Ron S. Weening

Chronic Granulomatous Disease: The European Experience

CGD is an immunodeficiency caused by deletions or mutations in genes that encode subunits of the leukocyte NADPH oxidase complex. Normally, assembly of the NADPH oxidase complex in phagosomes of certain phagocytic cells leads to a “respiratory burst”, essential for the clearance of phagocytosed micro-organisms. CGD patients lack this mechanism, which leads to life-threatening infections and granuloma formation. However, a clear picture of the clinical course of CGD is hampered by its low prevalence (∼1∶250,000). Therefore, extensive clinical data from 429 European patients were collected and analyzed. Of these patients 351 were males and 78 were females. X-linked (XL) CGD (gp91phox deficient) accounted for 67% of the cases, autosomal recessive (AR) inheritance for 33%. AR-CGD was diagnosed later in life, and the mean survival time was significantly better in AR patients (49.6 years) than in XL CGD (37.8 years), suggesting a milder disease course in AR patients. The disease manifested itself most frequently in the lungs (66% of patients), skin (53%), lymph nodes (50%), gastrointestinal tract (48%) and liver (32%). The most frequently cultured micro-organisms per episode were Staphylococcus aureus (30%), Aspergillus spp. (26%), and Salmonella spp. (16%). Surprisingly, Pseudomonas spp. (2%) and Burkholderia cepacia (<1%) were found only sporadically. Lesions induced by inoculation with BCG occurred in 8% of the patients. Only 71% of the patients received antibiotic maintenance therapy, and 53% antifungal prophylaxis. 33% were treated with γ-interferon. 24 patients (6%) had received a stem cell transplantation. The most prominent reason of death was pneumonia and pulmonary abscess (18/84 cases), septicemia (16/84) and brain abscess (4/84). These data provide further insight in the clinical course of CGD in Europe and hopefully can help to increase awareness and optimize the treatment of these patients.

Leukocyte adhesion deficiency type 1 (LAD-1)/variant. A novel immunodeficiency syndrome characterized by dysfunctional beta2 integrins.

Leukocyte adhesion deficiency (LAD) is characterized by the inability of leukocytes, in particular neutrophilic granulocytes, to emigrate from the bloodstream towards sites of inflammation. Infectious foci are nonpurulent and may eventually become necrotic because of abnormal wound healing. LAD-1 is characterized by the absence of the beta2 integrins (CD11/CD18) on leukocytes. When expression is completely absent, patients often die within the first year. However, low levels of beta2 expression may result in a milder clinical picture of recurrent infection, which offers a better prognosis. In this paper, we describe the in vivo and in vitro findings on a patient with clinical features of a mild LAD-1 disorder, i.e., suffering from bacterial infections without apparent pus formation in the presence of a striking granulocytosis, showing no delayed-type hypersensitivity reaction upon skin testing, no specific antibody generation, but normal in vitro T cell proliferation responses after immunization. Expression levels of CD11/CD18 proteins were completely normal, but leukocyte activation did not result in CD11/ CD18 activation and high-avidity ligand-binding. In vitro chemotaxis and endothelial transmigration of the neutrophils as well as leukocyte aggregation responses were almost absent. On the other hand, beta1 and beta3 integrin-mediated adhesion functions were completely normal. During follow-up, a bleeding tendency related to decreased beta3 activation became clinically apparent, different from previously described cellular adhesion molecule variants. Therefore, this is the first well-documented case of a clinical combined immunodeficiency syndrome that results from nonfunctional CD11/CD18 molecules, and thus designated LAD-1/ variant.

Protection of Human Neutrophils by Endogenous Catalase: STUDIES WITH CELLS FROM CATALASE-DEFICIENT INDIVIDUALS

To investigate the importance of catalase as a protecting enzyme against oxidative damage in phagocytic leukocytes, we have tested the functional capacity of neutrophils from two individuals homozygous for Swiss-type acatalasemia and from two individuals heterozygous for this deficiency. In the former cells, 25-30% of residual activity of catalase was present. In the latter cells, the values were close to normal. Chemotaxis towards casein, release of lysosomal enzymes and hydrogen peroxide during phagocytosis of zymosan, and intracellular killing of Staphylococcus aureus were normal in all cells tested. Inhibition of heme enzymes with azide (2 mM) enhanced the respiration and hexose monophosphate shunt activity of normal, but not of homozygous acatalasemic, neutrophils. This indicates that the enhancement in normal cells is, at least in part, due to catalase inhibition. After 15 min preincubation with an H(2)O(2)-generating system (glucose plus glucose oxidase), the respiratory response to zymosan phagocytosis was strongly depressed in the homozygous acatalasemic and in normal, azide-treated neutrophils, but not in normal, untreated cells. Under these conditions, the release of lysosomal enzymes was depressed and that of lactate dehydrogenase enhanced, in catalase-deficient and in catalase-inhibited, but not in normal, neutrophils. During prolonged incubation with the H(2)O(2)-generating system (30-60 min), the reduction level of intracellular glutathione remained high and the hexose monophosphate shunt continued to operate normally in all cells tested. Thus, although the function of neutrophils without catalase activity was depressed by extracellular hydrogen peroxide, the H(2)O(2) degradation via the glutathione redox system remained operative. The results indicate that the glutathione redox system by itself efficiently protects phagocytosing neutrophils against their own oxidative products. During heavy external oxidative stress, however, both catalase and the glutathione redox system are needed for adequate protection.

A Point Mutation in gp91-phox of Cytochrome b558 of the Human NADPH Oxidase Leading to Defective Translocation of the Cytosolic Proteins p47-phox and p67-phox

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patients neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp91-phox inhibited NADPH oxidase activity in the cell-free assay (IC50 about 10 microM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox.

Limitations on the use of dihydrorhodamine 123 for flow cytometric analysis of the neutrophil respiratory burst

Intracellular oxidation of dihydrorhodamine 123 (DHR) to the fluorescent compound rhodamine 123 (Rho123) was used to detect the production of oxygen metabolites in activated neutrophils. Total leukocyte preparations can be used in this assay, which is a great advantage when priming of the respiratory burst is studied. We have defined the conditions that should be taken into account when priming is studied with this assay. We found that neither the extent nor the kinetics of DHR oxidation match those of NADPH oxidase activity. In addition, DHR oxidation is influenced by the absolute and relative number of neutrophils in the leukocyte suspension, by the DHR concentration and by myeloperoxidase availability. The results presented in this study emphasize the need for carefully designed experiments when DHR is used to study the respiratory burst in neutrophils.

Continuous therapy with sulfamethoxazole-trimethoprim in patients with chronic granulomatous disease

In a retrospective study, the effect of long-term treatment with sulfamethoxazole-trimethoprim was evaluated in nine male patients with chronic granulomatous disease. During this treatment, a marked reduction was observed in the number of infectious episodes, the number of causative agents, and the number of surgical interventions. Furthermore, a significant reduction in days of hospitalization per year was found. The mean observation period was six years before and four years during treatment. Transient alopecia was observed in one patient during therapy. We conclude that prophylactic treatment with sulfamethoxazole-trimethoprim is beneficial in patients with chronic granulomatous disease.

Phagocytosing human neutrophils inactivate their own granular enzymes.

During phagocytosis, neutrophils generate reactive oxygen metabolites and release lysosomal enzymes into the extracellular medium. We have investigated the possibility that these enzyme are inactivated by the oxygen compounds. Phagocytosing neutrophils from 12 patients with chronic granulomatous disease, which do not generate these oxygen metabolites, released two to three times more activity of lysozyme and beta-glucuronidase than did normal neutrophils. This difference proved to be due to a decrease of approximately 20% of the total activity of these enzymes in normal neutrophils, but not in neutrophils of patients with chronic granulomatous disease. This inactivation of enzymes took place during phagocytosis of opsonized zymosan particles as well as during stimulation of normal cells with phorbol myristate acetate. The inactivation was not due to formation of inhibitors. The lysosomal enzymes were not activated when the neutrophils were stimulated under anaerobic conditions. Addition of catalase, superoxide dismutase, or albumin gave no protection against the oxidative damage; reduced glutathione gave partial protection. The oxidative inactivation was more pronounced in the presence of azide. Measurement of the activity and the amount of protein of acid alpha-glucosidase in the cells showed that the specific activity of this enzyme decreased by approximately 50% during 30 min of phagocytosis. This indicates that the inactivation of the lysosomal enzymes takes place in the phagolysosomes, before the enzymes have leaked into the extracellular medium.

A phosphoprotein of Mr 47,000, defective in autosomal chronic granulomatous disease, copurifies with one of two soluble components required for NADPH:O2 oxidoreductase activity in human neutrophils

The NADPH:O2 oxidoreductase (NADPH oxidase) of human neutrophils is converted from a dormant to an active state upon stimulation of the cells. We have studied the soluble fraction that is required for NADPH oxidase activation in a cell-free system. Human neutrophils were separated in a membrane-containing and a soluble fraction. The soluble fraction was separated on carboxymethyl (CM) Sepharose in 10 mM 4-morpholino-ethanesulfonic acid buffer of pH 6.8. Reconstitution of the NADPH oxidase activity, measured as O2 consumption, was only found when the membrane fraction was combined with the flowthrough of the CM Sepharose column as well as with a fraction that eluted at 125 mM NaCl. This result indicates that at least two soluble components are necessary for reconstitution of the NADPH oxidase activity: one that does not bind to CM Sepharose and one that does bind. These components were designated soluble oxidase component (SOC) I and SOC II, respectively. Boiling destroyed the activity in both fractions. In the soluble fraction of human lymphocytes and thrombocytes neither SOC I nor SOC II activity was found. SOC II copurified with a 47-kD phosphoprotein, previously found defective in patients with the autosomal form of chronic granulomatous disease (CGD). Inactive soluble fractions of cells from autosomal CGD patients were reconstituted with a SOC II fraction from control cells. The result of this experiment indicates that autosomal CGD patients are normal in SOC I but defective in SOC II.

Clinical differences in chronic granulomatous disease in patients with cytochrome b-negative or cytochrome b-positive neutrophils+

1. Tamborlane WV, Sherwin RS, Genel M, Felig P: Outpatient treatment of juvenile-onset diabetes with a preprogrammed portable subcutaneous insulin infusion system. Am J Med 68:190, 1980. 2. Mecklenburg RS, Benson JW, Becker NM, Brazel LP, Fredland PN, Metz R J, Nielsen RL, Sannac CA, Steenrod W J: Clinical use of the insulin infusion pump in 100 patients with Type [ diabetes. N Engl J Med 307:513, 1982. 3. Minuchin S, Posman BL, Baker L, et al: A conceptual model of psychosomatic illness in children. Arch Gen Psychiatry 32:1031, 1975. 4. Riley W J, Silverstein JH, Rosenbloom AL, Spillar R, McCallum MH: Ambulatory diabetes management with pulsed subcutaneous insulin using a portable pump. Clin Pediatr 19:609, 1980. 5. Barbosa J, Menth L, Eaton J, Sutherland D, Freier EF, Najarian J: Long-term, ambulatory subcutaneous insulin infusion versus multiple daily injections in brittle diabetic patients. Diabetes Care 4:269, 1981. 6. Keen H, Pickup JC, Viberti GC, Bilous R, Williams G: Aspects of continuous subcutaneous insulin infusion in diabetes. Diabetes Care 4:54, 1981. 7. Selam JL, Slingeneyer A, Hedon B, Mares P, Beraud J J, Mirouze J: Long-term ambulatory peritoneal insulin infusion of brittle diabetes with portable pumps: Comparison with intravenous and subcutaneous route. Diabetes Care 6:105, 1983. 8. Nathan DM: Successful treatment of extremely brittle, insulin-dependent diabetes with a novel-subcutaneous insulin pump regimen. Diabetes Care 5:105, 1982. 9. Schiffrin A, Desroslers M, Moffatt M, Belmonte MM: Feasibility of strict diabetes control in insulin-dependent diabetic adolescents. J PEDIATR 103:522, 1983.

The management of chronic granulomatous disease

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease which results from absence of the NADPH oxidase in the professional phagocytic cells [13] neutrophils, monocytes, macrophages and eosinophils. Deficiency of this oxidase renders the patient liable to infection by bacteria and fungi, and, as the name of the disease suggests, to chronic granulomatous inflammation. These patients present with a great variety of infections and other complications of their disease, which often tax the clinical and therapeutic skill of the doctors responsible for their care. Collectively we look after, or advise on the management of, over 100 of these subjects, and have developed experience in the diagnosis and management of the infections and other clinical problems they present. We thought that it might be timely to provide guidelines for their management based upon this experience. The numbers of patients are still relatively small, and the clinical presentations very varied, so it is immpossible to provide clear statistical proof of the veracity of this advice. It does, however, reflect the working practise of the physicians caring for many of these patients in Europe.