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(Journal of Leukocyte Biology. 2001;69:191-196.)
© 2001 by Society for Leukocyte Biology

Chronic granulomatous disease: more than the lack of superoxide?

Miklós Geiszt, András Kapus and Erzsébet Ligeti

Department of Physiology, Semmelweis University, H-1444 Budapest, P.O. Box 259, Hungary

Correspondence: Dr. Miklós Geiszt, Bldg. 10, Rm. 11N106, National Institutes of Health, Bethesda, MD 20892. E-mail: mgeiszt{at}nih.gov


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ABSTRACT
 
Chronic granulomatous disease (CGD) is an inherited disease characterized by severe and recurrent bacterial and fungal infections manifested in most cases in early childhood. Phagocytic cells of CGD patients are unable to produce superoxide anions, and their efficiency in bacterial killing is significantly impaired. Recent work has shown alterations in the electrophysiological properties of CGD granulocytes, which might contribute to the pathogenesis of the disease. The new aspects that we discuss in this review concern the proton channel function of gp91phox (the electron-transporting subunit of the NADPH oxidase) and the electrogenic activity of the active enzyme complex, which can affect the transmembrane trafficking of several ions. Based on the reviewed data, we also propose a hypothesis that the absence of a functional NADPH oxidase in CGD neutrophils could result in altered ion compositions within intracellular and intraphagosomal spaces during the process of phagocytosis.

Key Words: neutrophils • NADPH oxidase • proton channel • calcium • membrane potential


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INTRODUCTION
 
Chronic granulomatous disease (CGD) is an inherited immunodeficiency in which the patients’ phagocytes are unable to produce superoxide and its derivates [1 , 2 ]. Patients with CGD suffer from recurrent bacterial and fungal infections presented in the form of pneumonia, abscesses, and lymphadenitis. Most of the patients are diagnosed at early childhood (prevalence is 1:200,000), however some patients remain undiagnosed until adulthood. The failure of superoxide production in CGD is because of the defective function of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex. NADPH oxidase is a multicomponent enzyme that is dormant in resting cells but becomes highly active during the phagocytosis of invading pathogens [3 , 4 ]. Once activated, the enzyme transfers one electron from NADPH to molecular oxygen, resulting in the formation of superoxide anion (O2-). The enzyme is present in all professional phagocytes (neutrophils, eosinophils, macrophages) and also in B-lymphocytes, where its function remains elusive. The activated NADPH oxidase is composed of five components: the membrane-bound flavocytochrome b558; the cytosolic factors p47phox, p67phox, and p40phox; and the small GTPase Rac2 (Fig. 1 ). Cytochrome b558 itself is a complex of two subunits, gp91phox and p22phox [4 ]. There are basically four different molecular defects, which can cause the CGD phenotype [1 ]. In about two-thirds of the patients, the gene encoding gp91phox is defective, and because of its chromosomal localization, this form is inherited in an X-linked manner, affecting mainly boys. Although in this form of the disease, the gene for the smaller subunit p22phox is intact, its expression is also decreased, leading to the complete absence of the whole cytochrome complex. The same, cytochrome-deficient phenotype results if the gene encoding p22phox is defective. However, this form is inherited in an autosomal recessive manner, and it is responsible for only 5% of all CGD cases. Inheritance of deficiency in cytosolic proteins p47phox and p67phox is autosomal-recessive and responsible for 20% and 5% of CGD cases, respectively. The diagnosis of CGD is a multistep procedure, starting with simple methods detecting the failure of superoxide production (dihydrorhodamine fluorescence assay, photometric measurement of cytochrome c reduction) and followed by the identification of the absent component with the use of specific antibodies. The final step of the diagnostic process now includes the characterization of the genomic defect responsible for the CGD phenotype [1 ]. Thanks to the intensive work of many laboratories, an extensive database now exists documenting the various mutations responsible for the CGD phenotype [5 ].



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  		Figure 1. The active NADPH oxidase is composed of five subunits. The enzyme complex drives electrons from NADPH to molecular oxygen, resulting in strong depolarization of the plasma membrane.


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UNUSUAL FINDINGS IN CGD
 
It is generally accepted that phagocytes of CGD patients fail to kill pathogens because of their inability to produce superoxide and its derivates. However, some observations suggest that other factors may also contribute to the failure of phagocytic killing in this disease.

One early observation is that despite the common finding in all forms of CGD, i.e., the absence or serious reduction of superoxide production, the p47phox-deficient form of CGD usually follows a less severe clinical course than cytochrome b558-deficient forms of the disease [6 , 7 ]. Residual superoxide production observed in p47phox-deficient CGD neutrophils [8 , 9 ] may be responsible for the above-mentioned difference, however a role for other factors, including an additional function of cytochrome b558, cannot be excluded.

Superoxide anions themselves are only weakly anti-pathogenic, and for efficient killing of pathogens, first, superoxide has to be converted to more toxic metabolites. One of the key enzymes involved in this metabolic process is myeloperoxidase (MPO), which catalyzes the formation of HOCl from H2O2. It is interesting that MPO deficiency—a more frequent condition than CGD (prevalence is 1:2000)—usually manifests only in mild infections or remains essentially asymptomatic [10 ]. Although increased superoxide production or processing of superoxide through alternative pathways (e.g., nitroso compounds) may partially compensate for the absence of MPO, this apparent contradiction remains to be clarified.

A strong argument supporting the central role of superoxide in phagocytic killing is the observation that CGD patients are more sensitive to catalase-positive organisms than to species without this enzyme. It has been proposed that spontaneous H2O2 formation in catalase-negative pathogens may compensate for the defective NADPH oxidase function during phagocytosis, whereas catalase-positive organisms lack this supportive mechanism because they rapidly metabolise H2O2. Although this explanation is generally accepted, its accuracy has never been tested. Moreover, recently Chang et al. [11 ] showed that the virulence of Aspergillus nidulans in p47phox knockout mice was not decreased when the animals were infected by catalase-deficient mutants of the fungus. These observations are particularly important from a clinical standpoint, because Aspergillus species are the most frequent fungal pathogens affecting CGD patients [12 ]. Hopefully, in the near future, similar experiments will focus on the role of catalase enzymes in bacteria.


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ALTERED pH-HOMEOSTASIS IN CGD
 
Considering other factors involved in the pathogenesis of CGD, Anthony Segal et al. [13 ] suggested in the early 1980s that abnormal pH regulation within the phagosome of CGD phagocytes might have a role in defective killing. The basis of this assumption is that the initiation of superoxide production is accompanied normally by phagosomal alkalization as a result of the proton-acceptor function of superoxide anions. This pH change is proposed to be essential for the activation of granule enzymes within the phagosome. In CGD, however, the oxidase function is absent, and alkalization does not occur, resulting in impairment of the killing mechanisms. Segal et al. showed that correction of phagocytic pH to more physiological values restored the ability of CGD neutrophils to kill Staphylococcus aureus.

Some more recent findings suggest that intracellular/intraphagosomal pH regulation of CGD neutrophils might be abnormal not only because of the absent superoxide production but also because of altered H+ transport through the plasma membrane. Superoxide production is accompanied normally by a robust intracellular H+ production resulting from the oxidation of NADPH to NADP+ + H+. Neutrophil granulocytes possess three pathways for the removal of protons from the intracellular milieu: Na+-H+ exchange, H+-adenosinetriphosphatase (ATPase), and electrogenic proton transport [14 ]. This latter mechanism was first described by Henderson et al. [15 , 16 ]. They suggested that a proton-conducting channel might be responsible for the transfer of protons through the plasma membrane during activation of superoxide production. The proton transport was activated by low amounts of arachidonic acid and inhibited by divalent cations such as Zn2+ or Cd2+. Subsequent work from our laboratory demonstrated that phorbol 12-myristate 13-acetate (PMA), the most effective activator of superoxide production, is also a potent stimulus of this proton conductance [17 ]. The proton-conducting pathway of phagocytes was characterized by patch-clamp measurements, proving its electrogenic feature [18 19 20 ], however the molecular nature of the conductance remained elusive. Henderson et al. [21 ] could show that gp91phox functions as a proton channel when expressed in Chinese hamster ovary (CHO) cells, and even an N-terminal truncated form of the protein seems to be functional [22 ]. On the contrary, it was also demonstrated in patch-clamp experiments that gp91phox-deficient CGD monocytes still express a proton-conducting pathway [23 ], however PMA activation seemed to be disturbed in the CGD cells [24 ]. Furthermore, arachidonate activatable Zn2+- and Cd2+-sensitive, but PMA-resistant, electrogenic H+ transport was detected in T lymphocytes, where gp91phox protein was absent [25 ]. In a recent work, DeCoursey et al. [26 ] studied NADPH oxidase-related proton and electron currents in PMA-stimulated human neutrophils. The authors did not find any correlation between the PMA-induced electron current and proton conductance; therefore, they concluded that NADPH oxidase probably did not mediate the observed proton current. They also suggested that PMA stimulation probably modulates pre-existing proton channels rather than introducing new ones into the plasma membrane. On the contrary, a study by Banfi et al. [27 ] showed that two separate, proton-conducting pathways exist in phagocytes. The "basal" H+ conductance opens when the membrane potential becomes more positive than the H+ equilibrium potential and shows definite outward rectification, whereas the oxidase-associated conductance opens upon activation of the enzyme (e.g., by PMA) at a more negative potential than the H+ equilibrium potential and allows the movement of protons in both directions, following their electrochemical gradient.

Concerning the structural requirements for proton transport by gp91phox, site-directed mutagenesis experiments by Henderson and Meech [28 ] highlighted the importance of three histidine residues at positions 111, 115, and 119 in the third transmembrane domain of gp91phox. One of these histidines, His115, is also implicated in heme binding of gp91phox. The authors proposed a mechanism for proton transport through gp91phox, which includes the protonation-deprotonation of the histidine residue at 115, and residues at positions 111 and 119 are responsible for voltage-gating.

The importance of the missing H+-channel function in gp91phox-deficient CGD is unknown, however it might be an additional factor in the previously described difference between p47phox- and gp91phox-deficient CGD phenotypes.

The significance of gp91phox as a regulator of pH homeostasis was strengthened recently by the discovery of the mitogenic oxidase (Mox1), a homologue of gp91phox [29 ]. Mox1 is 56% identical to gp91phox and contains the structural elements, which are important in the electron transport activity of its phagocytic counterpart. Mox1 is expressed in the colon, prostate, uterus, and vascular smooth muscle and increases superoxide production and cell proliferation when overexpressed in NIH3T3 fibroblasts. Transfection of aortic smooth-muscle cells with antisense Mox1 decreased superoxide production and inhibited serum-dependent growth of the cells.

It is interesting that an alternatively spliced variant of the Mox1 transcript was also described [30 ], encoding a protein named NOH-1S (NADPH oxidase homolog 1, short variant), which is a 191 amino acid protein, containing the first three transmembrane domains of Mox1 but having a different fourth transmembrane region followed by a short C-terminal sequence. The three conserved, histidine residues, which are implicated in the proton channel function of gp91phox, are located at the same position in NOH-1S (positions 111, 115, and 119), however in the absence of the fifth transmembrane domain, His115 probably does not bind heme but might serve solely as proton translocator. When NOH-1S is expressed in HEK-293 cells, it shows voltage-dependent H+ channel activity, which is sensitive to Zn2+. It is interesting that aside from the tissues where Mox1 is expressed, the NOH-1S message was also detected in human leukaemia (HL-60) cells and leukocytes. The fact that the H+ channel function can be dissociated from the oxidase activity in the form of a separate protein also highlights the importance of the H+ channel activity of gp91phox.

Recently another novel homologue of gp91phox, Renox (Renal oxidase), was isolated and found to be expressed at high levels in kidney tubular cells [31 ]. Renox is a 578 amino acid-long protein showing 40% identity and 57% similarity to gp91phox. Renox shares all the structural features of gp91phox and Mox1, which are considered to be essential for NADPH oxidase function. NIH-3T3 fibroblasts overexpressing Renox showed increased superoxide production and in contrast to Mox1-transfected fibroblasts, showed reduced proliferation rate and signs of cellular senescence. Based on its localization and enzymatic function, Renox is a likely candidate for oxygen-sensor function in the kidney and might be responsible for the regulation of erythropoietin synthesis. When considering a possible proton channel function of Renox, it is noteworthy that Renox contains a phenylalanine residue in the position corresponding to His111 of gp91phox, although the other histidine residues are tightly conserved. Based on this structural difference, Renox offers an exciting experimental tool in the analysis of structural requirements for the proton transport function of gp91phox and its homologues.


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THE ELECTROGENIC NATURE OF THE NADPH OXIDASE
 
Recent studies raise the possibility of further functional abnormalities of CGD phagocytes that were not considered earlier to play a role in the pathogenesis of the disease. It was described in the early 1980s that stimulation of neutrophils by certain agonists leads to depolarization of the cells [32 ]. It is interesting that this membrane-potential response was absent in CGD neutrophils, however this defect was not coupled to the absent oxidase function. On the contrary, some early works suggested that the lack of superoxide production in CGD might be because of the lack of depolarization. It soon appeared that depolarization of neutrophils by other means does not stimulate superoxide production [33 ], and researchers’ interest in the membrane potential changes of phagocytes vanished for a time. The idea of an electrogenic oxidase first came from the work of Henderson et al. [15 ]. They showed that the extent of depolarization following the addition of PMA was reduced by DPI, an inhibitor of the NADPH oxidase. This observation, together with the fact that CGD neutrophils do not depolarize, suggested that superoxide production is responsible for the membrane potential response. The action of the NADPH oxidase is thought to be electrogenic, because the enzyme transfers electrons from the intracellular space to the extracellular/intraphagosomal milieu, and protons cannot follow electrons in equivalent amounts (Fig. 1) .

These findings argued strongly for an electrogenic, NADPH oxidase, however this property was not directly assessed until recently. The first direct evidence comes from the work of Schrenzel et al. [34 ], who were able to detect the electron current generated by the NADPH oxidase by patch-clamp measurements. Working on human eosinophils, they detected an inward current, which was inhibited by DPI and was absent in CGD eosinophils. Furthermore, they could demonstrate the absolute dependence of the current on the availability of NADPH on the cytosolic side. They also described the activation of the current by N-formyl-methionyl-leucyl-phenylalanine (fMLP), a well-known stimulus of the NADPH oxidase. It is interesting that the current was inward over the entire tested voltage range (-100 mV–+60 mV), indicating an exceptionally strong charge-separator capability of the oxidase. Previously, the oxidase-generated depolarization was measured only by fluorescent techniques, which do not allow accurate calibration. However, even with these traditional techniques, it seemed possible that granulocytes depolarize to positive membrane-potential values during the activation of the oxidase. According to the electrophysiological measurements of Schrenzel et al. [34 ], this value is +25 mV–+30 mV, which is comparable with the membrane potential response of excitable cells producing action potentials. The most recent experiments carried out with an improved fluorescent technique showed that the membrane potential of PMA-activated neutrophils could reach +58 mV [35 ].


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EVIDENCE FOR DISTURBED Ca2+ SIGNALING IN CGD NEUTROPHILS
 
What are the possible consequences of this huge depolarization, and what could happen if this response was completely absent during phagocytosis? Although these questions cannot be answered fully as yet, recent studies from our laboratory indicate that the absence of agonist-induced depolarization in CGD granulocytes may have a profound effect on the Ca2+ signaling of these cells [36 ].

When neutrophils are stimulated by various receptor agonists, an increase of intracellular calcium concentration ([Ca2+]i) occurs. This Ca2+ transient has an important role in the transduction of extracellular signals, leading to the stimulation of different effector responses (e.g., degranulation, superoxide production) of the cells [37 ]. The agonist-induced Ca2+ signal of neutrophils is composed of two components: a rapid, transient increase of [Ca2+]i as a result of Ca2+ release from the internal stores and a more sustained elevation of [Ca2+]i because of Ca2+ influx from the extracellular space. Neutrophil granulocytes do not express voltage-dependent Ca2+ channels, but the inositol triphosphate (IP3)-mediated emptying of internal Ca2+ stores induces Ca2+ influx from the extracellular space [38 ]. This mechanism of calcium entry is referred to as capacitative or store-operated Ca2+ entry [39 ], which seems to be a major Ca2+-uptake pathway in almost every mammalian cell type. In spite of intensive efforts, the exact mechanism of capacitative Ca2+ influx is still unknown, however there is growing evidence that IP3-induced emptying of the internal stores might result a conformational change of the IP3-receptor structure, which then activates, either directly or indirectly, a plasma membrane Ca2+ channel [40 ]. Other models of capacitative Ca2+ influx propose that emptying the internal stores leads to channel activation through a soluble messenger substance released from the empty stores. In human neutrophils, agonists of various chemotactic receptors [e.g., fMLP and platelet-activating factor (PAF)] initiate Ca2+ influx by emptying the internal stores. It is interesting that fMLP but not PAF inhibits capacitative Ca2+ influx by a poorly understood mechanism [41 ]. Thus, it appears that the net calcium entry into neutrophils following the fMLP stimulus results from the concurrent presence of activating and inhibiting mechanisms. The protein kinase C activator PMA was also shown to inhibit the capacitative pathway of neutrophils [42 ]. fMLP and PMA are well-known, effective stimuli of the superoxide-producing, NADPH oxidase; therefore, we asked whether a connection exists between inhibition of the calcium influx and stimulation of superoxide production.

Generation of superoxide by the NADPH oxidase results in depolarization of the cells because of transmembrane electron transfer from the intracellular space to the extracellular milieu. Depolarization was shown to inhibit Ca2+ influx in many cell types; therefore, we studied if a relationship exists between the inhibition of Ca2+ influx and NADPH oxidase-induced depolarization. Pharmacologically induced manipulation of the membrane potential revealed a close relationship between depolarization and inhibition of Ca2+ influx [36 ] (Fig. 2 A ). We have also demonstrated that the agonist-mediated inhibition of capacitative calcium influx is absent in the neutrophils of patients suffering from chronic granulomatous disease (Fig. 2B) . As a consequence of this regulatory defect, CGD neutrophils start to accumulate calcium from the extracellular space at an earlier time-point than healthy neutrophils, and the internal Ca2+ stores of CGD neutrophils become refilled more rapidly following receptor stimulation. The pathophysiological significance of this finding and its contribution to the CGD phenotype are currently unknown, however capacitative Ca2+ influx was shown to have a role in regulation of cytokine production of neutrophils [43 ], and the calcium content of internal stores might control the activity of phospholipase A2, an enzyme that has a key role in the inflammatory response [44 ].



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  		Figure 2. (A) In healthy cells, the NADPH oxidase-induced depolarization inhibits Ca2+ influx through the capacitative pathway. (B) In CGD neutrophils, the absence of oxidase-induced depolarization results in increased Ca2+ entry into the cells.


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POSSIBLE ALTERATION OF THE INTRACELLULAR AND INTRAPHAGOSOMAL ENVIRONMENT IN CGD
 
Furthermore, we suppose that the absence of an electrogenic, NADPH oxidase in CGD neutrophils probably does not affect Ca2+ homeostasis solely but may result in an anomaly in the transport of other ions as well. It is a well-known fact that the movement of ions across biological membranes is determined by their charge, concentration (chemical) gradient, the membrane’s permeability, and the actual membrane potential. In neutrophils, an agonist, such as fMLP, which turns on the oxidase and depolarizes the cells, also induces the activation of several ion conductances. The existence of fMLP-activated K+ and Cl- conductances is well-documented in human neutrophils [45 , 46 ], and the presence of other electrogenic transporters, such as the Na+-Ca2+ exchanger was also shown [47 ]. Because the activation of these transport processes parallels the activation of the NADPH oxidase, it is likely that the net movement of these ions is affected largely by the depolarization generated by the NADPH oxidase (Fig. 3 ). If this is the case, then the movement of these ions is probably altered in CGD neutrophils, resulting in a complex abnormality of the intracellular milieu of the cells.



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  		Figure 3. NADPH oxidase probably affects the movement of several ions among the extracellular, intracellular, and intraphagosomal spaces. Depending on its charge and concentration gradient, the oxidase-induced depolarization can increase (thick arrows) or decrease (thin arrows) the net transport of a particular ion.

One further interesting possibility becomes apparent if we consider that in vivo the activation of superoxide production occurs mainly during phagocytosis when stimuli (formyl peptides, immune complexes) that activate the oxidase act on the phagosomal membrane. During this process, NADPH oxidase becomes assembled in the phagosomal membrane and directs electrons in the form of superoxide to the interior of the phagosome [2 ]. The charge separation between the two compartments (intraphagosomal and intracellular) probably affects the movement of other ions across the phagosomal membrane. If the activation of different ion transport mechanisms occurs in the absence of a functional charge separator, as it happens in CGD, then it can result in abnormal intraphagosomal concentration of several ions (Fig. 3) ; e.g., extracellular Ca2+ trapped in the phagosome may escape, lowering the effective concentration around the phagocytosed particle. Thus, the absence of an active NADPH oxidase might be fatal not only because of the absence of reactive oxygen species but also because of the altered intraphagosomal environment. The importance of appropriate ion concentration in antimicrobial, peptide-defensin functioning was highlighted recently in the case of another inherited disease, cystic fibrosis. It was suggested that the often-fatal respiratory tract infections observed in cystic fibrosis might result from the malfunctioning of defensins in the abnormal, high salt-containing fluid film covering the respiratory epithelium [48 ]. Although in cystic fibrosis the change in total ionic strength seems to be responsible for the observed abnormalities, it illustrates the importance of appropriate ion environment for efficient microbial killing.

We believe that the ion homeostasis of CGD granulocytes should be analyzed carefully in future experiments so that we might gain a better understanding of the pathogenesis and effective treatment [49 ] of chronic granulomatous disease.


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ACKNOWLEDGEMENTS
 
Experimental work in the authors’ laboratory has been supported by the Hungarian National Fund (OTKA), the Ministry of Welfare (ETT), and the Ministry of Education (FKFP). We thank Dr. Thomas L. Leto and Dr. Steven M. Holland for inspiring discussions and helpful suggestions.


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FOOTNOTES
 
Current address of András Kapus: Department of Surgery, Toronto General Hospital and the University of Toronto, Toronto, Ontario M5G 2C4, Canada.

Received June 22, 2000; revised October 18, 2000; accepted October 20, 2000.


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