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 (gp91 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 cinterferon. 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. Citation: van den Berg JM, van Koppen E, Åhlin A, Belohradsky BH, Bernatowska E, et al. (2009) Chronic Granulomatous Disease: The European Experience. PLoS ONE 4(4): e5234. doi:10.1371/journal.pone.0005234 Editor: Andy Alspaugh, Duke University, United States of America Received June 16, 2008; Accepted November 12, 2008; Published April 21, 2009 Copyright: 2009 van den Berg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support was provided for data collection by the CGD trust, UK, a non-profit organization. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: j.m.vandenberg@amc. nl Introduction Chronic granulomatous disease (CGD) is an uncommon inherited immunodeficiency, occurring in about one in 250,000 individuals. Although the genetic basis for this disease is wellknown, the expected clinical course and outcome have only partially been defined, owing to its sporadic occurrence [1–3]. There are extreme differences in presentation between patients, varying form a relatively mild presentation late in life to fatal septicemia in infancy. This renders it difficult for clinicians to inform CGD patients and their parents what to expect, and to find a proper balance between risks, costs and benefits of various treatments, ranging from long-term prophylactic antibiotic medication to stem cell transplantation and gene therapy [4]. CGD is caused by a defect in the burst of oxygen consumption that normally accompanies phagocytosis in myeloid cells (i.e. neutrophils, eosinophils, monocytes, and macrophages). The ‘‘respiratory burst’’ involves the catalytic conversion of molecular oxygen to the oxygen free-radical superoxide (O2 ), which in turn gives rise to hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and hydroxyl radical (OH). These oxygen derivatives play a critical role in the killing of certain pathogenic bacteria and fungi. As a result of the failure to mount a respiratory burst in their phagocytes, the majority of CGD patients suffers from severe recurrent infections and also from dysregulated Th-17-lymphocyte-controled inflammation [5] Therefore, CGD patients can develop diffuse granulomas that can become sufficiently large to PLoS ONE | www.plosone.org 1 April 2009 | Volume 4 | Issue 4 | e5234 cause obstructive or painful symptoms in the esophagus, stomach, ureters, or urinary bladder, or dysfunctional disorders secondary to extensive fibrosis of the different systems (pulmonary, gastrointestinal, genitourinary, central nervous system) [6–9]. Also, autoimmune phenomena have been reported to occur at an increased incidence rate in CGD patients and in female Xlinked carriers [10–12]. The enzyme that catalyzes the respiratory burst, the leukocyte NADPH oxidase, consists of subunits, four of which are important for CGD (designated phox for phagocyte oxidase): gp91 (or Nox2) and p22, located in membranes, as well as two cytosolic oxidase components, p47 and p67. CGD is caused by a defect in any of these four components. Mutations in the gp91 gene (CYBB on chromosome Xp21.1) cause the X-linked recessive form of the disease that affects the majority of CGD patients (,70%). As expected from the genetics, the overwhelming majority of X-linked patients are males. The remaining 30% of cases has inherited the disease in an autosomal recessive manner, in which males and females are equally affected. These patients have mutations in the genes encoding p47 (NCF1 on chromosome 7q11.23), p67 (NCF2 on chromosome 1q25), or p22 (CYBA on chromosome 16q24). In contrast to p47 deficiency [13], most CGD patients with any of the other three forms have mutations unique to their families [14]. Apart from the description of several smaller cohorts, the first characterization of a large group of CGD patients was published by Winkelstein et al., who reported on a national American registry of 368 patients [1]. To further improve knowledge of the course of CGD, clinical data from European patients were collected. We now report our findings in 429 European patients, the largest cohort of CGD patients to date.

OBJECTIVE:The aim of this study was to analyze mutations in DNA from patients with leukocyte adhesion deficiency (LAD), an immunodeficiency caused by absence of the beta(2) subunit (CD18) of the leukocyte integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18), and CR4 (CD11d/CD18). METHODS:We developed genomic DNA PCR sequencing to detect mutations not only in exons but also in introns. RESULTS:Eight LAD patients were analyzed, of which five had homozygous mutations, i.e., a 0.8-kb deletion, a branchpoint mutation in intron 5 causing mRNA missplicing, a nonsense mutation, and two missense mutations. Four of these mutations are novel. We cotransfected the two mutant CD18 proteins with normal CD11a, b, or c in COS cells. This resulted in absence of all three beta(2) integrins on the surface of cells transfected with CD18(252Arg). However, CD18(593Cys) supported some LFA-1 and p150,95 formation in COS cells. The other three patients were compound heterozygotes in which only one allele had previously been characterized, because the other alleles were undetectable at the cDNA level. We identified the unknown mutations as a novel two-nucleotide deletion, a nonsense mutation, and a single nucleotide deletion. CONCLUSION:Our method allows identification of mutations in CD18 from genomic DNA. This opens the possibility of early prenatal diagnosis of LAD and reliable carrier detection.

The superoxide-forming nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase of human phagocytes comprises membrane-bound and cytosolic proteins, which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients with chronic granulomatous disease (CGD) are defective in one of the phagocyte oxidase (phox) components, p47-phox or p67-phox, which reside in the cytosol of resting phagocytes, or gp91-phox or p22-phox, which constitute the membrane-bound cytochrome b(558). In four X-linked CGD patients we have identified novel missense mutations in CYBB, the gene encoding gp91-phox. These mutations were associated with normal amounts of nonfunctional cytochrome b(558) in the patients' neutrophils. In phorbol-myristate-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of p47-phox and p67-phox with the membrane fraction of the cells with Cys369-->Arg, Gly408-->Glu, and Glu568--> Lys substitutions was strongly disturbed. Only a Thr341-->Lys substitution, residing in a region of gp91-phox involved in flavin adenine dinucleotide (FAD) binding, supported a normal translocation. Thus, the introduction or reversal of charge at residues 369, 408, and 568 in gp91-phox destroys the correct binding of p47-phox and p67-phox to cytochrome b(558). Based on mutagenesis studies of structurally related flavin-dependent oxidoreductases, we propose that the Thr341-->Lys substitution results in impaired hydride transfer from NADPH to FAD. Because we found no electron transfer in solubilized neutrophil plasma membranes from any of the four patients, we conclude that all four amino acid replacements are critical for electron transfer. Apparently, an intimate relation exists between domains of gp91-phox involved in electron transfer and in p47/p67-phox binding. (Blood. 2000;95:666-673)

gamma-Glutamylcysteine synthetase (GCS) catalyzes the initial and rate-limiting step in the biosynthesis of glutathione. gamma-GCS consists of a heavy and a light subunit encoded by separate genes. Hereditary deficiency of GCS has been reported in 6 patients with hemolytic anemia and low erythrocyte levels of glutathione and gamma-glutamylcysteine, In addition, 2 patients also had generalized aminoaciduria and developed neurologic symptoms. We have examined a Dutch kindred with 1 suspected case of GCS deficiency. The proband was a 68-year-old woman with a history of transient jaundice and compensated hemolytic anemia. One of her grandchildren was also GCS deficient; he was 11 years old and had a history of neonatal jaundice. The enzyme defect was confirmed and GCS activity was found to be less than 2% of normal in the erythrocytes of both patients. The complementary DNA (cDNA) for the heavy subunit of GCS was sequenced in these patients and in several members of the family. The proband and her GCS-deficient grandson were identified as homozygous for a 473C --> T substitution, changing codon 158 from CCC for proline into CTC for leucine, Several family members with half-normal GCS activity in their erythrocytes were heterozygous for the mutation. (Blood, 2000;95:2193-2197) (C) 2000 by The American Society of Hematology.

Chronic granulomatous disease (CGD) is a clinical syndrome of recurrent bacterial and fungal infections caused by a rare disorder of phagocytic cells. In CGD, the phagocytes are unable to generate oxygen radicals after stimulation of these cells, due to a defect in the NADPH oxidase system. This NADPH oxidase is a multicomponent enzyme of at least four subunits, of which the beta-subunit of cytochrome b558, gp91-phox, is encoded by an X-linked gene (called CYBB). We report here five patients from two families; in each family we found a different mutation in the promoter region of CYBB. Both mutations prevented the expression of gp91-phox in the patients' neutrophils and thus caused inability of these cells to generate oxygen radicals. However, the mutations left the gp91-phox expression and the function of the NADPH oxidase in the patients' eosinophils intact. The relatively mild course of the CGD in these patients can probably be attributed to the fact that the eosinophils have retained their oxidative capacity. Furthermore, our results indicate that neutrophils and eosinophils differ in their regulation of gp91-phox expression.

Neutrophils have a crucial function in the defense against bacteria and fungi. Indeed, during chronic, severe neutropenia and in case of severe neutrophil dysfunctions, the patients may suffer recurrent and sometimes life-threatening infections. This article describes the clinical symptoms, the theory behind the antimicrobial systems of neutrophils, the methods to diagnose the various aberrations, and the possibilities for treating these patients. A few of the most common causes of neutropenia and neutrophil dysfunctions are described in detail, including recent genetic information regarding the cause of these diseases.

We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C-->T mutation, predicting a Pro172-->Ser replacement. Analysis of G6PD RNA from the patient's leukocytes and fibroblasts showed only transcripts with the 514C-->T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient's daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability.

HCA2 is a widely used monoclonal antibody, thought to be highly selective for HLA-A and -G heavy chains. We demonstrate here that it also shows affinity to HLA-B73 and HLA-E molecules on intact cells. By comparing the differences in the amino acid (AA) sequences of several HLA class I alleles that are either recognised or not recognised by HCA2, a likely epitope of HCA2 has been deduced. It extends from position 76 to position 83 of the α1-domain. In intact cells, the solvent-exposed AA in positions 76 (Ala, Val, or Met), 80 (Asn or Thr) and 83 (Gly) are likely to constitute the recognition region. Inhibition experiments with peptides spanning the region of the α1-domain from position 74 to 85 of various HLA class I heavy chains prove that HCA2 recognizes a broadly shared epitope on HLA-E, -F and -G molecules as well as selected HLA-A, -B and -C antigens.

A 12 year old boy suffering from p67-phox deficient chronic granulomatous disease presented with a bullous skin disease and a lung infection with paecilomyces species. The histopathology of a bullous lesion showed subepidermal blister formation and microabcesses containing eosinophils in the dermal papillae. By direct immunofluorescence, linear staining of IgA at the dermal-epidermal junction was detected which confirmed the clinical diagnosis of chronic bullous disease of childhood (linear IgA dermatosis).

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.

Neutropenia is one of the risk factors for severe therapy-related morbidity in childhood malignancies. We have studied the potential of GM-CSF to shorten the neutropenic period after normal-dose chemotherapy in children who were treated for solid tumors. Patients with osteosarcomas, with Ewing sarcomas, or with rhabdomyosarcomas received 10 daily subcutaneous doses GM-CSF (Leucomax, 5 micrograms/kg) after a course of normal-dose chemotherapy in an open-label study. Because these patients were treated with different combinations of chemotherapeutic agents, they were randomized before each pair of identical courses of chemotherapy to receive GM-CSF after the first or after the second course. Fourteen such combinations could be evaluated in eight patients. The results show that GM-CSF significantly reduced the mean duration of the chemotherapy-induced neutropenia (mean reduction +/- SEM in days: 2.2 +/- 0.6, P = .003). There was no significant difference between the mean number of days with fever in either group. GM-CSF was well tolerated by all patients. We conclude that GM-CSF reduced the mean neutropenic period in children with solid tumors who were treated with standard-dose chemotherapy.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are frequently used in the clinical management of neutropenia. These cytokines not only enhance the proliferation of myeloid precursor cells but also influence the function of mature leukocytes. In a previous study, we found that the in vivo effects of G-CSF on neutrophils differed from those in vitro. In the present study, we investigated the effects of a single dose of recombinant GM-CSF (7.5 microg/kg, subcutaneously) on neutrophils, eosinophils, and monocytes in healthy volunteers. We analyzed leukocyte kinetics, phenotypical changes, neutrophil degranulation, and systemic cytokine production. After GM-CSF injection, phenotypical changes included upregulation of CD11b on all three cell types and a decreased expression of L-selectin and Fc(gamma)RIII on neutrophils. Neutrophil degranulation was evident from the increased plasma concentrations of lactoferrin and elastase. GM-CSF induced the release of interleukin-8 (IL-8), but not of IL-6 or tumor necrosis factor alpha. In comparison to the results from our previous study with G-CSF in healthy volunteers, GM-CSF induced a stronger activation of mature neutrophils but had a much less pronounced effect on the production and maturation of neutrophil precursors. These data may help to guide the choice between the two cytokines in different clinical situations.

During the international placebo-controlled trial on the efficacy of interferon-gamma (IFN-gamma) in chronic granulomatous disease (CGD), 19 patients entered the study via our Institute. One patient stopped treatment shortly thereafter. RNA was purified from the mononuclear cells of the remaining 18 CGD patients before and during this placebo-controlled trial. The mRNA levels for the NADPH oxidase components were subsequently analyzed. Compared with the placebo-treated CGD patients, the mRNA levels for p47-phox were significantly increased in the IFN-gamma-treated CGD patients (P < 0.002). No significant changes were observed in the mRNA levels of the other oxidase components. These findings are in agreement with observations in vitro and indicate that IFN-gamma is active on the NADPH oxidase in vivo as well. However, it remains questionable whether these effects in vivo can explain the observed reduction of infections in these patients.

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.

An 8‐year‐old boy had been suffering from chronic autoimmune neutropenia for more than 5 years. The neutropenia proved to be resistant to high‐dose steroids and intravenous (either low‐ or high‐dose) immunoglobulin (Ig) therapy. The chronic autoimmune thrombocytopenia and recurrent phases of autoimmune haemolytic anaemia did, however, respond to high‐dose prednisone. Other signs of immune dysregulation in this patient consisted of insulin‐dependent diabetes mellitus type I (IDDM) and an acquired hypogammaglobulinaemia, most compatible with common variable immunodeficiency (CVI). Prior to rhG‐CSF therapy the child had suffered for more than 2 years from recurrent life‐threatening bacterial infections.Anti‐neutrophil autoantibodies had pan‐FcγRIII (CD16, NA1/NA2) specificity. The neutropenia as well as the anti‐neutrophil autoantibodies disappeared when subcutaneous rhG‐CSF therapy was started. Upon tapering rhG‐CSF, anti‐FcγRIII antibodies reappeared together with an absolute neutropenia. Renewed administration resulted again in the normalization of symptoms.Soluble FcγRIII (sFcγRIII) antigen levels in plasma increased dramatically during rhG‐CSF treatment. These high levels of sFcγRIII together with increased numbers as well as decreased apoptotic reactions of neutrophils apparently result in adsorption of the autoantibodies in vivo, contributing to the normalization of autoimmune‐mediated neutropenia upon rhG‐CSF treatment. Long‐term administration of rhG‐CSF represents an alternative in the treatment of autoimmune neutropenia.

HRONIC granulomatous disease (CGD) is an immunoC deficiency syndrome characterized clinically by severe recurrent bacterial and fungal infections that are difficult to treat by conventional means. These infections predominate in the lymph nodes, the subcutaneous tissues, the lungs, the liver and the bones. Gastrointestinal manifestations include diarrhea, perianal abscesses, and obstructions caused by granuloma formation. The disease is usually recognized in young children below the age of 2, but sometimes it is recognized later.’ Biochemically, CGD is characterized by the inability of phagocytic leukocytes (neutrophils, eosinophils, monocytes, and macrophages) to generate reactive oxygen compounds, which are needed for intracellular killing of phagocytized microorganisms.’ Therefore, the most common pathogens encountered in CGD patients are catalase-positive organisms, because catalase prevents the CGD phagocytes from using microbial-generated hydrogen peroxide for killing these pathogens. Predominant are Staphylococcus uureus, Aspergillus species, and a variety of Gram-negative enteric bacilli. CGD is a rare disease, with an estimated incidence of 1 in about 250,000 individuals, without any ethnic preference. Clinically and biochemically, CGD is a very heterogenous disorder due to the existence of many antimicrobial systems that can partially compensate for the defect in oxygen-dependent killing systems and to the complicated genetic origin of CGD. In this review, a short overview will be given of our present knowledge of the NADPH oxidase, the enzyme that is responsible for the production of antimicrobial oxygen compounds in leukocytes. This will be followed by a summary of all known mutations in the genes that encode the various NADPH oxidase components, and thus lead to the manifestation of CGD. This knowledge is of importance for a better understanding of the functional domains within the oxidase components, for improved prenatal and postnatal diagnosis of CGD, and for advances in therapy, including the future use of gene replacement.

Src homology 3 (SH3) domains have been suggested to play an important role in the assembly of the superoxide-forming nicotinamide adenine dinucleotide phosphate (NADPH) oxidase upon activation of phagocytes, which involves the association of membrane-bound and cytosolic components. We studied the translocation of the cytosolic proteins to the plasma membrane in neutrophils of a patient with a point mutation in the gene encoding the light chain of cytochrome b558. This mutation leads to a substitution at residue 156 of a proline into a glutamine in a putative SH3 binding domain of p22-phox (Dinauer, M., E. A. Pierce, R. W. Erickson, T. Muhlebach, H. Messner, R. A. Seger, S. H. Orkin, and J. T. Curnutte. 1991. Proc. Natl. Acad. Sci. 88:11231). In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient's neutrophils was virtually absent. In contrast, when solubilized membranes of the patient's neutrophils were activated with phospholipids in the absence of cytosol (Koshkin, V., and E. Pick. 1993. FEBS [Fed. Eur. Biochem. Soc.] Lett. 327:57), the rate of NADPH-dependent oxygen uptake was observed at a rate similar to that of control membranes. We suggest that the binding of an SH3 domain of p47-phox to p22-phox, and thus activation of the oxidase, does not occur in the neutrophils of this patient, although under artificial conditions, electron flow from NADPH to oxygen in cytochrome b558 is possible.

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 patient's 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.