Eur J Pediatr (1992) 151 : 279-282
European Journal of
Pediatrics
9 Springer-Verlag1992
Cytochrome b positive X-linked chronic granulomatous disease: a normal cell surface expression of cytochrome b H. Azuma 1, H. Oomi, D. Ueda, K. Sasaki, Y. Makita, K.Tomizawa, Y. Sakiyama, K. Fujita, H. Yoshioka, and A. Okuno 1Department of Paediatrics, Asahikawa Medical College, Nishikagura 4-5, Asahikawa, Hokkaido, Japan 078 2Department of Paediatrics Hokkaido University, Hokkaido, Japan Received September 7, 1990 / Accepted August 14, 1991
Abstract. The polymorphonuclear (PMN) cells from a patient with cytochrome b positive Xqinked chronic granulomatous disease ( X b + C G D ) were studied using flow cytometry. Both the cell surface expression of monoclonal antibody defined cytochrome b and the superoxide production (intracellular 2' ,7'-Dichlorofluorescin Diacetate oxidation) were investigated at a single cell level. Flow cytometry clearly demonstrated the complete absence of superoxide production in the patient's PMN cells, the mosaicism in his mother's PMN cells and also indicated the normal cell surface expression of cytochrome b. The results obtained by Western blot analysis and reducedminus-oxidized spectra confirmed the presence of functional and normal amounts of cytochrome b. We concluded that this is a case of X b + C G D with a normal cell surface expression of cytochrome b.
Key words: Chronic granulomatous disease - Cytochrome b - Cell surface expression - 7D5 - Monoclonal antibody
Introduction Patients with chronic granulomatous disease (CGD) have recurrent severe bacterial infections because of a defective N A D P H oxidase system that fails to generate superoxide. This oxidase system is composed of both membrane and cytosolic components [2, 3, 8, 12, 13]. Cytochrome b is one of the membrane components and consists of two subunits; gp91-phox (heavy chain) and p22phox (light chain) [19-21]. In most cases with X-linked form of C G D , cytochrome b is absent due to a defect of the gene encoding its heavy chain ( X b - ) [9, 23, 24]. The existence of a rare X-linked form of C G D with positive Offprint requests to: H. Azuma Abbreviations: CGD = chronic granulomatous disease; NBT
= nitroblue tetrazdium; PMN = polymorphonuclear; Xb+ = X-linked cytochrome b positive
cytochrome b ( X b + ) has been reported by several investigators [1, 7, 17, 18, 22]. One cause of the X b + type was shown to be a missense mutation of cytochrome b heavy chain gene [10]. However, it is not clear whether cytochrome b is normally expressed on the cell surface of polymorphonuclear (PMN) cells from X b + C G D or not. In this paper we report a case of X b + C G D with a normal cell surface expression of cytochrome b and stress the usefulness of flow cytometry to establish the diagnosis.
Case report The patient is at present 13 years old. He was admitted to hospital with pneumonia at the age of 1 month. From 1 to 3 years, he suffered from perianal abscesses and swelling of the superficial lymph nodes. At the age of 3.5 years, he had multiple liver abscesses. Daily intake of a trimethoprim-suphisoxazole compound was started when he was 5 years old. After that, his condition was well controlled and he went to school without any major problems. Surprisingly, there is no need for daily intake of prophylactic antibiotics at present. His three brothers are all dead. Two of them died of bacterial infection when they were young and the diagnosis of CGD had been made based on a nitroblue tetrazolium (NBT) reduction test. His only sister, parents and his aunts appear to be healthy.
Material and method Heparinized whole blood was mixed with 6% dextran saline and allowed to stand for 15 min. The leucocyte rich plasma was overlayed on the top of a two layer Percoll solution and the PMN cell fraction was separated according to Hj orth et al. [11]. The remaining red blood cells were removed by using lysing buffer (0.87% NH4C1). After washing three times with 0.1% gel Hanks Balanced Salt Solution, the cells were suspended in the same solution. A quantitative NBT reduction test was performed according to Baehher and Nathan [4] without sodium azide. An endotoxin coated NBT slide test was done according to Ochs and Igo [16] using lipopolysaccharide B derived from Escherichia coli 055:B5 (Difco, Detroit, M[). Luminol dependent chemiluminescence was measured with a chemiluminometer (Picolight, Packard, Zurich, Switz). Briefly, the standard reaction mixture contained 200 gl of 0.1% Gel Hanks Balanced Salt Solution, 25gl of luminol solution (2 x 10-4M)
280 and 100 gl of PMN suspension (1 • 107/ml) and 100 ~tl of formylmethionyl-leucyl-phenylalanine(Sigma, St. Louis, MO) (1 • 10-7M). The relative chemiluminescence intensity was expressed as counts per minute. Superoxide anion liberation was measured kinetically by cytochrome C reduction using intact PMN cells. The slope at the inflexion point of the tracing was taken as the rate of cytochrome C reduction was described by Borregaard et al. [6]. Flow cytometric analysis of superoxide production by neutrophils was performed basically according to the method described by Bass et al. [5]. The monoclonal antibody specific for the light chain of cytochrome b (previously designated as 7D5 [15]) was used to evaluate the cell surface expression of cytochrome b. Fluoroscein isothiocyanate-labelled goat anit-mouse IgG was used as the second antibody. The assay of cytochrome b in PMN cells was carried out by means of the dithionite reduced-minus-oxidized difference spectrum, recorded with a spectrophotometer (Hitachi 557 Spectrophotometer, Tokyo, Japan) according to Ohno et al. [17]. Western blot analysis of the heavy and light chains of cytochrome b was performed using antibodies raised in rabbits immunized by synthetic peptides [corresponding to their carboxyl-terminal regions [21, 23] as haptens (ImajohOhmi et al. unpublished)].
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250
200
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Results T h e results o f t h e q u a n t i t a t i v e N B T r e d u c t i o n t e s t o n t h e p a t i e n t , m o t h e r a n d c o n t r o l was 0.028, 0.198 a n d 0.346, LU I-U3
Table 1. PMN cells data NBT reduction (AOD)
FMLP stimu- Cytochrome C lated chemi- reduction luminescence (nmole/min/106 cells) (Peak value,
0
50
:
1
:
I
100
150
cpm) Control Patient Mother Sister
0.346 0.028 0.198 N.T.
319 765 3 401 54 499 452248
9.7 _+3.4 (n = 5) N.D. 4.5 13.3
N.D., Not detected; N.T., not tested; FMLP = Formyl-methionyl-leucyl-phenylalanine; OD = optical density
Fig. 2. Flow cytometric analysis of superoxide production. The PMA (phorbol myristate acetate) solution used was 25 gg/ml. Only the results obtained after PMA stimulation are shown. In the patient's PMN cells, the peak observed before PMA stimulation (negative peak) did not shift to the right after stimulation. The mother's PMN cells showed two peaks. The negative peak did not shift to the right even when 100 gg/ml of PMA solution was employed. The sister's PMN cells showed normal pattern
Fig. 1. Endotoxin Coated slide test. (a) patient; (b) mother; (e) father; (d) control
281 400-
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5
1
2
3
4
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(pmol/10 6cells) 2o i1[1
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9 Control D Patient
OD = 0.011
a b Fig. 4. Western blot analysis. Membrane fractions from normal subjects (lane 1-3), this patient (Xb+) (lane 4), and patient (Xb-) (lane 5) were electrophoresed, electroblotted to polyvinylidene difluoride membranes, where heavy and light chains of cytochrome b were detected by antibodies (panel a, antiheavy chain; panel b, anti-light chain). The amounts of membrane fractions were as follow: lane 1, 4,5 from 105 cells; lane 2, from 5 • 1 0 4 cells; lane 3, from 2 • 1 0 4 cells. Membranes from normal neutrophils were diluted with erythrocyte lysates
Fig. 5, Reduced-minus-oxidized difference spectra. The patient's PMN cells gave a peak at 424 nm and 558 nm indicating the presence of functional cytochrome b. The amount of cytochrome b was measured by using the peak at 424nm. The level of cytochrome b in patient's PMN cells was 16.1 pmole/106 cells. The normal range was 10.5 + 3.7pmole/106 cells. OD, Optical density
respectively (Table 1). The p e a k value of formyl-methionyMeucyl-phenylalanine stimulated chemiluminescence of the patient, his m o t h e r and his sister's P M N cells was 3401cpm, 54499cpm, 452248cpm, respectively, compared with the contol value of 319765 cpm. Almost the same results were obtained when opsonized zymosan particles or phorbol myristate acetate were employed as a stimulant. The rate of cytochrome C reduction by the patient's P M N cells was not detectable. His m o t h e r and sister's P M N cells showed 4.5 nmole/min per 106 cells, 13.3 nmole/min per 106 cells, respectively as c o m p a r e d with the control value of 9.7 + 3.4nmole/min per 106 cells (n = 5). The result of the endotoxin coated slide test
(Fig. 1) showed that none of the patient's P M N cells reduced N B T at all, but the mother's P M N ceils showed a mosaic pattern. The flow cytometric analysis clearly demonstrated the mosaicism of P M N cells derived from his m o t h e r (Fig. 2). The flow cytometric study of the cell surface expression of cytochrome b suggested the normal expression of cytochrome b on both the patient and his m o t h e r ' s P M N cells (Fig. 3). The Western blot analysis of cytochrome b subunits revealed the presence of both a heavy and light chain at a normal level (Fig. 4). The spectroscopical study showed the p e a k at 424 nm and 558 nm suggesting the presence of a functional h a e m group of cytochrome b at a normal level (Fig. 5).
I
1
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400
500
600
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Mean = 10.5_+3.7
Wavelength (nm)
282 Discussion T h e superoxide g e n e r a t e d f r o m the patient's P M N cells was n o t detectable by any of the assay m e t h o d s used. In addition, P M N cells from the m o t h e r showed intermediate values in every e x p e r i m e n t and s h o w e d the mosaic pattern in b o t h the e n d o t o x i n c o a t e d slide test (Fig. 1) and the flow cytometric study (Fig. 2). All these l a b o r a t o r y findings and the family history strongly suggested that this case was X-linked C G D . I n addition, it should be stressed that the findings o b t a i n e d by the flow c y t o m e t r y were useful in confirming the diagnosis. M i z u n o et al. [14] have r e p o r t e d the absence of 7D5 defined c y t o c h r o m e b o n the surface of P M N cells f r o m patients with X b - C G D . W e have s h o w n here that in the case of X b + C G D , c y t o c h r o m e b might be present on the cell surface at the n o r m a l level. Thus, the detection of cell surface c y t o c h r o m e b might have diagnostic values in distinguishing b e t w e e n X b + and X b - C G D . It was not clear w h e t h e r the h e a v y chain which is linked to X - c h r o m o s o m e was expressed n o r m a l l y on the cell surface b e c a u s e 7D5 was specific for the light chain. B u t b o t h the W e s t e r n blot analysis and the spectroscopic analysis of c y t o c h r o m e b suggested n o r m a l c y t o c h r o m e b function (Figs. 4, 5). Thus, the patient's c y t o c h r o m e b a p p e a r e d to be quite intact and m u s t be expressed o n the cell surface. T h e r e f o r e , it is m o s t likely that there is a point m u t a t i o n in the h e a v y chain gene affecting only the electron transport [9] leaving the c y t o c h r o m e b (including its cell surface expression) functionally intact. T h e p r o b a b l e point m u t a t i o n o f the gene coding the h e a v y chain is n o w u n d e r investigation. In conclusion: 1. W e present a case of X b + C G D with n o r m a l cell surface expression of c y t o c h r o m e b. 2. T h e detection of c y t o c h r o m e b on the cell surface using flow c y t o m e t r y appears to be useful for the diagnosis of X b + C G D . 3. A point m u t a t i o n affecting only electron transport could be suggested to explain the defective superoxide p r o d u c t i o n in the patient's P M N cells.
Acknowledgements. This work was supported in part by a grant from The Ministry of Health and Hygiene. The authors wish to thank Dr. S. Kanegasaki and S. Oomi at The Institute of Medical Science, The University of Tokyo for the Western blot analysis of cytochrome b, Dr.M. Nakamura for the gift of 7D5 monoclonal antibody and Yoshimi Kiya for preparing the manuscript. References 1. Ament ME, Ochs HD (1973) Gastrointestinal manifestation of chronic granulomatous disease. N Engl J Med 288: 382-387 2. Babior BM (1988) The respiratory burst oxidase. Hematol Oncol Clin North Am 2 : 201-212 3. Babior BM, Kuver R, Curnutte JT (1988) Kinetics of activation of the respiratory burst oxidase in a fully soluble system from human neutrophils. J Biol Chem 263 : 1713-1718 4. Baehner RL, Nathan DG (1968) Quantitative nitroblue tetrazolium test in chronic granulomatous disease. N Engl J Med 278 : 971-976 5. Bass DA, Parce JW, Dechatelet LR, Szeijda P, Seeds MC, Thomas M (1983) Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stimulation. J Immunol 130 : 1910-1917 6. Borregaard N, Johansen KS, Esmann V (1979) Quantitation of superoxide production in human polymorphonuclear leuko-
cytes from normals and 3 types of chronic granulomatous disease. Biochem Biophys Res Commun 90 : 214-219 7. Clark RA, Malech HL, Gallin JI, Nunoi H, Volpp BD, Pearson DW, Nauseef WM, Curnutte JT (1989) Genetic variants of chronic granulomatous disease: prevalence of deficiency of two cytosolic components of the NADPH oxidase system. N Eng J Med 321 : 647-652 8. Curnutte JT (1988) Classification of chronic granulomatous disease. Hematol Oncol Clin North Am 2 : 241-252 9. Dinauer MC, Orkin SH, Brown R, Jesaitis AJ, Parkos CA (1987) The glycoprotein encoded by the X-linked chronic granulomatous disease locus in a component of the neutrophil cytochrome b complex. Nature 327: 717-720 10. Dinauer MC, Curnutte JT, Rosen H, Orkin SH (1989) A missense mutation in the neutrophil cytochrome b heavy chain in cytochrome-positive X-linked chronic granulomatous disease. J Clin Invest 84: 2012-2016 11. Hjorth R, Jonsson AK, Vretblad P (1981) A rapid method for purification of human granulocytes using Percoll. A comparison with dextran sedimentation. Immunol Method 43 : 95-101 12. Lehrer RI, Ganz T, Selsted ME, Babior BM, Curnutte JT (1988) Neutrophils and host defense. Ann Intern Med 109: 127-142 13. McPhail LC, Shirley PS, Clayton CC, Snyderman R (1985) Activation of the respiratory burst enzyme from human neutrophils in a cell-free system: evidence for a soluble cofactor. J Clin Invest 75 : 1735-1739 14. Mizuno Y, Hara T, Nakamura M, Ueda K, Minakami S, Take H (1988) Classification of chronic granulomatous disease on the basis of monoclonal antibody-defined cytochrome b deficiency. J Pediatr 113 : 458-462 15. Nakamura M, Sendo S, Zwieten RZ, Koga T, Roos D, Kanegasaki S (1988) Immunocytochemical discovery of the 22- to 23-Kd subunit of cytochrome b558at the surface of human peripheral phagocytes. Blood 72:1550-1552 16. Ochs HD, Igo RP (1973) The NBT slide test: a simple screening method for detecting chronic granulomatous disease and female carriers. J Pediatr 83 : 77-82 17. Ohno Y, Buescher ES, Roberts R, Metcalf JA, Galin JI (1986) Reevaluation of cytochrome b and flavin adenine dinucleotide in neutrophils from patients with chronic granulomatous disease and description of a family with probable autosomal recessive inheritance of cytochrome b deficiency. Blood 67:1132-1138 18. Okamura N, Malawista SE, Roberts RL, Rosen H, Ochs HD, Babior BM, Curnutte JT (1988) Phosphorylation of the oxidase-related 48K phosphoprotein family in the unusual autosomal cytochrome-negative and X-linked cytochrome-positive types of chronic granulomatous disease. Blood 72: 811-816 19. Parkos CA, Allen RA, Cochrane CG, Jesaitis AJ (1987) Purified cytochrome b from human granulocyte plasma membrane is composed of two polypeptide with relative molecular weights of 91,000 and 22,000. J Clin Invest 80 : 732-742 20. Parkos CA, Alien RA, Cochrane CG, Jesaitis AJ (1988) The quarternary structure of the plasma membrane b type cytochrome of human granulocytes. Biochim Biophys Acta 932: 71-83 21. Parkos CA, Dinauer MC, Walker LE, Allen RA, Jesaitis AJ, Orkin SH (1988) Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b. Proc Natl Acad Sci USA 85 : 3319-3323 22. Rosen H, Klebanoff SJ (1976) Chemiluminescence and superoxide production by myeloperoxidase-deficient leukocytes. J Clin Invest 58 : 50-60 23. Royer-Pokora B, Kunkel LM, Monaco AP, Doff SC, Newburger PE, Baehner RL, Cole FS, Curnutte JT, Orkin SH (1986) Cloning the gene for an inherited human disorder chronic granulomatous disease - on the basis of its chromosomal location. Nature 322 : 32-38 24. Segal AW (1987) Absence of both cytoehrome b-245 subunits from neutrophils in X-linked chronic granulomatous disease. Nature 326 : 88-91
European Journal of
Pediatrics
9 Springer-Verlag1992
Cytochrome b positive X-linked chronic granulomatous disease: a normal cell surface expression of cytochrome b H. Azuma 1, H. Oomi, D. Ueda, K. Sasaki, Y. Makita, K.Tomizawa, Y. Sakiyama, K. Fujita, H. Yoshioka, and A. Okuno 1Department of Paediatrics, Asahikawa Medical College, Nishikagura 4-5, Asahikawa, Hokkaido, Japan 078 2Department of Paediatrics Hokkaido University, Hokkaido, Japan Received September 7, 1990 / Accepted August 14, 1991
Abstract. The polymorphonuclear (PMN) cells from a patient with cytochrome b positive Xqinked chronic granulomatous disease ( X b + C G D ) were studied using flow cytometry. Both the cell surface expression of monoclonal antibody defined cytochrome b and the superoxide production (intracellular 2' ,7'-Dichlorofluorescin Diacetate oxidation) were investigated at a single cell level. Flow cytometry clearly demonstrated the complete absence of superoxide production in the patient's PMN cells, the mosaicism in his mother's PMN cells and also indicated the normal cell surface expression of cytochrome b. The results obtained by Western blot analysis and reducedminus-oxidized spectra confirmed the presence of functional and normal amounts of cytochrome b. We concluded that this is a case of X b + C G D with a normal cell surface expression of cytochrome b.
Key words: Chronic granulomatous disease - Cytochrome b - Cell surface expression - 7D5 - Monoclonal antibody
Introduction Patients with chronic granulomatous disease (CGD) have recurrent severe bacterial infections because of a defective N A D P H oxidase system that fails to generate superoxide. This oxidase system is composed of both membrane and cytosolic components [2, 3, 8, 12, 13]. Cytochrome b is one of the membrane components and consists of two subunits; gp91-phox (heavy chain) and p22phox (light chain) [19-21]. In most cases with X-linked form of C G D , cytochrome b is absent due to a defect of the gene encoding its heavy chain ( X b - ) [9, 23, 24]. The existence of a rare X-linked form of C G D with positive Offprint requests to: H. Azuma Abbreviations: CGD = chronic granulomatous disease; NBT
= nitroblue tetrazdium; PMN = polymorphonuclear; Xb+ = X-linked cytochrome b positive
cytochrome b ( X b + ) has been reported by several investigators [1, 7, 17, 18, 22]. One cause of the X b + type was shown to be a missense mutation of cytochrome b heavy chain gene [10]. However, it is not clear whether cytochrome b is normally expressed on the cell surface of polymorphonuclear (PMN) cells from X b + C G D or not. In this paper we report a case of X b + C G D with a normal cell surface expression of cytochrome b and stress the usefulness of flow cytometry to establish the diagnosis.
Case report The patient is at present 13 years old. He was admitted to hospital with pneumonia at the age of 1 month. From 1 to 3 years, he suffered from perianal abscesses and swelling of the superficial lymph nodes. At the age of 3.5 years, he had multiple liver abscesses. Daily intake of a trimethoprim-suphisoxazole compound was started when he was 5 years old. After that, his condition was well controlled and he went to school without any major problems. Surprisingly, there is no need for daily intake of prophylactic antibiotics at present. His three brothers are all dead. Two of them died of bacterial infection when they were young and the diagnosis of CGD had been made based on a nitroblue tetrazolium (NBT) reduction test. His only sister, parents and his aunts appear to be healthy.
Material and method Heparinized whole blood was mixed with 6% dextran saline and allowed to stand for 15 min. The leucocyte rich plasma was overlayed on the top of a two layer Percoll solution and the PMN cell fraction was separated according to Hj orth et al. [11]. The remaining red blood cells were removed by using lysing buffer (0.87% NH4C1). After washing three times with 0.1% gel Hanks Balanced Salt Solution, the cells were suspended in the same solution. A quantitative NBT reduction test was performed according to Baehher and Nathan [4] without sodium azide. An endotoxin coated NBT slide test was done according to Ochs and Igo [16] using lipopolysaccharide B derived from Escherichia coli 055:B5 (Difco, Detroit, M[). Luminol dependent chemiluminescence was measured with a chemiluminometer (Picolight, Packard, Zurich, Switz). Briefly, the standard reaction mixture contained 200 gl of 0.1% Gel Hanks Balanced Salt Solution, 25gl of luminol solution (2 x 10-4M)
280 and 100 gl of PMN suspension (1 • 107/ml) and 100 ~tl of formylmethionyl-leucyl-phenylalanine(Sigma, St. Louis, MO) (1 • 10-7M). The relative chemiluminescence intensity was expressed as counts per minute. Superoxide anion liberation was measured kinetically by cytochrome C reduction using intact PMN cells. The slope at the inflexion point of the tracing was taken as the rate of cytochrome C reduction was described by Borregaard et al. [6]. Flow cytometric analysis of superoxide production by neutrophils was performed basically according to the method described by Bass et al. [5]. The monoclonal antibody specific for the light chain of cytochrome b (previously designated as 7D5 [15]) was used to evaluate the cell surface expression of cytochrome b. Fluoroscein isothiocyanate-labelled goat anit-mouse IgG was used as the second antibody. The assay of cytochrome b in PMN cells was carried out by means of the dithionite reduced-minus-oxidized difference spectrum, recorded with a spectrophotometer (Hitachi 557 Spectrophotometer, Tokyo, Japan) according to Ohno et al. [17]. Western blot analysis of the heavy and light chains of cytochrome b was performed using antibodies raised in rabbits immunized by synthetic peptides [corresponding to their carboxyl-terminal regions [21, 23] as haptens (ImajohOhmi et al. unpublished)].
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Results T h e results o f t h e q u a n t i t a t i v e N B T r e d u c t i o n t e s t o n t h e p a t i e n t , m o t h e r a n d c o n t r o l was 0.028, 0.198 a n d 0.346, LU I-U3
Table 1. PMN cells data NBT reduction (AOD)
FMLP stimu- Cytochrome C lated chemi- reduction luminescence (nmole/min/106 cells) (Peak value,
0
50
:
1
:
I
100
150
cpm) Control Patient Mother Sister
0.346 0.028 0.198 N.T.
319 765 3 401 54 499 452248
9.7 _+3.4 (n = 5) N.D. 4.5 13.3
N.D., Not detected; N.T., not tested; FMLP = Formyl-methionyl-leucyl-phenylalanine; OD = optical density
Fig. 2. Flow cytometric analysis of superoxide production. The PMA (phorbol myristate acetate) solution used was 25 gg/ml. Only the results obtained after PMA stimulation are shown. In the patient's PMN cells, the peak observed before PMA stimulation (negative peak) did not shift to the right after stimulation. The mother's PMN cells showed two peaks. The negative peak did not shift to the right even when 100 gg/ml of PMA solution was employed. The sister's PMN cells showed normal pattern
Fig. 1. Endotoxin Coated slide test. (a) patient; (b) mother; (e) father; (d) control
281 400-
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a b Fig. 4. Western blot analysis. Membrane fractions from normal subjects (lane 1-3), this patient (Xb+) (lane 4), and patient (Xb-) (lane 5) were electrophoresed, electroblotted to polyvinylidene difluoride membranes, where heavy and light chains of cytochrome b were detected by antibodies (panel a, antiheavy chain; panel b, anti-light chain). The amounts of membrane fractions were as follow: lane 1, 4,5 from 105 cells; lane 2, from 5 • 1 0 4 cells; lane 3, from 2 • 1 0 4 cells. Membranes from normal neutrophils were diluted with erythrocyte lysates
Fig. 5, Reduced-minus-oxidized difference spectra. The patient's PMN cells gave a peak at 424 nm and 558 nm indicating the presence of functional cytochrome b. The amount of cytochrome b was measured by using the peak at 424nm. The level of cytochrome b in patient's PMN cells was 16.1 pmole/106 cells. The normal range was 10.5 + 3.7pmole/106 cells. OD, Optical density
respectively (Table 1). The p e a k value of formyl-methionyMeucyl-phenylalanine stimulated chemiluminescence of the patient, his m o t h e r and his sister's P M N cells was 3401cpm, 54499cpm, 452248cpm, respectively, compared with the contol value of 319765 cpm. Almost the same results were obtained when opsonized zymosan particles or phorbol myristate acetate were employed as a stimulant. The rate of cytochrome C reduction by the patient's P M N cells was not detectable. His m o t h e r and sister's P M N cells showed 4.5 nmole/min per 106 cells, 13.3 nmole/min per 106 cells, respectively as c o m p a r e d with the control value of 9.7 + 3.4nmole/min per 106 cells (n = 5). The result of the endotoxin coated slide test
(Fig. 1) showed that none of the patient's P M N cells reduced N B T at all, but the mother's P M N ceils showed a mosaic pattern. The flow cytometric analysis clearly demonstrated the mosaicism of P M N cells derived from his m o t h e r (Fig. 2). The flow cytometric study of the cell surface expression of cytochrome b suggested the normal expression of cytochrome b on both the patient and his m o t h e r ' s P M N cells (Fig. 3). The Western blot analysis of cytochrome b subunits revealed the presence of both a heavy and light chain at a normal level (Fig. 4). The spectroscopical study showed the p e a k at 424 nm and 558 nm suggesting the presence of a functional h a e m group of cytochrome b at a normal level (Fig. 5).
I
1
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400
500
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Mean = 10.5_+3.7
Wavelength (nm)
282 Discussion T h e superoxide g e n e r a t e d f r o m the patient's P M N cells was n o t detectable by any of the assay m e t h o d s used. In addition, P M N cells from the m o t h e r showed intermediate values in every e x p e r i m e n t and s h o w e d the mosaic pattern in b o t h the e n d o t o x i n c o a t e d slide test (Fig. 1) and the flow cytometric study (Fig. 2). All these l a b o r a t o r y findings and the family history strongly suggested that this case was X-linked C G D . I n addition, it should be stressed that the findings o b t a i n e d by the flow c y t o m e t r y were useful in confirming the diagnosis. M i z u n o et al. [14] have r e p o r t e d the absence of 7D5 defined c y t o c h r o m e b o n the surface of P M N cells f r o m patients with X b - C G D . W e have s h o w n here that in the case of X b + C G D , c y t o c h r o m e b might be present on the cell surface at the n o r m a l level. Thus, the detection of cell surface c y t o c h r o m e b might have diagnostic values in distinguishing b e t w e e n X b + and X b - C G D . It was not clear w h e t h e r the h e a v y chain which is linked to X - c h r o m o s o m e was expressed n o r m a l l y on the cell surface b e c a u s e 7D5 was specific for the light chain. B u t b o t h the W e s t e r n blot analysis and the spectroscopic analysis of c y t o c h r o m e b suggested n o r m a l c y t o c h r o m e b function (Figs. 4, 5). Thus, the patient's c y t o c h r o m e b a p p e a r e d to be quite intact and m u s t be expressed o n the cell surface. T h e r e f o r e , it is m o s t likely that there is a point m u t a t i o n in the h e a v y chain gene affecting only the electron transport [9] leaving the c y t o c h r o m e b (including its cell surface expression) functionally intact. T h e p r o b a b l e point m u t a t i o n o f the gene coding the h e a v y chain is n o w u n d e r investigation. In conclusion: 1. W e present a case of X b + C G D with n o r m a l cell surface expression of c y t o c h r o m e b. 2. T h e detection of c y t o c h r o m e b on the cell surface using flow c y t o m e t r y appears to be useful for the diagnosis of X b + C G D . 3. A point m u t a t i o n affecting only electron transport could be suggested to explain the defective superoxide p r o d u c t i o n in the patient's P M N cells.
Acknowledgements. This work was supported in part by a grant from The Ministry of Health and Hygiene. The authors wish to thank Dr. S. Kanegasaki and S. Oomi at The Institute of Medical Science, The University of Tokyo for the Western blot analysis of cytochrome b, Dr.M. Nakamura for the gift of 7D5 monoclonal antibody and Yoshimi Kiya for preparing the manuscript. References 1. Ament ME, Ochs HD (1973) Gastrointestinal manifestation of chronic granulomatous disease. N Engl J Med 288: 382-387 2. Babior BM (1988) The respiratory burst oxidase. Hematol Oncol Clin North Am 2 : 201-212 3. Babior BM, Kuver R, Curnutte JT (1988) Kinetics of activation of the respiratory burst oxidase in a fully soluble system from human neutrophils. J Biol Chem 263 : 1713-1718 4. Baehner RL, Nathan DG (1968) Quantitative nitroblue tetrazolium test in chronic granulomatous disease. N Engl J Med 278 : 971-976 5. Bass DA, Parce JW, Dechatelet LR, Szeijda P, Seeds MC, Thomas M (1983) Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stimulation. J Immunol 130 : 1910-1917 6. Borregaard N, Johansen KS, Esmann V (1979) Quantitation of superoxide production in human polymorphonuclear leuko-
cytes from normals and 3 types of chronic granulomatous disease. Biochem Biophys Res Commun 90 : 214-219 7. Clark RA, Malech HL, Gallin JI, Nunoi H, Volpp BD, Pearson DW, Nauseef WM, Curnutte JT (1989) Genetic variants of chronic granulomatous disease: prevalence of deficiency of two cytosolic components of the NADPH oxidase system. N Eng J Med 321 : 647-652 8. Curnutte JT (1988) Classification of chronic granulomatous disease. Hematol Oncol Clin North Am 2 : 241-252 9. Dinauer MC, Orkin SH, Brown R, Jesaitis AJ, Parkos CA (1987) The glycoprotein encoded by the X-linked chronic granulomatous disease locus in a component of the neutrophil cytochrome b complex. Nature 327: 717-720 10. Dinauer MC, Curnutte JT, Rosen H, Orkin SH (1989) A missense mutation in the neutrophil cytochrome b heavy chain in cytochrome-positive X-linked chronic granulomatous disease. J Clin Invest 84: 2012-2016 11. Hjorth R, Jonsson AK, Vretblad P (1981) A rapid method for purification of human granulocytes using Percoll. A comparison with dextran sedimentation. Immunol Method 43 : 95-101 12. Lehrer RI, Ganz T, Selsted ME, Babior BM, Curnutte JT (1988) Neutrophils and host defense. Ann Intern Med 109: 127-142 13. McPhail LC, Shirley PS, Clayton CC, Snyderman R (1985) Activation of the respiratory burst enzyme from human neutrophils in a cell-free system: evidence for a soluble cofactor. J Clin Invest 75 : 1735-1739 14. Mizuno Y, Hara T, Nakamura M, Ueda K, Minakami S, Take H (1988) Classification of chronic granulomatous disease on the basis of monoclonal antibody-defined cytochrome b deficiency. J Pediatr 113 : 458-462 15. Nakamura M, Sendo S, Zwieten RZ, Koga T, Roos D, Kanegasaki S (1988) Immunocytochemical discovery of the 22- to 23-Kd subunit of cytochrome b558at the surface of human peripheral phagocytes. Blood 72:1550-1552 16. Ochs HD, Igo RP (1973) The NBT slide test: a simple screening method for detecting chronic granulomatous disease and female carriers. J Pediatr 83 : 77-82 17. Ohno Y, Buescher ES, Roberts R, Metcalf JA, Galin JI (1986) Reevaluation of cytochrome b and flavin adenine dinucleotide in neutrophils from patients with chronic granulomatous disease and description of a family with probable autosomal recessive inheritance of cytochrome b deficiency. Blood 67:1132-1138 18. Okamura N, Malawista SE, Roberts RL, Rosen H, Ochs HD, Babior BM, Curnutte JT (1988) Phosphorylation of the oxidase-related 48K phosphoprotein family in the unusual autosomal cytochrome-negative and X-linked cytochrome-positive types of chronic granulomatous disease. Blood 72: 811-816 19. Parkos CA, Allen RA, Cochrane CG, Jesaitis AJ (1987) Purified cytochrome b from human granulocyte plasma membrane is composed of two polypeptide with relative molecular weights of 91,000 and 22,000. J Clin Invest 80 : 732-742 20. Parkos CA, Alien RA, Cochrane CG, Jesaitis AJ (1988) The quarternary structure of the plasma membrane b type cytochrome of human granulocytes. Biochim Biophys Acta 932: 71-83 21. Parkos CA, Dinauer MC, Walker LE, Allen RA, Jesaitis AJ, Orkin SH (1988) Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b. Proc Natl Acad Sci USA 85 : 3319-3323 22. Rosen H, Klebanoff SJ (1976) Chemiluminescence and superoxide production by myeloperoxidase-deficient leukocytes. J Clin Invest 58 : 50-60 23. Royer-Pokora B, Kunkel LM, Monaco AP, Doff SC, Newburger PE, Baehner RL, Cole FS, Curnutte JT, Orkin SH (1986) Cloning the gene for an inherited human disorder chronic granulomatous disease - on the basis of its chromosomal location. Nature 322 : 32-38 24. Segal AW (1987) Absence of both cytoehrome b-245 subunits from neutrophils in X-linked chronic granulomatous disease. Nature 326 : 88-91
