed stimulated O2-.-release rates that were indistinguishable from those of fibroblasts obtained from healthy persons. Since the genetic mutation responsible for this type of CGD results in the absence of cytochrome b-558 in PMNs, fibroblasts should be affected in the same way if both cytochrome species were identical. These results suggest therefore that the low-potential b-type cytochromes in PMNs and fibroblasts are structurally and genetically distinct.
Neutrophils from 50 pediatric patients with normal phagocyte functions, from 150 healthy adults, from 10 chronic granulomatous disease (CGDI-patients (4 CGD,), and from 18 X-linked carriers for CGD have been tested for their production of H20z using staining with dihydrorhodamine 123 and subsequent flow cytometry. Additionally, neutrophils from three patients with myeloperoxidase deficiency were assessed. Cells were activated to produce H202 by the phorbol ester phorbol-myristate-acetate (PMA) and by phagocytosis of Escherichia coli bacteria. To evaluate the sensitivity of the method, H,O,-production by neutrophils which was inhibited by different concentrations of diphenyljodonium (DPI) was measured. The results were compared to those from other methods (NBT-testing, cytochrome c-reduction, and especially chemiluminescence). Normal values and ranges of scatter profile were evaluated in terms of peak channel fluorescence: 97% > 700, x = 840 f 59 (S.D.), 97% < 890, for pediatric patients. Normal quantitative values also resulted from small blood samples of infants (<1 year, n = 6, x = 830 2 52). For CGD, (n = 4) the results were clearly far below the normal range. In indicating decreased production of reactive oxygen intermediates the method was at least as sensitive as lucigenin enhanced chemiluminescence. Cytochrome bSs8-expression of neutrophils from patients and healthy controls was established by flow cytometry following staining with the monoclonal antibody 705. The normal range was 97% > 485, 97% -=c 680, peak channel fluorescence. We conclude that flow cytometric routine diagnostics of CGD can easily enhance the reliability of recognition and the yield of information about this disease compared to conventional methods.
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