Deficiency of NADPH oxidase activity in chronic granulomatous disease

February 1977 The Journal o f P E D I A T R I C S

213

Deficiency of NADPH oxidase activity in chronic granulomatous disease NADPH oxidase activity was examined in paired 27,000 • g granule fractions isolated from normal polymorphonuclear leukocytes and leukocytes from patients with chronic granulomatous disease. A t O.17 mM NADPH, the oxidase activity was not measurable in normal resting cells but was activated by phagocytosis. This activation was absent in CGD cells. At higher levels o[ NADPH, activity was present in cells from patients with CGD, although it was lower than normal, and no difference in activity was found between resting and phagocytizing cells. Granule fractions from phagocytizing normal cells exhibited higher activity than granule fractions from resting normal cells at all levels of NADPH. These results suggest that NA DPH oxidase activity is defective in chronic granulomatous disease, and further that the defect is not the absence of the enzyme but rather a faihtre to activate it.

Linda C. McPhail, Lawrence R. DeChatelet,* Pamela S. Shirley, Catherine Wilfert, Richard B. Johnston, Jr., and Charles E. McCall, Winston-Salem

a n d D u r h a m , N . C . , a n d B i r m i n g h a m , A la.

CHRONIC GRANULOMATOUS DISEASE is a condition which usually appears during childhood and is characterized by recurrent and persistent bacterial infections. These infections are secondary to the inability of the patient's neutrophils to destroy certain bacteria? Granulocytes from these patients fail to exhibit the burst of metabolic activity which normally accompanies phagocytosis. The study of these neutrophils has enabled researchers to pinpoint many of the metabolic events occurring in the cell which may be responsible for its bactericidal activity. Much of this work has been summarized in a recent review.'-' One of the problems yet to be satisfactorily resolved is

From the Departments of Biochemistry and Medicine, The Bowman Gray School of Medicine, the Department of Pediatrics, Duke University Medical Center, and the Departments of Pediatrics and Microbiology, University of Alabama Medical Center Supported in part by a grant from the Forsyth Cancer Service and by United States Public Health Service Grants AI-10732, A1-10286, CA-12197, CA-13148, and CA-16673 from the National Institutes of Health. *Reprint address: Department of Biochemistry, Bowman Grav School of Medicine, Winston-Salem, NC 27103.

the identity of the enzyme which triggers the respiratory burst in normal cells and is defective in chronic granulomatous disease. Various candidates have been proposed, but primary emphasis has been on an oxidase which utilizes NADH, NADPH, or both. Controversy has arisen over which pyridine nucleotide is the important substrate physiologically, and evidence has been presented for both Abbreviations used CGD: chronic granulomatous disease PMNL: polymorphonuclear leukocytes PBS: phosphate-buffered saline NADH and NADPH. Baehner and Karnovsky:' have described a soluble NADH oxidase which was found to have depressed activity in leukocytes from patients with chronic granulomatous disease. They also have recently reported a plasma membrane-associated NADH oxidase activity which was increased upon phagocytosis.' On the other hand, a granule-associated NADPH oxidase which was activated by phagocytosis has been reported in guinea pig =' and human .... leukocytes. This activity has been demonstrated to be severely depressed in patients with CGD. ~.~

Vot 90, No. 2, pp. 213-217

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McPhail et al.

The Journal of Pediatrics February 1977

Table I. Description of patients studied Patient

Sex

Age

Mode of inheritance

J.P. A.C. B.C. S.C. J.R. T.S. T.B. R.S. R.D.

M F M F M F M M M

I 1 yr 6 yr 21 yr 19 yr 9 mo 2 yr 16 yr 16 yr 7 yr

Autosomal recessive Autosomal recessive Autosomol recessive Autosomal recessive X-linked recessive Autosomal recessive Autosomal recessive Autosomal recessive X-linked recessive

Table II. N A D P H oxidase activity in granules isolated from CGD and normal cells NADPH Oxidase C.P.M. Condition

Normal

[

Spontaneous 2,323 _+ 349 (12) oxidation Resting granule 2,028 + 335 (14) fraction Phagocytizing 7,239 _+ 1.484 (14) granule fraction

CGD 2,323 _+ 349 (12) 1,484 _+ 117 (12) 1,884 _+ 411 (14)

Concentrationof NADPH was 0.17 mM. Numbers in parentheses denote number of experiments. Values are the mean _+ SE. Experiments were carried oul as described in Methods. Normal and CGD samples were paired in each experiment and were assayed in triplicate. Spontaneous oxidation of NADPH was determined in each paired experiment and tabulated in each column.

One criticism of the work with N A D P H oxidase has been the requirement of manganese ion for maximal activity. Two recent reports presented evidence that manganese, in the presence of superoxide anion, actually stimulated a nonenzymatic oxidation of N A D P H : " Neutrophils have been shown to produce superoxide'"; thus the true enzymatic activity of N A D P H oxidase is likely to be obscured in the presence of manganese. This report is directed toward the examination of N A D P H oxidase activity without added manganese in normal neutrophils and in neutrophils from patients with chronic granulomatous disease. MATERIALS

AND METHODS

Nine patients with chronic granulomatous disease were studied in a total of 14 separate experiments. A brief description of each is given in Table [. Patients R. S.) ~ T. B.? z A. C., and B. CY ~have been described previously. Patients A. C., B. C., and S. C. are siblings. Diagnosis in each patient was based on clinical symptoms and metabolic studies on the isolated ceils. These studies included

NBT reduction, oxygen consumption, hexose monophosphate shunt activity, superoxide production, and bactericidal activity. Two patients, J. R. and R. D., were determined to have the X-linked recessive mode of inheritance by intermediate values of the metabolic tests in the mother and grandmother of each, respectively. The other patients appeared to have the non-X-linked, presumably autosomal recessive, form of the disease. Only three patients had no signs of infection at the time of the study, although the presence of infection seemed to have no effect on results obtained. Glutathione peroxidase activity was measured in six patients (including one of the patients with the X-linked disease) and was found to be normal. In each study, blood samples were also obtained from one or two normal adult volunteers and these were treated exactly as the blood from the patients. Experiments were performed according to the principles of the Declaration of Helsinki and informed consent was obtained. Polymorphonuclear leukocytes were isolated from heparinized venous blood as previously described: Cell preparations routinely contained 85 to 90% PMNL's. The N A D P H oxidase activity was assayed in a 27,000 x g particulate fraction (termed the granule fraction) prepared from isolated neutrophils. The procedures for preparation of granule fraction and assay of N A D P H oxidase activity have been described in detail in a previous publication/ Briefly, isolated PMNL's were incubated for three minutes with phosphate-buffered saline and designated as resting cells, or with zymosan (ICN Nutritional Biochemical Div.) opsonized with serum and designated as phagocytizing ceils. Incubation mixes were homogenized in 0.34 M sucrose for five minutes in order to obtain greater than 90% cell breakage. Homogenates were centrifuged at 500 x g for ten minutes to sediment unbroken cells, nuclei, and large debris. Supernates from this centrifugation were then recentrifuged at 27,000 x g for 15 minutes. Final pellets were resuspended in 0.34 M sucrose, and protein concentration was determined by the method of Lowry and colleagues. 1~ Before use in each assay, granule fractions were adjusted to the same level of protein. N A D P H oxidase activity was assayed in a two-step incubation procedure. NADP ~ was first generated in a reaction mixture containing 0.1 M potassium phosphate buffer, pH 5.5, 2.0 m M KCN, 0.1 to 0.2 mg granule fraction, and concentrations of N A D P H varying from 0.17 to 2.5 mM. Controls, in which the granule fraction was omitted, were included in each experiment in order to obtain a measure of the spontaneous oxidation of NADPH. The incubation was stopped after 30 minutes at 37 ~ C, and the amount of N A D P ~ produced was quanti-

Volume 90 Number 2

tated in a second incubation utilizing 0.1 ~Ci [1-'4C] 6phosphogluconate (New England Nuclear Corp.) and 1 unit 6-phosphogluconate dehydrogenase (Sigma Chemical Co.). "CO_, was generated in an amount directly proportional to the amount of NADP- originally produced. This was trapped and counted as previously described.':' Certain modifications were made in the original procedureT: (1) manganese ion was eliminated from the initial reaction mixture and (2) isotope with a 10fold higher specific activity (0.4 tzCi/>mole) was substituted in the second incubation.

?CAD P H oxidase activity in CGD

40.0 --

2!5

~

NORMAL

32.0

P R

C

240

16.0 8o

RESULTS NADPH oxidase activity was examined in paired granule preparations isolated from CGD cells and normal cells. Summarized in Table II are the accumulated data at 0.17 mM NADPH. No activity was apparent in granule fractions obtained from either normal resting cells or resting cells from patients with CGD. In fact, resting preparations from CGD patients actually seemed to inhibit the spontaneous oxidation of NADPH. The amount of this inhibition varied from patient to patient and was also apparent a times with resting normal cells. In every case, activity was present in preparations isolated from phagocytizing normal cells, but none was measurable in those obtained from phagocytizing CGD cells. Thus, in normal cells, NADPH oxidase activity was activated by phagocytosis, but this activation did not seem to occur in cells from patients with CGD. Further experiments were carried out at higher levels of NADPH to investigate the possibility that the enzyme might simple be inactive in CGD cells at low substrate concentrations. Fig. 1 is a summary of data obtained by varying the NADPH concentration from 0.17 mM to 2.5 raM. NADPH oxidase activity found in granule fractions from resting and phagocytizing normal cells is shown in the upper panel. Measurable activity was absent in resting cells at 0.17 mM NADPH but was apparent at higher concentrations. Activity was present in granules from phagocytizing cells at all levels of NADPH; this activity was higher than that found in resting cells. The lower panel exhibits results obtained from the patients with CGD. NADPH oxidase activity was not apparent in granules from either resting or phagocytizing cells at 0.17 mM NADPH. Activity appeared at higher levels of substrate, although it was lower than normal. In contrast to normal cells, there seemed to be little difference in activity between the resting and the phagocytizing CGD cells. Although NADPH oxidase activity was always absent in CGD cells at 0.17 mM NADPH, we observed some variation in the amount of activity expressed at higher