Form Of Chronic Granulomatous Disease Research Articles

Detection of carriers of the autosomal form of chronic granulomatous disease

The NADPH:O2 oxidoreductase catalyzing the respiratory burst in activated phagocytes from healthy individuals is not operative in phagocytes from patients with chronic granulomatous disease (CGD). In a microscopic slide test using the dye nitroblue tetrazolium (NBT), carriers of X-linked CGD can be recognized by a mosaic pattern of NBT- positive and NBT-negative cells, governed by the expression of an unaffected or an affected X chromosome, respectively. Until now, it has not been possible to detect carriers of the autosomal form of CGD (other than by family studies) because all cells of these carriers stain positive in the NBT test. We have investigated whether neutrophils from carriers of autosomal CGD can be recognized by measurement of the rate of oxygen uptake upon stimulation of the cells. It was found that with the phorbol ester PMA as a stimulus, the respiratory burst is significantly lower in autosomal CGD carriers. With serum-treated zymosan as a stimulus, no difference between controls and carriers was observed. The addition of f-Met-Leu-Phe (1 microM) to PMA-activated neutrophils of control donors caused a transient increase in oxygen consumption of about 40%. Under these conditions, an increase of more than 100% was observed in neutrophils from carriers of autosomal CGD. These findings provide a simple method for the detection of carriers of the autosomal form of CGD.

Purified protein kinase C phosphorylates a 47-kDa protein in control neutrophil cytoplasts but not in neutrophil cytoplasts from patients with the autosomal form of chronic granulomatous disease.

The neutrophil activators phorbol 12-myristate 13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine, serum-treated zymosan, and IgG-coated latex cause an increase in protein phosphorylation in human neutrophil cytoplasts, concomitantly with an increase in oxygen consumption. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, phosphorylation was apparent in many proteins, must abundantly in 42-, 47-, 50-, 60-, and 80-kDa proteins. In neutrophil cytoplasts from autosomal chronic granulomatous disease (CGD) patients that were stimulated with PMA, the phosphorylation of a 47-kDa protein is absent. The localization of this protein in PMA-activated control cytoplasts is mainly in the cytosol and, to a lower and more variable extent, in the membrane. After addition of purified protein kinase C to lysates of nonstimulated control cytoplasts, phosphorylation occurred at the 47-kDa level in both the cytosol and the membrane fraction. With lysates of autosomal CGD cytoplasts, in vitro phosphorylation of the 47-kDa protein was completely absent. After separation of cytoplast proteins on a sodium dodecyl sulfate-polyacrylamide gel and excision of the 47-kDa protein(s), phosphorylation of the isolated 47-kDa band was observed in the presence of purified protein kinase C. This reaction was again absent when autosomal CGD cytoplasts were used as starting material. Our studies have identified the 47-kDa protein in neutrophil cytoplasts as a true substrate for protein kinase C and indicate that the defect in phosphorylation at the 47-kDa level in autosomal CGD cytoplasts is due to a defective protein.

Chronic granulomatous disease due to a defect in the cytosolic factor required for nicotinamide adenine dinucleotide phosphate oxidase activation.

The superoxide-generating enzyme of human neutrophils, NADPH oxidase, is present in a dormant state in unstimulated neutrophils. It can be converted to an active form in a cell-free system if both the plasma membrane and cytosol fractions are incubated together in the presence of arachidonic acid. This system was used to determine the nature of the biochemical defect in seven patients with the autosomal recessive, cytochrome b-positive form of chronic granulomatous disease (CGD). A severe deficiency in the cytosol factor was identified in each patient. The defective activity was not caused by the presence of an inhibitor, nor could it be restored to normal by combining cytosol fractions from different patients. In contrast, the membrane fractions from all seven patients contained normal levels of NADPH oxidase when activated in the presence of control cytosol. Of family members tested (obligate heterozygotes for this disorder), seven of eight had intermediate levels of cytosol factor activity. The respiratory burst defect in this form of CGD is caused by an abnormality in the cytosolic factor required for NADPH oxidase activation.

Activation of neutrophil NADPH oxidase in a cell-free system. Partial purification of components and characterization of the activation process.

The superoxide-generating enzyme of human neutrophils, NADPH oxidase, is converted from an inactive to an active form upon stimulation of the neutrophil. This activation process was examined using a recently developed cell-free system in which dormant oxidase is activated by arachidonic acid in the presence of a soluble factor from the neutrophil (Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743). NADPH oxidase from unstimulated human neutrophils was detected only in the membrane fraction. The soluble activation factor was localized entirely to the cytosolic fraction and exhibited two peaks of activity when partially purified under nondenaturing conditions: a major peak with a molecular mass of approximately 250 kDa and a variable minor peak with a mass of approximately 40 kDa. Both forms activated NADPH oxidase in a similar manner and did not exhibit synergy when combined. The cytosolic factor is not protein kinase C (or another kinase) as both peaks of factor activity could be resolved from the protein kinase C peak and neither required calcium or ATP to activate the oxidase. Activation of NADPH oxidase did require the simultaneous presence of the membrane fraction, the cytosolic factor, arachidonic acid, and magnesium. Following activation, however, only the membrane fraction was then required for O2- production. Cytosolic factor levels were normal in five patients with either X-linked or autosomal recessive cytochrome b-negative chronic granulomatous disease. In contrast, the membrane fractions from each failed to generate O2-, indicating that the defects in these two genetic forms of chronic granulomatous disease reside either in the oxidase itself or in a membrane component required for activation.

The influence of phagocytic stimuli on the expression of HLA-DR antigens; role of reactive oxygen intermediates.

Human peripheral blood mononuclear cells show after one day of culture with opsonized zymosan a decreased expression of HLA-DR (but not HLA-A, B, C) antigens, which can be prevented by the scavenger of reactive oxygen intermediates beta-carotene, or superoxide dismutase or indomethacin. Mononuclear cells of a patient with a heterozygous form of chronic granulomatous disease show no alterations in HLA-DR antigen expression after culture with opsonized zymosan. Possible roles of reactive oxygen intermediates or prostaglandins in the modulation of HLA-DR antigens are discussed.

Cytochrome b deficiency in an autosomal form of chronic granulomatous disease. A third form of chronic granulomatous disease recognized by monocyte hybridization

Cytochrome b deficiency in an autosomal form of chronic granulomatous disease. A third form of chronic granulomatous disease recognized by monocyte hybridization

Cytochrome b deficiency in an autosomal form of chronic granulomatous disease. A third form of chronic granulomatous disease recognized by monocyte hybridization.

Three patients (two sisters and a brother) in one family are described with chronic granulomatous disease. The granulocytes of these patients did not respond with a metabolic burst to various stimuli and failed to kill catalase-positive microorganisms. The magnitude of the cytochrome b signal in the optical spectrum of the patients' granulocytes was less than 4% of the normal value, whereas the amount of noncovalently bound flavin in these cells was normal. The mode of inheritance of the genetic defect in this family is autosomal because the granulocytes of both parents (first cousins) and a nonaffected sister of the patients expressed 70-80% of the normal cytochrome b signal, showed low-normal or subnormal oxidative reactions during stimulation, and did not display mosaicism in the stimulated nitroblue-tetrazolium slide test. Somatic cell hybridization was performed between the monocytes from the affected boy in this family with monocytes from either a cytochrome b-negative male patient with X-linked chronic granulomatous disease or a cytochrome b-positive male patient with the classic autosomal form of this disease. In both combinations, monocyte hybrids were observed with nitroblue tetrazolium reductase activity after stimulation with phorbol myristate acetate. This complementation of the oxidase activity required protein synthesis. Our results prove that the defect in this family is genetically distinct from that in the other two forms of chronic granulomatous disease. Moreover, our results also indicate that the expression of cytochrome b in human phagocytes is coded by at least two loci, one on the X chromosome and one on an autosome.

Spectrally abnormal cytochrome-b in a male patient with chronic granulomatous disease

Cytochrome-b and flavin adenine dinucleotide (FAD) were measured in neutrophils from a male patient and his family with the X-linked form of Chronic Granulomatous Disease (CGD). The membrane fraction of the patient's neutrophils contained 93pmol/mg protein of FAD (control 102±20, mean±sd), and 32pmol/mg protein of cytochrome-b (control 88±20, mean±sd). In the spectrophotometric study, the maximal absorbance peak of cytochrome-b was at 421nm, as that from control was at 427nm. Cytochrome-b in neutrophils from his mother and a sister, who were thought to be carriers, had their maximal absorbance peak at 425nm. These findings suggest that the spectral abnormality of cytochrome-b contribute to the pathogenesis of CGD in this case.

Complementation in monocyte hybrids revealing genetic heterogeneity in chronic granulomatous disease.

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.

Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patients with chronic granulomatous disease

Cytoplasmic free calcium concentration (Ca2+)i was measured in neutrophils from patients with the classical X-linked form of chronic granulomatous disease (CGD) by trapping the fluorescent calcium indicator Quin 2 in intact cells. CGD neutrophils do not produce superoxide and are only slightly depolarized upon stimulation by the chemotactic peptide. N-formyl-methionyl-leucyl-phenylalanine (FMLP). The resting levels, as well as (Ca2+)i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal cells. We conclude that the defect in CGD cells is distal to, or independent of, the changes in (Ca2+)i induced by FMLP stimulation and that normal membrane depolarization does not seem to be necessary for receptor-mediated rise in free cytosolic calcium in human neutrophils.

The association of FAD with the cytochrome b-245 of human neutrophils.

A plasma membrane fraction prepared from human neutrophils had a fluorescence resembling that of a fluorescent flavoprotein, with emission maximum near 520nm and excitation maxima near 380 and 460nm. The fluorescence emission and excitation properties of Triton N-101-solubilized membrane fraction resembled those of FAD. FAD was present in the membranes at a concentration of 417pmol/mg of protein and cytochrome b(-245) at a concentration of 407pmol/mg of protein. In a 110-fold purified preparation of cytochrome b(-245) the ratio of FAD:cytochrome b was 1:1. Analytical gradient centrifugation of neutrophil homogenates shows a coincidence of two cytochrome b peaks and two peaks of fluorescence, corresponding with plasma membrane and specific granule fractions; most of the FAD was non-fluorescent and located in fractions lighter than the plasma membrane. Plasma membrane fractions prepared from neutrophils of patients suffering from the X-linked form of chronic granulomatous disease lacked cytochrome b and contained 194pmol of FAD/mg of protein; plasma membrane fractions prepared from neutrophils of patients with the autosomal recessive form of chronic granulomatous disease contained both cytochrome b(-245) and FAD in the normal range of concentrations in a ratio of 1:1. Phagocytic vesicles were prepared from normal neutrophils and found to contain FAD and cytochrome b in a ratio 2.22:1, suggesting that activation of neutrophils many involve the incorporation of an additional flavin into the membrane. Under anaerobic conditions in the presence of EDTA to act as an electron donor to a flavin, the cytochrome b(-245) of neutrophil membranes was partly (12%) photoreducible, an effect increased to 100% by the addition of FMN. The extent of reduction of cytochrome b in an anaerobic neutrophil homogenate containing NADH increased from 30% to 70% on illumination. We suggest that these results indicate a close association between FAD and cytochrome b(-245) and support a scheme for electron transport thus: [Formula: see text]

The enzymatic basis for O-.2 production by human neutrophils.

O-.2 production is the first step in the generation of a group of powerful microbicidal oxidants by neutrophils. The production of O-.2 is catalyzed by a membrane-bound, NADPH-preferring flavoprotein oxidase, a conclusion supported by much evidence including the discovery of a new form of chronic granulomatous disease caused by a mutation affecting that oxidase directly. Also involved in the O-.2-forming reaction is a b-type cytochrome; the role of this cytochrome is as yet undefined, though it does not appear to be on the direct route of electron transfer between NADPH and oxygen. It has been postulated that quinones too participate in the O-.2-forming reaction, but further work is necessary to define their role more fully.

Chronic granulomatous disease, Kx negative neutrophils and linkage with Xg.

Individuals afflicted with chronic granulomatous disease (CGD) suffer from recurrent infections due to the inability of their neutrophils to kill ingested microorganisms. Defective microbicidal function is a consequence of the inability of these cells to generate metabolites of oxygen which are toxic to ingested bacteria. This defect in oxidative activity is believed to be due to the absence or dysfunction of a membrane bound oxidase. In 1975, Marsh and co-workers (1) reported that neutrophils from patients with X-linked CGD, but not other forms of CGD, lacked a surface antigen called Kx. Kx is an X-linked, Kell-related antigen normally present on the surface of both red cells and granulocytic white cells. Its distribution on these two cell types is determined by any of four alleles that have been described at the Xk locus (2). Erythrocytic Kx is converted to the appropriate Kell antigen by an autosomally determined enzyme. Thus, Kx is believed to serve as a precursor substance for Kell antigens on red cells, whereas on granulocytes it remains unaltered.KeywordsChronic Granulomatous DiseaseNormal NeutrophilInfectious Disease DepartmentHemagglutination ReactionClinical Research DepartmentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

CYTOCHROME b IS PRESENT IN NEUTROPHILS FROM PATIENTS WITH CHRONIC GRANULOMATOUS DISEASE

Analysis of dithionite difference spectra demonstrated that cytochrome b was present in neutrophil homogenates from a 17-year-old girl and her 25-year-old brother who had the autosomal recessive form of chronic granulomatous disease, and from an 18-year-old boy with the X-linked form of chronic granulomatous disease. These results indicate that the postulated importance of cytochrome b in the oxygen burst during phagocytosis is questionable.

Chronic granulomatous disease of childhood in a 23-year-old man

A 23-year-old man with a history of recurrent bacterial and fungal infections since age 12 was shown to have a functional leukocyte defect similar to that found in children with chronic granulomatous disease. A liver biopsy revealed discrete granulomata. The documentation of this case calls attention to the possibility that there may be a mild form of chronic granulomatous disease and that recurrent bacterial infection in the adult may be due to a functional disorder of polymorphonuclear leukocytes.

Chronic Granulomatous Disease of Childhood in a 23-Year-Old Man

A 23-year-old man with a history of recurrent bacterial and fungal infections since age 12 was shown to have a functional leukocyte defect similar to that found in children with chronic granulomatous disease. A liver biopsy revealed discrete granulomata. The documentation of this case calls attention to the possibility that there may be a mild form of chronic granulomatous disease and that recurrent bacterial infection in the adult may be due to a functional disorder of polymorphonuclear leukocytes.

Accelerated decay of glucose 6-phosphate dehydrogenase activity in chronic granulomatous disease.

Extract: Glucose 6-phosphate dehydrogenase (EG. 1.1.1.49) (Glc-6-PD) activity was measured in the lcukocytes and red cells of three patients with the classic form of chronic granulomatous discase (CGD) and in controls. Leukocyte homogenates from all three children revealed a decreased Glc-6-PD activity. The activities in both white cells and red cells, measured at timed intervals under conditions of storage at 0° or heating at 38° revealed a more rapid rate of decay in cells of patients than that seen in cells of control subjects. Studies measuring the activities of Glc-6-PD in the presence of 10 μM NADP, or 14 mM 2-mercaptoethanol, or both, disclosed a stabilizing effect with these reagents. Measurement of activity of another NADP-dependent enzyme, 6-phosphogluconic dehydrogenase (EC. 1.1.1.43) (6-PGD), in leukocytes were within normal limits. A correlation coefficient of 0.86 (P < 0.01) was found between Glc-6-Pd activity and the quantitative nitrobluc tetrazolium (NBT) test used to diagnose CGD. Speculation: An increased rate of decay in activity of glucose 6-phosphate dehydrogenase was detected in the leukocytes and red cells of patients with one form of chronic granulomatous disease. The finding of a functional abnormality in an X-linked enzyme is consonant with the known X-linked recessive mode of transmission of this form of the entity. The metabolic derangement involving leukocyte Glc-6-PD activity may provide a basis for the study of other abnormalities of the leukocyte, the socalled neutrophil dysfunction syndrome [7].