Monoclonal antibodies (MoAbs) were raised against cytochrome b558, a membrane-bound component of the NADPH:O2 oxidoreductase in human neutrophils. This cytochrome consists of a low-molecular-weight (low- mol-wt) subunit of 22 to 23 Kd, probably encoded by an autosomal gene, and a high-mol-wt subunit of 75 to 90 Kd, encoded on the X-chromosome. MoAb 449 reacts with the low-mol-wt subunit and MoAb 48 with the high- mol-wt subunit on Western blots of purified cytochrome b558 and on blots of whole neutrophil extracts. In extracts of neutrophils from patients with chronic granulomatous disease (CGD) in which cytochrome b558 is not detectable by spectrophotometric methods, the low-mol-wt subunit is present, albeit in a much smaller amount. The high-mol-wt subunit is not detected by MoAb 48 in neutrophils of patients with X- linked CGD and in neutrophils of patients with the autosomal, cytochrome-b558-negative form of the disease. These results can be explained by a marked instability of these subunits when the synthesis of either of the two is disturbed. In differentiated HL-60 cells, the high-mol-wt subunit appears to be present in a different form. Cloning of the low-mol-wt subunit with the help of MoAb 449 suggests the presence of a heme-binding site on this subunit. By comparison of the binding characteristics of MoAb 449 to intact and permeabilized neutrophils with those of MoAb 7D5, recently isolated by Nakamura et al (Blood 69:1404, 1987), the low-mol-wt subunit was established as a transmembrane protein.

The microscopic nitroblue tetrazolium (NBT) slide test, used to score the ability of individual phagocytic leukocytes to produce superoxide, was improved according to the following procedure. Purified granulocyte suspensions are incubated with NBT and fixed in suspension, thereafter centrifuged on microscope slides and stained with nuclear fast red. This method precludes stimulation and selection of cells by adherence and washing. The number of formazan grains per cell can be judged in a semiquantitative way. In parallel incubations, the cells are stained with May-Grünwald/Giemsa, allowing identification of formazan-positive and -negative cells. The test discriminates well between cells from normal individuals, cells from patients with chronic granulomatous disease (CGD), and cells from heterozygotes for the X-linked form of CGD. Several patients and heterozygotes with an autosomal or variant form of CGD were detected with decreased NBT-reducing activity in their neutrophils and/or eosinophils. The relation between NBT-reductase activity of the phagocytes and the clinical situation is discussed.

Chronic granulomatous disease (CGD) is a rare syndrome, found predominantly in male children and characterized by life-threatening, recurrent infections. The superoxide (O2-)/hydrogen peroxide (H2O2) generating system in the granulocytes and monocytes of CGD patients is completely defective. Furthermore, a novel type of cytochrome b, detected by the optical spectrum of phagocytes from healthy subjects, is lacking in those of most male CGD patients. In female CGD patients, the cytochrome b is present, but cannot, as in normal cells, be reduced on metabolic stimulation of the phagocytes in anaerobic conditions. Here, to demonstrate the importance of cytochrome b in this system and to investigate the genetic background of the various forms of CGD, we have hybridized monocytes from a cytochrome b negative, X-linked male CGD patient with monocytes from a cytochrome b positive, male CGD patient with unknown genetic background. Monocytes were used because they are the only blood phagocytes that show an active protein synthesis, whereas fibroblasts or lymphocytes do not express the O2-/H2O2 generating system. The heterologous hybrids were positive in the nitroblue tetrazolium (NBT) slide test, indicating the complementation of the O2-/H2O2 generating system, whereas the homologous hybrids remained negative, as did the non-fused cells of these patients. We thus conclude that cytochrome b is part of the O2-/H2O2 generating system and that somatic cell hybridization experiments with monocytes provide a means of studying the genetic background of CGD patients. We believe this to be the first report of genetic complementation by somatic cell hybridization experiments using monocytes instead of fibroblasts.