Unusual forms of an uncommon disease (chronic granulomatous disease)
defined as a syndrome of recurrent purulent infections, most commonly of the skin, subcutaneous tissues, and reticuloendothelial organs, associated with a particular defect of phagocyte function: Patients' cells can ingest bacteria and fungi normally but cannot kill catalasepositive organisms that do not effectively produce hydrogen peroxide (H20 0 ) Underlying the bactericidal defect is an inability of patients' phagocytes to convert oxygen into metabolites that are required to kill most bacteria and to reduce nitroblue tetrazolium dye. Establishing the basic abnormality of C G D has substantiated recent findings regarding normal phagocyte function. More specifically, the CGD cell, with its clinically important bactericidal defect, has been an invaluable means of determining the interrelationships between the oxidative metabolism of neutrophils and monocytes and their special role in host defense. A current conceptual model of these interrelationships is diagrammed in Fig. 1. The burst of oxidative metabolic activity that occurs with phagocytosis apparently begins when initial contact is made between an opsonized microbe (coated with antibody or C3 or both) and the phagocytic cell membrane. This contact activates a cell surface-associated enzyme (oxidase) that then catalyzes the transfer of one electron from an undetermined substrate to oxygen. The resultant product is oxygen with an extra electron, an unstable free radical termed superoxide anion (OD2 Two superoxide radicals interact spontaneously to generate H~O~ and another unstable variant of oxygen, singlet oxygen ('0~). ~ H20~ and O~ can interact to form hydroxyl radical (~ OH), ~ ' a potent oxidizing agent, since it needs an electron to achieve a more stable state. The energy inherent in singlet oxygen can be released in oxidation
From the Departments of Pediatrics and Microbiology University o f Alabama Medical Center. Supported by United State& Public Health Service grants A I 10286, CA 13148, CA 16673, and RR 00032.
The Journal of P E D I A T R I C S Vol. 88, No. 1, pp. 172-174.
reactions or as light, and this agent is thought to cause the luminescence that accompanies phagocytosis/. 4 Transfer of the "extra" electrons from superoxide anions appears responsible for most of the NBT reduction of phagocytic cells.4. See related article, p. 63.
Abbreviations used CGD: chronic granulomatous disease NBT: nitroblue tetrazolium G-6-PD: glucose-6-phosphate dehydrogenase NADH: nicotinamide adenine dinucleotide reduced NADPH: nicotinamide adenine dinucleotide reduced phosphate Inasmuch as a portion of the cell membrane forms the boundaries of the phagocytic vacuole, after phagocytosis a mechanism for converting molecular oxygen to these highly reactive species would be located between the captured bacteria and the cytoplasm of the cell. Thus these agents would accumulate in the vacuole where they could act on the captured microorganisms without interference from their inactivators, superoxide dismutase and catalase, in the cytoplasm. Yet these enzymes would protect critical cellular components from the destructive effects ofsuperoxide and H202, respectively, and their byproducts. Lysosomal degranulation would introduce digestive hydrolytic enzymes, cationic proteins, and myeloperoxidase into the vacuole. The last has been well shown to boost the bactericidal and fungicidal effects of H~O~ in conjunction with chloride or iodide ions 6 and could stimulate further free-radical or singlet oxygen formation from H~O~.'.6 Cells from patients with CGD do not undergo the normal phagocytosis-associated increase in oxygen consumption, superoxide and HzO~ generation, NBT reduction, or chemiluminescence. In this issue Biggar, Buron, and Holmes describe a case of CGD with unusual clinical and genetic features, which raises the question of variants of the disease. This important question is currently impossible to answer since so
Volume 88 Number 1
tittle is known about the basic molecular defect of CGD, presumably deficient activity of an enzyme responsible for conversion of oxygen to bactericidal species. N A D H oxidase, NADPH oxidase, and glutathione peroxidase have each been proposed as the enzyme deficient in CGD, but none have been clearly proved as such. v' 8 Deficiency of G-6-PD involving leukocytes as well as erythrocytes represents t h e first enzyme defect shown clearly to cause CGD, as defined above. When G-6-PD levels are less than 1% of normal, the patient suffers a clinical syndrome that mimics CGD. 9 These patients' cells do not reduce NBT or generate H~O2, which has been attributed to a lack ofsubstrate (NADH and N A D P H ) for the enzymatic conversion of oxygen. TM Only a small percentage of CGD cases can be attributed to this particular abnormality. There are at least two different types of C G D if one considers genetic transmission. Evidence that the disorder can be passed in families as an X-linked recessive trait 'is strong: The transmission pattern is typical, mothers and sisters can be identified as carriers b y phagocytic bactericidal and biochemical assays, and at least two mothers have delivered affected offspring with different fathers. The disease also occurs in girls, however, and in sisters, siblings of both sexes, TM 12and some boys without demonstrable leukocyte defects in either parent. Transmission by an autosomal recessive gene has seemed most likely in these cases, but rarer mechanisms are possible in at least some of the families. The existence of other "varieties" of C G D is less definite. For example, a patient reported to have a neutrophil bactericidal defect confined to staphylococci on the basis of in vitro studies had septicemia and a paravertebral abscess due to Serratia marcescens and died o f Pseudomonas pneumonia and septicemia. Variable susceptibility of different bacterial strains to killing by oxygen metabolites 1~ could explain such a discrepancy. Patients have been reported who had C G D in association with Klinefelter syndrome, IgA deficiency, a serum inhibitor of chemotaxis, defective leukocyte motility, serum inhibitors of lymphocyte transformation, or cryoglobulinemia. These abnormalities could have occurred by chance or as secondary phenomena. Three sisters with the neutrophil biochemical and bactericidal defects of C G D but a milder course and absence of granuloma formation," and two brothers with mild disease and a partial defect in phagocyte function 18 could well represent variant disorders. Separation of any of these unusual cases as a distinct entity, however, will require identification of a precise molecular defect that can explain the biochemical and functional abnormalities. Considering the fact that C G D is an uncommon condi-
-'- H202 + ~
+ PEROXlDASE 9
MICROBIAL DEATH Fig. 1. Schematic diagram of the conversion of oxygen by the phagocytic cell to by-products involved in killing ingested microorganisms, according to a conceptual model described in detail elsewhere:~ tion, the effort spent in evaluating, describing, publishing, and reading about its various aspects may seem disproportionate. Can this effort be justified? Other than improving diagnosis and management of patients, which is obviously important, what has been (and can be) gained from continued careful analysis of this disease?: 1. CGD is a model disease. Even though the fundamental enzyme defect is not known, cause-and-effect relationships between the metabolic abnormality (failure to convert oxygen to toxic metabolites), the functional cellular defect (inability to kill phagocytized bacteria), the laboratory abnormalities (compensatory leukocytosis and hypergammaglobulinemia), the clinical picture (chronic, indolent infections at sites of phagocytic cell accumulation), and the pathology (granulomas) are beautifully defined. The pedagogic importance of these interrelationships is immeasurable, not just in the area of host defense but also from the standpoint of analysis of basic disease mechanisms. That is, C G D reminds us all that there is a logic in the development of disease that can be deciphered, at least in part, and it encourages us to analyze sensitively the origin and steps in development of a patient's particular clinical and laboratory abnormalities, rather than merely to name his disease. 2. CGD has taught us about the normal. It has shown that phagocytic bactericidal activity Cannot be replaced in host defense by antibody, complement, or other factors, that a commensal relationship with many bacteria of low virulence is maintained only if phagocyte function is normal, that phagocytic oxygen metabolism is essential for the killing of many; if not most, bacteria a n d fungi, and that lysosomal degranulation is not enough by itself to accomplish this. Anyone who has tried to fix a
The Journal of Pedialrics January 1976
mechanical device will recognize the necessity of understanding the normal if he is to deal effectively with the abnormal, and this principle seems reasonably applied to medicine. 3. The X-linked form of CGD has been important to the field of genetics by confirming the hypotheses that only one X chromosome remains active in an individual cell and that inactivation of the "extra" X-chromosome is a random event. This has been accomplished with a histoehemical assay that permits assessment of the ability of individual neutrophils to reduce NBT when stimulated by endotoxin or phagocytosis, as described by Biggar and colleagues. Carriers of X-linked CGD consistently have had 35-60% normal neutrophils by this assay, One might predict, however, that inactivation of the normal X chromosome, if truly random, could deviate even further from 50%. Solid evidence that this can occur in CGD comes from studies of a sister of the patient reported in this issue: Only about 20% of her neutrophil population has retained the normal X chromosome. Her cells behaved almost as abnormally as those of the patient in bactericidal and metabolic assays, demonstrating the importance of using the histochemical NBT assay when abnormal phagocyte function is found in a subject with few infections. 4. The occurrence of granulomas in CGD has made it clear that these structures result from the prolonged intraphagocytic residence of materials not easily digested, whether the indigestion is due to the nature of the material itself or to a faulty cell. Awareness of this relationship allows the clinician or pathologist to narrow possible causative agents of granulomatosis to particles and parasites that could resist intracellular digestion. The utility of describing unusual aspects of uncommon diseases in general medical journals has been questioned. Yet the fact that a disease is uncommon is of little solace to affected families, and, of course, they stand to profit from any improvement in the understanding of their disease. What may be even more important to us all, however, is the less tangible but ultimately greater benefit of new insight into the balance between health and disease.
Richard B. Johnston, Jr., M.D. Associate Professor of Pediatrics and Microbiology The University of Alabama in Birmingham Birmingham, .Ala. 35294
1. Quie PG, et al: in, Bellanti JA, and Dayton DH, editors: The phagocytic ceU in host resistance, New York, 1975, Raven Press, p 225. 2. Ffidovich I: Oxygen: Boon and bane, Am Sci 63:54, 1975. 3. Allen RC, Stjernholm RL, and Steele RH: Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity, Biochem Biophys Res Commun 47:679, 1972. 4. Johnston RB Jr, Kecle BB Jr, Misra HP, Lehmeyer JE, Webb LS, Baehner RL, and Rajagopalan KV: The role of superoxide anion generation in phagocytic bactericidal activity: Studies with normal and chronic granulomat0us disease leukocytes, J Clin Invest 55:1357, 1975. 5. Baehner RL, Murrmann SK, Davis J, and Johnston RB Jr: The role of superoxide anion and hydrogen peroxide in phagocytosis-associated oxidative metabolic reactions, J Clin Invest 56:(in press). 6. Klebanoff SJ: Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes, Semin Hematol 12:117, 1975. 7. Karnovsky ML:-Chronic granutomatous disease-pieces of a cellular and molecular puzzle, Fed Proc 32:1527, 1973. 8. DeChatelet LR: Oxidative bactericidal mechanisms ofpolymorphonuclear leukocytes, J Infect Dis 131:295, 1975. 9. Gray GR, Klebanoff SJ, Stamatoyannopoulos G, Austin T, Naiman SC; Yoshida A, Kliman MR, and Robinson GCF: Neutrophil dysfunction, chronic granulomatous disease, and non-sperocytichaemolytic anaemia caused by complete deficiency of glucose-6,phosphate dehydrogenase, Lancet 2:530, 1973. 10. Baehner RL, Johnston RB Jr, and Nathan DG: Comparative study of the metabolic and bactericidal characteristics of severely glucose-6-phosphate dehydrogenase-deficient polymorphonuclear leukocytes and leukocytes from children with chronic granulomatous disease, J Reticuloendothel Soc 12:150, 1972. 11. Malawista SE, and Gifford RH: Chronic granulomatous disease of childhood with leukocyte glutathione peroxidase deficiency in a brother and sister: A likely autosomal recessive inheritance, Clin Res 23:416A, 1975. 12. Johnston RB Jr, Wilfert CM, Buckley RH, Webb LS, DeChatelet LR, and McCall CE: Enhanced bactericidal activity of phagocytes from patients with chronic granulomatous disease in the presence of sulphisoxazole, Lancet 1:824, 1975. 13. Mandell GL: Bactericidal activity of aerobic and anaerobic polymorphonuclear neutrophils, Infect Immun 9:337, 1974. 14. KodeyGE, Park BH, Ford DK, Holmes-Gray B, and Good RA:-Defective bactericidal activity of peripheral blood leukocytes in lipochrome histiocytosis, Am J Med 49:322, 1970. 15. Ochs HD, and Igo RP: The NBT slide test: A simple screening method for detecting chronic granulomatous disease and female carriers, J PBDIATI~83:77, 1973.