Chronic granulomatous disease

INTRODUCTION

A clinical syndrome characterized by recurrent life-threatening Staphylococcus aureus, Proteus or Pseudomonas, hypergammaglobulinaemia, and widespread chronic granulomatous infiltration was first recognized in the paediatric literature between 1954 and 1960 [1–3]. The pathological mechanisms responsible for this condition became evident when it was demonstrated that neutrophils collected from a male patient were unable to kill S. aureus in vitro, and that there was a primary abnormality of neutrophil function [4]. In the same year, it was shown that neutrophils from patients with this familial granulomatosis, now called chronic granulomatous disease (CGD), failed to exhibit a characteristic increase of oxidative metabolism, called the ‘respiratory burst’, during phagocytosis [5].

IDENTIFICATION OF THE NAPDH–OXIDASE

The enhanced ‘respiration’ of leucocytes was first described as a small but significant increase in the oxygen consumption of canine neutrophils during phagocytosis of bacteria [6]. This metabolic response was attributed to increased generation of energy during phagocytosis, and was known as ‘the extra respiration of phagocytosis’, but was later shown to be resistant to conventional inhibitors of mitochondrial respiration [7]. It was also shown that the necessary energy for phagocytosis and cytoplasmic degranulation was provided by the glycolytic pathway. The function of the ‘respiratory burst’ remained obscure until it became apparent that the ability of phagocytic cells to kill certain bacteria in vitro was markedly diminished under anaerobic conditions, and that cells obtained from patients with CGD, which were unable to mount this metabolic response, exhibited the same microbicidal deficiency in the presence of oxygen [4]. The identity of the substrate for the reaction was also the subject of considerable speculation [8,9], but the sharp increase in oxidation of glucose via the hexose monophosphate shunt (the purpose of which is to maintain cellular NADPH levels, and the activity of which is controlled by the rate of oxidation of NADPH) coincident with neutrophil activation, suggested that NADPH was the most likely candidate molecule.

COMPONENTS OF THE NAPDH–OXIDASE

The NAPDH–oxidase catalyses the formation of superoxide, which is a precursor for the generation of potent oxidant compounds, by transmembrane passage of electrons from NADPH to molecular O₂. It is most abundant in phagocytic cells, particularly neutrophils, eosinophils and cells of the monocyte/macrophage lineage, consisting of a membrane-bound flavocytochrome b₅₅₈ and four cytosolic factors, p47^phox, p67^phox, p40^phox and p21rac, which translocate to the membrane on activation of the cell (the suffix phox represents ph agocyte ox idase) [10]. Activation is initiated classically by opsonized particles, but also by many soluble inflammatory mediators. More recently, components of the NAPDH–oxidase and low level enzymatic activity have been detected in other cell types, although their biological functions are unclear and will not be discussed further here.

The redox centre of the oxidase is the flavocytochrome b₅₅₈, the midpoint potential for which is sufficiently low to induce direct reduction of oxygen to superoxide. The flavocytochrome consists of two proteins with apparent molecular weights of 23 kD (p22^phox, α-subunit) and 76–92 kD (gp91^phox, β-subunit), respectively, and are arranged as a 1:1 heterodimer [11–14]. The larger β-subunit migrates on SDS–PAGE as a broad band, an electrophoretic property characteristic of glycoproteins, and comprises about 21% carbohydrate, predominantly of the N-linked high-lactosamine complex type [15]. Both p22^phox and gp91^phox are missing in cells derived from most CGD patients with a molecular lesion of either subunit, indicating that mutual interaction is necessary for assembly of the mature complex [16–18]. A 65-kD biosynthetic gp91^phox intermediate precursor with high-mannose type oligosaccharide side chains has been detected in membrane fractions (which includes endosomal compartments) derived from patients with p22^phox-deficient CGD, which is processed to a mature terminally glycosylated form only if the deficiency of the smaller subunit protein is restored [19]. Biosynthesis of gp91^phox glycoprotein is also dependent on the incorporation of two non-identical haem groups for each heterodimer within the membrane-spanning α helices, one predicted to lie near the inner face, and the other towards the outer face of the cell [18]. The locations of the binding sites for the substrate NADPH, and for the electron carrier flavin adenine dinucleotide (FAD), are now known to lie within the C-terminal region of gp91^phox itself (although an additional NADPH-binding site may exist on p67^phox). Final transfer of electrons across the cell membrane is probably mediated by the two associated haem groups. The flavocytochrome b₅₅₈ therefore comprises the complete electron transporting apparatus of the NAPDH–oxidase. The membrane-spanning N-terminal region of the flavocytochrome has recently been identified as the site of a charge-compensating H + conductance during activation of the respiratory burst, and may also therefore be responsible for maintaining intracellular (preventing deleterious acidification) and phagosomal pH (for optimal activity of proteolytic enzymes) [20–26].

Fig. 1.

Schematic representation of the active NAPDH–oxidase complex. The flavocytochrome b₅₅₈ consists of two components, gp91^phox and p22^phox, which reside in the membrane as a heterodimer. On activation of the cell, a complex of cytosolic components (see text) assembles at the membrane and induces electron transport from NADPH to molecular oxygen via flavin adenine dinucleotide (FAD) and haem groups. gp91^phox has also recently been shown to act as a voltage-gated H + channel which could participate in charge compensation. Termination of activity may be regulated by the GTPase, p21rac.

The first abnormalities defined for autosomal recessive CGD (AR-CGD) were deficiencies of two cytosolic proteins p47^phox and p67^phox[27]. Phosphorylation of both proteins coincides with activation of the NAPDH–oxidase, and is likely to be necessary for regulated assembly of the functional complex in vivo, as it is not necessary for activation in cell-free systems [28–34]. p40^phox exists in a complex with p47^phox and p67^phox in resting cytosol, and translocates to the membrane on cell activation, although the function of this component is not yet known. All three cytosolic phox proteins contain src-homology 3 (SH3) domains which provide considerable opportunity for intramolecular and intermolecular interaction between themselves, with the cytoplasmic tail of p22^phox, and with other signalling or cytoskeleton-associated molecules (reviewed in [36]). The Rho family GTPase p21rac was co-purified with rho-GDI (GDP-dissociation inhibitor) from a cytosolic fraction prepared from guinea pig macrophages, and found to be essential for cell-free activation of the NAPDH–oxidase [37]. In contrast to p21rac1, which is ubiquitously expressed, p21rac2 is restricted to myeloid lineage cells. However, studies in p21rac2-deficient mice generated by gene targeting indicate that activation of the NAPDH–oxidase by p21rac2 is stimulus-specific [38]. This raises the possibility that other GTPases, including p21rac1, could activate the system in other circumstances. Another GTP-binding protein, Rap1A, has been shown to co-purify with the flavocytochrome b₅₅₈, although its role in NAPDH–oxidase function has not been clarified [39,40].

ACTIVATION OF ELECTRON TRANSPORT

The flavocytochrome b₅₅₈ almost certainly comprises the complete electron transporting system and forms the membrane docking site for the cytosolic components. In resting neutrophils, the plasma membrane is devoid of flavocytochrome b₅₅₈ which resides almost exclusively in specialized light density intracellular vesicles and within the membranes of specific granules [41,42]. When the cell is activated the plasma membrane invaginates to form the phagocytic vacuole with which vesicles containing flavocytochrome b₅₅₈ fuse. The cytosolic components form an activation complex which translocates to the membrane to associate with the flavocytochrome b₅₅₈. Cytosolic residues on both p22^phox and gp91^phox appear to be important for this interaction, which is thought to be mediated predominantly by p47^phox[43,44]. p67^phox probably localizes by virtue of interaction with p47^phox, as it does not do so in its absence. p21rac has been shown to interact directly with p67^phox, and they may therefore translocate together [45–47]. In contrast, translocation of p21rac2 has been shown to occur in the absence of either p47^phox or p67^phox, suggesting that alternative mechanisms exist for oxidase activation [48]. Assembly of the complete NAPDH–oxidase complex may induce conformational changes in flavocytochrome b₅₅₈ which permit binding of the substrate NADPH, and which are energetically favourable for electron transport. Other proteins, for example p40^phox, are not essential in cell-free systems, and are therefore more likely to be important for the stabilization of individual component proteins or for their initial assembly into an activation complex at the cell membrane.

The assembled NAPDH–oxidase transfers electrons to molecular oxygen, which is reduced to a free radical superoxide anion O₂⁻ within the phagocytic vacuole. O₂⁻ participates in a series of reactions that result in generation of hydrogen peroxide, hypohalous acids, and possibly other more reactive oxygen radicals, including those derived from nitric oxide. The discovery of an intrinsic NAPDH–oxidase hydrogen ion conductance also lends support to the idea that a major function of the system is to regulate phagosomal pH, and therefore the activity of secreted granule proteins [20–26,49]. NAPDH–oxidase activity occurs transiently after phagocytosis and is dependent upon continued receptor occupancy, and continued association of oxidase components at the membrane. Termination of the response is not simply mediated by the release of p47^phox and p67^phox into the cytosol, as these remain in association with the membrane well after the burst is over [34]. One potential regulator of termination is the GTP-binding protein p21rac, the activity of which is governed by the phosphorylation state of the guanine nucleotide.

ANIMAL MODELS

Murine models have been developed for CGD (gp91^phox and p47^phox) by gene targeting [50,51]. Affected mice lack phagocyte superoxide production, and are susceptible to infection with S. aureus and Aspergillus fumigatus, reflecting a similar phenotype to that seen in human CGD. These have therefore become useful models for study of NAPDH–oxidase function in both phagocytic and non-phagocytic cells, and for development of novel therapies. To investigate the contribution of mechanisms other than generation of reactive oxygen or nitrogen species to the killing of bacteria, mice have been generated that are deficient in both gp91^phox and inducible nitric oxide synthase (NOS2) [52]. These mice develop massive abscesses containing mainly commensal enteric bacteria, even when reared under pathogen-free conditions. Interestingly, though susceptible to virulent Listeria, doubly deficient animals retained partial killing activity against S. typhimurium, Escherichia coli and attenuated Listeria, suggesting that alternative mechanisms exist. More recently, mice deficient in the granule serine proteases, elastase and cathepsin G, have been shown to be susceptible to fungal infection, indicating the importance of non-oxidative effector mechanisms in host defence [53]. Neutrophils obtained from mice deficient in the Rho family GTPase Rac2 display significant defects of chemotaxis, adhesion, superoxide production, and increased mortality when challenged with A. fumigatus [38]. The suggestion that expression of other genes may be important for the development of inflammatory complications is supported by studies showing that administration of sterilized aspergillus hyphae to X-CGD lungs in vivo is characterized by exaggerated production of IL-1β, tumour necrosis factor-alpha (TNF-α), and the chemokine KC [54]. Therefore, for reasons unrelated to active infection, the cytokine response in X-CGD mice is proinflammatory, and may reflect the development of sterile granulomatous inflammation seen in patients.

MOLECULAR PATHOLOGY

The genes encoding both subunits of the flavocytochrome b₅₅₈, and four cytosolic factors, p40^phox, p47 ^phox, p67^phox, and p21rac2, have been cloned, and molecular lesions identified in all but p40^phox[55–58]. The distribution of genetic lesions within CGD patients is shown in Table 1. Molecular lesions on the X chromosome at CYBB account for the majority of cases of CGD (X91⁰CGD). The mutations are heterogeneous, and are unique to individual families in over 90% of cases [59–62]. As expected, missense mutations result in considerable heterogeneity of phenotype and in some cases expression of a mutant flavocytochrome b₅₅₈ with residual biochemical activity (variant CGD). In rare cases, extreme patterns of X-inactivation can result in an X-CGD phenotype in girls. Molecular lesions at CYBA are also heterogeneous. The second most common cause of CGD is A47⁰CGD. In contrast to other forms of CGD, a GT dinucleotide deletion at a GTGT repeat at the boundary between the first intron and second exon is found in the majority of mutant alleles, resulting in a chain terminator at amino acid residue 51 [63]. This has now been found to arise from recombination events between the p47^phox gene and highly homologous pseudogenes, which contain the GT deletion [64]. For A47⁰CGD, carrier testing is therefore problematic as all normal individuals possess the deleted pseudogenes. Most recently, two patients with recurrent bacterial infections, and associated abnormalities of cell migration, degranulation, and O₂-production have been reported to have a dominant-negative mutation in p21rac2 [38].

Table 1.

Characteristics and distribution of gene defects in chronic granulomatous disease (CGD)

Component	gp91phox	p22phox	p47phox	p67phox	p40phox	p21rac2
Disease	X-linked	Autosomal	Autosomal	Autosomal	Not known	Autosomal
	X91 CGD*	recessive	recessive	recessive		dominant
		A22 CGD*	A47 CGD*	A67 CGD*
Results from USA and  European studies of  a total of 122 families†:	X91069 (56%)	A2207 (6%)	A47028 (23%)	A6707 (6%)	Not described
Numbers of affected	X91−8 (7%)	A22+1 (1%)
families, and incidence	X91+2 (2%)
Genetic locus	CYBB	CYBA	NCF-1	NCF-2
Chromosomal location	Xp21.1	16q24	7q11.23	1q25	22q13.1	22q12
Gene/mRNA size	30 kb/4·7 kb	8·5 kb/0·8 kb	15·2 kb/1·4 kb	37 kb/2·4 kb	18 kb/1·2 kb	18 kb/1·5 kb
Exons	13	6	11	16	10	7
Tissue specificity	Myeloid Low levels  in mesangial cells,  and some B lymphocytes.  Pulmonary  neuroepithelial bodies.	mRNA ubiquitous  Protein expression only  in presence of gp91phox	Myeloid	Myeloid	Myeloid	p21rac2,myeloid

^*Accepted classification of CGD, in which A or X denote inheritance pattern. This is followed by the molecular weight of the affected component in kD. The superscript refers to the level of detectable immunoreactive protein: (0) indicates no protein, (−) indicates diminished protein, and (+) indicates normal levels of defective protein.

^†Adapted from reference [140].

In a recent study of 129 patients with CGD, genetic modifiers (single nucleotide polymorphisms (SNP)) of disease severity were identified and linked to the development of inflammatory complications [65]. In particular, physiologically relevant polymorphisms were identified in myeloperoxidase and Fcγ RIIIb, and were strongly associated with an increased risk of gastrointestinal inflammation. The development of other inflammatory complications was also shown to be associated with polymorphisms in the mannose binding lectin gene (MBL), and less so with Fcγ RIIa genotype.

CLINICAL PRESENTATION

Estimates of the incidence of CGD vary geographically and range from 1 in 200 000 in the USA [66] and 1 in 287 000 in Japan [67] to 1 in 450 000 in Sweden [68]. It is likely that the disease continues to be underdiagnosed. The hallmark of the clinical presentation of CGD is recurrent infections at epithelial surfaces in direct contact with the environment such as the skin, lungs and gut. The majority of affected individuals are diagnosed before the age of 2 years [69,70], although patients may remain undiagnosed until adult life despite the early onset of symptoms [71]. Lymphadenitis is the most common presenting feature [72,73], followed by skin abscesses, pneumonia and hepatomegaly. Diarrhoea and sepsis syndromes may also be a presenting feature and CGD may be misdiagnosed as Crohn's disease when diarrhoea and colitis are the initial findings [74]. A registry established in the USA contains data on 368 patients and has documented the clinical complications [66]. The most commonly described complications in this cohort are pneumonia (79%), followed by lymphadenitis (53%), subcutaneous abscess (42%), liver abscess (27%), osteomyelitis (25%) and sepsis (18%). Long-term follow up of CGD has revealed that with improved survival or increasing age, symptoms of obstruction in hollow organs or inflammation not obviously associated with infection may become prominent [70]. These include colitis, gastric outlet obstruction secondary to granulomatous involvement of the stomach wall, urinary tract obstruction secondary to granulomatous cystitis, and oesophageal obstruction secondary to granulomatous involvement of the oesophageal wall. Other rare complications of unknown aetiology such as pericardial effusion [75] and chorioretinitis [76] are well described. Individuals with X-CGD are said to have a more severe clinical phenotype and increased mortality than those with A47⁰CGD[66].

The pathogens responsible for the majority of infections in CGD are characteristic bacteria and fungi. Catalase-positive bacteria are the most important and include S. aureus and the Gram-negative enterobacteriacea including Salmonella, Klebsiella, Aerobacter and Serratia. Pseudomonas (Burkholderia) cepacia is increasingly being recognized as an important pathogen [77]. Catalase-negative bacteria such as streptococci rarely cause problems, probably because small amounts of H₂O₂ are produced by the bacteria within the phagocytic vacuole. Aspergillus (predominantly fumigatus) is the fungus most commonly implicated in CGD, although reports of infections with other members of the Aspergillus family such as A. nidulans [78–79] and other fungi such as Candida albicans, Scedosporium apiospernum [80] and Chyrosporium zonatum [81] are prevalent.

Patients are often anaemic with an iron-deficient pattern, although this may remain stubbornly resistant to iron supplementation. Some patients with bowel disease may also malabsorb vitamin B₁₂. A raised erythrocyte sedimentation rate (ESR) can be found, even in the apparently uninfected patients, and probably reflects ongoing, subclinical inflammation. The level of C-reactive protein is rarely raised when the patient is apparently infection-free and thus remains a better marker of bacterial sepsis in the acutely ill patient.

DIAGNOSIS

The functional diagnosis of CGD can be made by demonstrating the inability of phagocytes from affected individuals to produce a normal respiratory burst. This is conveniently done by the phorbol myristate acetate-stimulated nitroblue tetrazolium (NBT) test [82]. In this test, incubation of activated neutrophils with the yellow dye NBT results in the accumulation of dark blue pigment, formazan, within normal phagocytes, although proper interpretation relies on an experienced observer. For X-CGD, carrier status can be determined by observing a mixed population of NBT-positive and NBT-negative cells (assuming that X-inactivation is random). A flow cytometric test for superoxide production was first described in 1985 [83], being subsequently refined by many groups [84], and is now available in a kit form (Bursttest (Phagoburst®); Orpogen, Heidelberg, Germany) [85]. It relies on the reduction of dihydrorhodamine by stimulated phagocytes in heparinized whole blood and provides a quick and convenient method for semiquantitatively determining NAPDH–oxidase function. It can also accurately detect carrier status in X-CGD. Immunoblotting for individual components of the NAPDH–oxidase can help identify the defective protein in the majority of cases (remembering that mutations in either subunit of flavocytochrome b₅₅₈ usually result in the absence of both), while confirmation of the molecular defect can be obtained by sequencing of the relevant gene. A database of CGD mutations has been set up [62] and can be accessed via the internet (http://http://www.uta.fi/laitokset/imt/bioinfo/XCGDbase/).

Prenatal diagnosis can be made on tissue obtained by chorionic villus sampling in the first trimester. This strategy is dependent on identification of specific family based mutations or on informative polymorphisms [86–88]. It may also be possible to detect the presence of individual NAPDH–oxidase components in chorion-derived macrophages with specific antibody [89]. Alternatively, prenatal diagnosis can be reliably determined by measurement of NAPDH–oxidase activity in fetal blood samples taken during the second trimester.

CLINICAL MANAGEMENT

Prophylactic treatments

Live bacterial vaccines such as bacille Calmette–Guérin (BCG) should be avoided. The mainstay of therapy is adequate anti-microbial prophylaxis. The most common antibiotic used for chemoprophylaxis is cotrimoxazole, which has broad activity against the pathogens encountered in CGD, is lipophilic and is thus concentrated inside cells, and is well tolerated because it does not affect anaerobic gut flora [90–92]. No randomized controlled trials of cotrimoxazole have been performed but a number of small studies have shown a reduction in the incidence of serious infections in patients on regular prophylaxis [93–96]. Anti-fungal prophylaxis can be achieved with itraconazole, a triazole anti-fungal with good activity against Aspergillus species. While no double blind, randomized, placebo controlled trials of itraconazole in CGD have been performed, a number of studies suggest it is efficacious in the prevention of Aspergillus infection [97,98]. Many anecdotal reports of its efficacy in the treatment of established Aspergillus infection have been published [99–101]. Interferon-gamma (IFN-γ) is an immunomodulatory cytokine which was shown to partially restore NAPDH–oxidase activity in the neutrophils and monocytes of selected patients with X-linked CGD [102–105]. On the basis of these preliminary results, IFN-γ was evaluated in a large multicentre study for its efficacy and potential toxicity in the prevention of infection in CGD [106]. While overall the study demonstrated significant efficacy in the IFN-γ arm, there was a powerful centre effect with the European study sites demonstrating similar rates of infection in the IFN-γ and control arms. Other European centres subsequently demonstrated incidence rates of infection using antibiotic prophylaxis that were lower than those seen in the IFN-γ arm in the USA sites [107]. In addition, there was a failure to demonstrate restoration of NAPDH–oxidase activity in patients enrolled in the clinical study [108,109]. While ongoing studies in Europe and the USA have demonstrated the safety of IFN-γ prophylaxis [110,111], it is not used universally.

Treatment of acute infection

Infections in CGD need to be managed promptly and aggressively with appropriate intravenous antibiotics or anti-fungal agents where necessary. In many sepsis syndromes, therapy must be empirical as a pathogen is rarely isolated. Ciprofloxacin is a useful first line agent with an appropriate spectrum of activity and an advantage in being lipophilic, thus achieving a high intracellular concentration [112,113]. Together with the Gram-positive cover provided by flucloxacillin, they form a useful first line empirical strategy for bacterial sepsis. Amphotericin is the mainstay of anti-fungal treatment; liposomal amphotericin at high dose (5 mg/kg) may be required to eradicate established Aspergillus infection [114] although itraconazole is also useful [99,100,115]. IFN-γ has been used in the treatment of deep seated infections (such as liver abscesses) that are refractory to conventional therapy, although efficacy remains anecdotal [99,115–119]. Leukophoresed normal granulocytes (matched for ABO and Rhesus antigens) are also useful in refractory situations [120,121], although the risk of alloimmunization remains significant [122].

The mainstay of therapy for inflammatory complications in CGD, including colitis and those leading to hollow organ obstruction, is immunomodulatory. Topical and systemic steroids have been used to treat colitis and obstructive complications [123] such as those encountered in the oesophagus [124] and bladder [125]. Some patients with mild enteritis or cystitis may be exquisitely sensitive to steroids (which may provide relief within the first 12 h of treatment), although relapse once off steroids may occur. A significant number of patients unfortunately remain dependent on steroids. In these cases, a variety of additional agents including sulphasalazine, cyclosporin [126], thalidomide [127] and granulocyte colony-stimulating factor [128] have been used.

OUTCOME

In 1967 only 21% of children with CGD were said to survive beyond 5 years of age, although the actuarial survival to 8 years had improved to 70% in a cohort born between 1967 and 1977 [93]. In this same study a cohort born after 1978 demonstrated an improved survival to 8 years of 92%. In a report by Finn et al. of CGD patients born over a 32-year period, 50% were alive through to the third decade [69]. A recent report from Japan has demonstrated an increase in the mean age of survivors from 8 years in 1985 to 16 years in 1998, although the overall mortality remained unchanged at 23·1% over the 13 years of the study [67]. The reasons for the failure to improve overall mortality are multifactorial, but probably include the problem of compliance with medication in adolescents with CGD.

CURATIVE THERAPY

Bone marrow transplantation has been shown to be potentially curative in CGD, although it has been associated with unacceptably high rates of morbidity, mortality and graft failure except in patients with an HLA-identical sibling donor [129]. Attempts to reduce conditioning-related toxicity are continuing using non-myeloablative regimens [130]. For patients without an HLA-identical donor, gene therapy may soon be a possibility. A number of preclinical studies have established the potential efficacy of this strategy [131–138]. However, in clinical studies reconstitution of NAPDH–oxidase activity in peripheral blood neutrophils has been transient [139]. It is therefore likely that some form of conditioning of the patients will be required for successful engraftment of corrected stem cells.

PATIENT SUPPORT

The CGD Research Trust and Support Group is an active group based in the UK who also raise money for, and fund research into CGD (http://www.cgd.org.uk). They are part of the International Patient Organization for Primary Immunodeficiency (IPOPI). Their website has useful links to other organizations (http://www.ipopi.org). In the USA, a number of websites provide information about CGD, including one at http://www.healthlinkusa.com/72ent.htm, which has links to a variety of other CGD-related websites.